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Sample records for a1d l-type ca

  1. L-type Ca2+ channels in the heart: structure and regulation.

    PubMed

    Treinys, Rimantas; Jurevicius, Jonas

    2008-01-01

    This review analyzes the structure and regulation mechanisms of voltage-dependent L-type Ca(2+) channel in the heart. L-type Ca(2+) channels in the heart are composed of four different polypeptide subunits, and the pore-forming subunit alpha(1) is the most important part of the channel. In cardiac myocytes, Ca(2+) enter cell cytoplasm from extracellular space mainly through L-type Ca(2+) channels; these channels are very important system in heart Ca(2+) uptake regulation. L-type Ca(2+) channels are responsible for the activation of sarcoplasmic reticulum channels (RyR2) and force of muscle contraction generation in heart; hence, activity of the heart depends on L-type Ca(2+) channels. Phosphorylation of channel-forming subunits by different kinases is one of the most important ways to change the activity of L-type Ca(2+) channel. Additionally, the activity of L-type Ca(2+) channels depends on Ca(2+) concentration in cytoplasm. Ca(2+) current in cardiac cells can facilitate, and this process is regulated by phosphorylation of L-type Ca(2+) channels and intracellular Ca(2+) concentration. Disturbances in cellular Ca(2+) transport and regulation of L-type Ca(2+) channels are directly related to heart diseases, life quality, and life span.

  2. Localized Calcineurin Confers Ca2+-Dependent Inactivation Upon Neuronal L-Type Ca2+ Channels

    PubMed Central

    Oliveria, Seth F.; Dittmer, Philip J.; Youn, Dong-ho; Dell’Acqua, Mark L.; Sather, William A.

    2012-01-01

    Excitation-driven entry of Ca2+ through L-type voltage-gated Ca2+ channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of CaV1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to CaV1.2 channels by A-kinase anchoring proteins (AKAPs). CaV1.2 channels also undergo negative autoregulation via Ca2+-dependent inactivation (CDI), which strongly limits Ca2+ entry. An abundance of evidence indicates that CDI relies upon binding of Ca2+/calmodulin (CaM) to an IQ motif in the carboxy tail of CaV1.2 L-type channels, a molecular mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. But our work reveals, in cultured hippocampal neurons and a heterologous expression system, that the Ca2+/CaM-activated phosphatase calcineurin (CaN) is scaffolded to CaV1.2 channels by the neuronal anchoring protein AKAP79/150 and that over-expression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX) impedes CDI. Interventions that suppress CaN activity—mutation in its catalytic site, antagonism with cyclosporine A or FK506, or intracellular perfusion with a peptide mimicking the sequence of the phosphatase’s autoinhibitory domain—interfere with normal CDI. In cultured hippocampal neurons from a ΔPIX knock-in mouse, CDI is absent. Results of experiments with the adenylyl cyclase stimulator forskolin and with the PKA inhibitor PKI suggest that Ca2+/CaM-activated CaN promotes CDI by reversing channel enhancement effectuated by kinases such as PKA. Hence our investigation of AKAP79/150-anchored CaN reconciles the CaM-based model of CDI with an earlier, seemingly contradictory model based on dephosphorylation signaling. PMID:23115171

  3. L-type Ca2+ channels in heart and brain

    PubMed Central

    Striessnig, Jörg; Pinggera, Alexandra; Kaur, Gurjot; Bock, Gabriella; Tuluc, Petronel

    2014-01-01

    L-type calcium channels (Cav1) represent one of the three major classes (Cav1–3) of voltage-gated calcium channels. They were identified as the target of clinically used calcium channel blockers (CCBs; so-called calcium antagonists) and were the first class accessible to biochemical characterization. Four of the 10 known α1 subunits (Cav1.1–Cav1.4) form the pore of L-type calcium channels (LTCCs) and contain the high-affinity drug-binding sites for dihydropyridines and other chemical classes of organic CCBs. In essentially all electrically excitable cells one or more of these LTCC isoforms is expressed, and therefore it is not surprising that many body functions including muscle, brain, endocrine, and sensory function depend on proper LTCC activity. Gene knockouts and inherited human diseases have allowed detailed insight into the physiological and pathophysiological role of these channels. Genome-wide association studies and analysis of human genomes are currently providing even more hints that even small changes of channel expression or activity may be associated with disease, such as psychiatric disease or cardiac arrhythmias. Therefore, it is important to understand the structure–function relationship of LTCC isoforms, their differential contribution to physiological function, as well as their fine-tuning by modulatory cellular processes. PMID:24683526

  4. Trifluoperazine enhancement of Ca2+-dependent inactivation of L-type Ca2+ currents in Helix aspersa neurons.

    PubMed

    Cruzblanca, H; Gamiño, S M; Bernal, J; Alvarez-Leefmans, F J

    1998-03-01

    The effects of trifluoperazine hydrochloride (TFP), a calmodulin antagonist, on L-type Ca2+ currents (L-type ICa2+) and their Ca(2+)-dependent inactivation, were studied in identified Helix aspersa neurons, using two microelectrode voltage clamp. Changes in [Ca2+]i were measured in unclamped fura-2 loaded neurons. Bath applied TFP produced a reversible and dose-dependent reduction in amplitude of L-type ICa2+ (IC50 = 28 microM). Using a double-pulse protocol, we found that TFP enhances the efficacy of Ca(2+)-dependent inactivation of L-type ICa2+. Trifluoperazine sulfoxide (50 microM), a TFP derivative with low calmodulin-antagonist activity, did not have any effects on either amplitude or inactivation of L-type ICa2+. TFP (20 microM) increased basal [Ca2+]i from 147 +/- 37 nM to 650 +/- 40 nM (N = 7). The increase in [Ca2+]i was prevented by removal of external Ca2+ and curtailed by depletion of caffeine-sensitive intracellular Ca2+ stores. Since TFP may also block protein kinase C (PKC), we tested the effect of a PKC activator (12-C-tetradecanoyl-phorbol-13-acetate) on L-type Ca2+ currents. This compound produced an increase in L-type ICa2+ without enhancing Ca(2+)-dependent inactivation. The results show that 1) TFP reduces L-type ICa2+ while enhancing the efficacy of Ca(2+)-dependent inactivation. 2) TFP produces an increase in basal [Ca2+]i which may contribute to the enhancement of Ca(2+)-dependent inactivation. 3) PKC up-regulates L-type ICa2+ without altering the efficacy of Ca(2+)-dependent inactivation. 4) The TFP effects cannot be attributed to its action as PKC blocker.

  5. Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca2+ channels

    PubMed Central

    González, G; Zaldívar, D; Carrillo, ED; Hernández, A; García, MC; Sánchez, JA

    2010-01-01

    BACKGROUND AND PURPOSE Pharmacological preconditioning (PPC) with mitochondrial ATP-sensitive K+ (mitoKATP) channel openers such as diazoxide, leads to cardioprotection against ischaemia. However, effects on Ca2+ homeostasis during PPC, particularly changes in Ca2+ channel activity, are poorly understood. We investigated the effects of PPC on cardiac L-type Ca2+ channels. EXPERIMENTAL APPROACH PPC was induced in isolated hearts and enzymatically dissociated cardiomyocytes from adult rats by preincubation with diazoxide. We measured reactive oxygen species (ROS) production and Ca2+ signals associated with action potentials using fluorescent probes, and L-type currents using a whole-cell patch-clamp technique. Levels of the α1c subunit of L-type channels in the cellular membrane were measured by Western blot. KEY RESULTS PPC was accompanied by a 50% reduction in α1c subunit levels, and by a reversible fall in L-type current amplitude and Ca2+ transients. These effects were prevented by the ROS scavenger N-acetyl-L-cysteine (NAC), or by the mitoKATP channel blocker 5-hydroxydecanoate (5-HD). PPC signficantly reduced infarct size, an effect blocked by NAC and 5-HD. Nifedipine also conferred protection against infarction when applied during the reperfusion period. Downregulation of the α1c subunit and Ca2+ channel function were prevented in part by the protease inhibitor leupeptin. CONCLUSIONS AND IMPLICATIONS PPC downregulated the α1c subunit, possibly through ROS. Downregulation involved increased degradation of the Ca2+ channel, which in turn reduced Ca2+ influx, which may attenuate Ca2+ overload during reperfusion. PMID:20636393

  6. 12-lipoxygenase regulates hippocampal long-term potentiation by modulating L-type Ca2+ channels

    PubMed Central

    DeCostanzo, Anthony J.; Voloshyna, Iryna; Rosen, Zev B.; Feinmark, Steven J.; Siegelbaum, Steven A.

    2010-01-01

    Although long-term potentiation (LTP) has been intensely studied, there is disagreement as to which molecules mediate and modulate LTP. This is partly due to the presence of mechanistically distinct forms of LTP that are induced by different patterns of stimulation and that depend on distinct Ca2+ sources. Here we report a novel role for the arachidonic acid-metabolizing enzyme 12-lipoxygenase (12-LO) in LTP at CA3-CA1 hippocampal synapses that is dependent on the pattern of tetanic stimulation. We find that 12-LO activity is required for the induction of LTP in response to a theta-burst stimulation (TBS) protocol, which depends on Ca2+ influx through both NMDA receptors and L-type voltage-gated Ca2+ channels. In contrast, LTP induced by 100 Hz tetanic stimulation, which requires Ca2+ influx through NMDA receptors but not L-type channels, does not require 12-LO. We find that 12-LO regulates LTP by enhancing postsynaptic somatodendritic Ca2+ influx through L-type channels during theta burst stimulation, an action exerted via 12(S)-HPETE, a downstream metabolite of 12-LO. These results help define the role of a long-disputed signaling enzyme in LTP. PMID:20130191

  7. Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate.

    PubMed

    Le Blanc, Catherine; Mironneau, Chantal; Barbot, Caroline; Henaff, Morgana; Bondeva, Tzvetanka; Wetzker, Reinhard; Macrez, Nathalie

    2004-08-06

    Modulation of voltage-gated L-type Ca2+ channels by phosphoinositide 3-kinase (PI3K) regulates Ca2+ entry and plays a crucial role in vascular excitation-contraction coupling. Angiotensin II (Ang II) activates Ca2+ entry by stimulating L-type Ca2+ channels through Gbeta-sensitive PI3K in portal vein myocytes. Moreover, PI3K and Ca2+ entry activation have been reported to be necessary for receptor tyrosine kinase-coupled and G protein-coupled receptor-induced DNA synthesis in vascular cells. We have previously shown that tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks are able to modulate vascular L-type Ca2+ channels. PI3Ks display 2 enzymatic activities: a lipid-kinase activity leading to the formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] and a serine-kinase activity. Here we show that exogenous PIP3 applied into the cell through the patch pipette is able to reproduce the Ca2+ channel-stimulating effect of Ang II and PI3Ks. Moreover, the Ang II-induced PI3K-mediated stimulation of Ca2+ channel and the resulting increase in cytosolic Ca2+ concentration are blocked by the anti-PIP3 antibody. Mutants of PI3K transfected into vascular myocytes also revealed the essential role of the lipid-kinase activity of PI3K in Ang II-induced Ca2+ responses. These results suggest that PIP3 is necessary and sufficient to activate a Ca2+ influx in vascular myocytes stimulated by Ang II.

  8. Surface L-type Ca2+ channel expression levels are increased in aged hippocampus

    PubMed Central

    Núñez-Santana, Félix Luis; Oh, Myongsoo Matthew; Antion, Marcia Diana; Lee, Amy; Hell, Johannes Wilhelm; Disterhoft, John Francis

    2014-01-01

    Age-related increase in L-type Ca2+ channel (LTCC) expression in hippocampal pyramidal neurons has been hypothesized to underlie the increased Ca2+ influx and subsequent reduced intrinsic neuronal excitability of these neurons that lead to age-related cognitive deficits. Here, using specific antibodies against Cav1.2 and Cav1.3 subunits of LTCCs, we systematically re-examined the expression of these proteins in the hippocampus from young (3 to 4 month old) and aged (30 to 32 month old) F344xBN rats. Western blot analysis of the total expression levels revealed significant reductions in both Cav1.2 and Cav1.3 subunits from all three major hippocampal regions of aged rats. Despite the decreases in total expression levels, surface biotinylation experiments revealed significantly higher proportion of expression on the plasma membrane of Cav1.2 in the CA1 and CA3 regions and of Cav1.3 in the CA3 region from aged rats. Furthermore, the surface biotinylation results were supported by immunohistochemical analysis that revealed significant increases in Cav1.2 immunoreactivity in the CA1 and CA3 regions of aged hippocampal pyramidal neurons. In addition, we found a significant increase in the level of phosphorylated Cav1.2 on the plasma membrane in the dentate gyrus of aged rats. Taken together, our present findings strongly suggest that age-related cognitive deficits cannot be attributed to a global change in L-type channel expression nor to the level of phosphorylation of Cav1.2 on the plasma membrane of hippocampal neurons. Rather, increased expression and density of LTCCs on the plasma membrane may underlie the age-related increase in L-type Ca2+ channel activity in CA1 pyramidal neurons. PMID:24033980

  9. The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry

    PubMed Central

    Bannister, Roger A.; Pessah, Isaac N.; Beam, Kurt G.

    2009-01-01

    The term excitation-coupled Ca2+ entry (ECCE) designates the entry of extracellular Ca2+ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca2+ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn2+. Here, we have examined the effects of these agents on the L-type Ca2+ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La3+, and Gd3+) and dantrolene all inhibited the L-type Ca2+ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca2+ channel displays permeability to Mn2+ in the absence of external Ca2+ and produces a Ca2+ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca2+ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca2+; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca2+-free solution and were unaffected by Ca2+ removal; and (3) after block of SR Ca2+ release by 200 μM ryanodine, normal myotubes still displayed a large Ca2+ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca2+ entry attributed to ECCE. PMID:19114636

  10. The skeletal L-type Ca(2+) current is a major contributor to excitation-coupled Ca(2+) entry.

    PubMed

    Bannister, Roger A; Pessah, Isaac N; Beam, Kurt G

    2009-01-01

    The term excitation-coupled Ca(2+) entry (ECCE) designates the entry of extracellular Ca(2+) into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca(2+) entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 microM), and is permeable to Mn(2+). Here, we have examined the effects of these agents on the L-type Ca(2+) current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La(3+), and Gd(3+)) and dantrolene all inhibited the L-type Ca(2+) current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 microM. Like ECCE, the L-type Ca(2+) channel displays permeability to Mn(2+) in the absence of external Ca(2+) and produces a Ca(2+) current that persists during prolonged ( approximately 10-second) depolarization. This current appears to contribute to the Ca(2+) transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca(2+); (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR alpha(1S) pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca(2+)-free solution and were unaffected by Ca(2+) removal; and (3) after block of SR Ca(2+) release by 200 microM ryanodine, normal myotubes still displayed a large Ca(2+) transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca(2+) entry attributed to ECCE.

  11. Tetrodotoxin blocks L-type Ca2+ channels in canine ventricular cardiomyocytes.

    PubMed

    Hegyi, Bence; Bárándi, László; Komáromi, István; Papp, Ferenc; Horváth, Balázs; Magyar, János; Bányász, Tamás; Krasznai, Zoltán; Szentandrássy, Norbert; Nánási, Péter P

    2012-08-01

    Tetrodotoxin (TTX) is believed to be the most selective inhibitor of voltage-gated fast Na(+) channels in excitable tissues, including nerve, skeletal muscle, and heart, although TTX sensitivity of the latter is lower than the former by at least three orders of magnitude. In the present study, the TTX sensitivity of L-type Ca(2+) current (I (Ca)) was studied in isolated canine ventricular cells using conventional voltage clamp and action potential voltage clamp techniques. TTX was found to block I (Ca) in a reversible manner without altering inactivation kinetics of I (Ca). Fitting results to the Hill equation, an IC(50) value of 55 ± 2 μM was obtained with a Hill coefficient of unity (1.0 ± s0.04). The current was fully abolished by 1 μM nisoldipine, indicating that it was really I (Ca). Under action potential voltage clamp conditions, the TTX-sensitive current displayed the typical fingerprint of I (Ca), which was absent in the presence of nisoldipine. Stick-and-ball models for Cav1.2 and Nav1.5 channel proteins were constructed to explain the differences observed between action of TTX on cardiac I (Ca) and I (Na). This is the first report demonstrating TTX to interact with L-type calcium current in the heart.

  12. Oestrogen directly inhibits the cardiovascular L-type Ca{sup 2+} channel Ca{sub v}1.2

    SciTech Connect

    Ullrich, Nina D. . E-mail: ullrich@pyl.unibe.ch; Koschak, Alexandra; MacLeod, Kenneth T.

    2007-09-21

    Oestrogen can modify the contractile function of vascular smooth muscle and cardiomyocytes. The negative inotropic actions of oestrogen on the heart and coronary vasculature appear to be mediated by L-type Ca{sup 2+} channel (Ca{sub v}1.2) inhibition, but the underlying mechanisms remain elusive. We tested the hypothesis that oestrogen directly inhibits the cardiovascular L-type Ca{sup 2+} current, I {sub CaL}. The effect of oestrogen on I {sub CaL} was measured in Ca{sub v}1.2-transfected HEK-293 cells using the whole-cell patch-clamp technique. The current revealed typical activation and inactivation profiles of nifedipine- and cadmium-sensitive I {sub CaL}. Oestrogen (50 {mu}M) rapidly reduced I {sub CaL} by 50% and shifted voltage-dependent activation and availability to more negative potentials. Furthermore, oestrogen blocked the Ca{sup 2+} channel in a rate-dependent way, exhibiting higher efficiency of block at higher stimulation frequencies. Our data suggest that oestrogen inhibits I {sub CaL} through direct interaction of the steroid with the channel protein.

  13. Molecular basis of proton block of L-type Ca2+ channels

    PubMed Central

    1996-01-01

    Hydrogen ions are important regulators of ion flux through voltage- gated Ca2+ channels but their site of action has been controversial. To identify molecular determinants of proton block of L-type Ca2+ channels, we combined site-directed mutagenesis and unitary current recordings from wild-type (WT) and mutant L-type Ca2+ channels expressed in Xenopus oocytes. WT channels in 150 mM K+ displayed two conductance states, deprotonated (140 pS) and protonated (45 pS), as found previously in native L-type Ca2+ channels. Proton block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-EIV) that form the locus of high affinity Ca2+ interaction. Glu(E)-->Gln(Q) substitution in either repeats I or III abolished the high-conductance state, as if the titration site had become permanently protonated. While the EIQ mutant displayed only an approximately 40 pS conductance, the EIIIQ mutant showed the approximately 40 pS conductance plus additional pH-sensitive transitions to an even lower conductance level. The EIVQ mutant exhibited the same deprotonated and protonated conductance states as WT, but with an accelerated rate of deprotonation. The EIIQ mutant was unusual in exhibiting three conductance states (approximately 145, 102, 50 pS, respectively). Occupancy of the low conductance state increased with external acidification, albeit much higher proton concentration was required than for WT. In contrast, the equilibrium between medium and high conductance levels was apparently pH-insensitive. We concluded that the protonation site in L-type Ca2+ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats I, II, and III, acting in concert. EIV lies to the cytoplasmic side of the site but exerts an additional stabilizing influence on protonation, most likely via electrostatic interaction. These findings are likely to hold for all voltage-gated Ca2+ channels and provide a simple molecular explanation

  14. Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca(2+) channels.

    PubMed

    Macrez, N; Mironneau, C; Carricaburu, V; Quignard, J F; Babich, A; Czupalla, C; Nürnberg, B; Mironneau, J

    2001-10-12

    Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

  15. Characterization of L-type Voltage-Gated Ca2+ Channel Expression and Function in Developing CA3 Pyramidal Neurons

    PubMed Central

    Morton, Russell A.; Norlin, Mackenzie S.; Vollmer, Cyndel C.; Valenzuela, C. Fernando

    2013-01-01

    Voltage gated calcium channels (VGCCs) play a major role during the development of the central nervous system (CNS). Ca2+ influx via VGCCs regulates axonal growth and neuronal migration as well as synaptic plasticity. Specifically, L-type VGCCs have been well characterized to be involved in the formation and refinement of the connections within the CA3 region of the hippocampus. The majority of the growth, formation, and refinement in the CNS occurs during the human third trimester. An equivalent developmental time period in rodents occurs during the first two weeks of post-natal life, and the expression pattern of L-type VGCCs during this time period has not been well characterized. In this study, we show that Cav1.2 channels are more highly expressed during this developmental period compared to adolescence (post-natal day 30) and that L-type VGCCs significantly contribute to the overall Ca2+ currents. These findings suggest that L-type VGCCs are functionally expressed during the crucial developmental period. PMID:23415785

  16. Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear

    PubMed Central

    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J.; Striessnig, Jörg; Singewald, Nicolas

    2008-01-01

    Dihydropyridine (DHP) L-type Ca2+ channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. CaV1.2 and CaV1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive CaV1.2 LTCCs (CaV1.2DHP−/− mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in CaV1.2DHP−/− mice, indicating that it is mediated by CaV1.2, but not by CaV1.3 LTCCs. Supporting this conclusion, CaV1.3-deficient mice (CaV1.3−/−) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral CaV1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in CaV1.2DHP−/− mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the CaV1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly CaV1.3) is not sufficient to accelerate extinction of conditioned fear in mice. PMID:18441296

  17. Na(+) permeates through L-type Ca(2+) channel in bovine airway smooth muscle.

    PubMed

    Sommer, Bettina; Flores-Soto, Edgar; Reyes-García, Jorge; Díaz-Hernández, Verónica; Carbajal, Verónica; Montaño, Luis M

    2016-07-05

    Membrane depolarization of airway smooth muscle (ASM) opens L-type voltage dependent Ca(2+) channels (L-VDCC) allowing Ca(2+) entrance to produce contraction. In Ca(2+) free conditions Na(+) permeates through L-VDCC in excitable and non-excitable cells and this phenomenon is annulled at µM Ca(2+) concentrations. Membrane depolarization also induces activation of Gq proteins and sarcoplasmic reticulum Ca(2+) release. In bovine ASM, KCl induced a transient contraction sensitive to nifedipine in Ca(2+)free medium, indicating an additional mechanism to the SR-Ca(2+) release. It is possible that Na(+) could permeate through L-VDCC in bovine ASM. KCl induced a transient contraction in Ca(2+) free medium with a fast intracellular Ca(2+) increment, reduced by TMB-8. This contraction was abolished by caffeine and CPA, diminished with nifedipine and augmented by Bay K8644. Increasing extracellular Na(+) concentration in tracheal myocytes, proportionally augmented the SBFI fluorescence ratio, suggesting an increment in the intracellular Na(+) concentration ([Na(+)]i). 50mM Na(+) with and without Ca(2+) induced a [Na(+)]i increment, enhanced by Bay K8644 and inhibited with D-600. In Ca(2+) free medium, KCl increased [Na(+)]i. Ba(2+) currents corresponding to L-VDCC were observed in myocytes and Na(+) permeated in the presence and absence of Ca(2+). SBFI-loaded myocytes in Na(+) and Ca(2+) containing Krebs stimulated with carbachol showed a Na(+) increment with a plateau. D-600 and 2-APB almost abolished the carbachol-induced Na(+) increment. RT-PCR demonstrated that CaV1.2 is the only L-VDCC subunit present in ASM. under physiological conditions, Na(+) permeates through L-VDCC in bovine ASM, probably contributing to sustain membrane depolarization during agonist stimulation. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Repetitive transcranial magnetic stimulation regulates L-type Ca(2+) channel activity inhibited by early sevoflurane exposure.

    PubMed

    Liu, Yang; Yang, Huiyun; Tang, Xiaohong; Bai, Wenwen; Wang, Guolin; Tian, Xin

    2016-09-01

    Sevoflurane might be harmful to the developing brain. Therefore, it is essential to reverse sevoflurane-induced brain injury. This study aimed to determine whether low-frequency repetitive transcranial magnetic stimulation (rTMS) can regulate L-type Ca(2+) channel activity, which is inhibited by early sevoflurane exposure. Rats were randomly divided into three groups: control, sevoflurane, and rTMS groups. A Whole-cell patch clamp technique was applied to record L-type Ca(2+) channel currents. The I-V curve, steady-state activation and inactivation curves were studied in rats of each group at different ages (1 week, 2 weeks, 3 weeks, 4 weeks and 5 weeks old). In the control group, L-type Ca(2+) channel current density significantly increased from week 2 to week 3. Compared with the control group, L-type Ca(2+) channel currents of rats in the sevoflurane group were significantly inhibited from week 1 to week 3. Activation curves of L-type Ca(2+) channel shifted significantly towards depolarization at week 1 and week 2. Moreover, steady-state inactivation curves shifted towards hyperpolarization from week 1 to week 3. Compared with the sevoflurane group, rTMS significantly increased L-type Ca(2+) channel currents at week 2 and week 3. Activation curves of L-type Ca(2+) channel significantly shifted towards hyperpolarization at week 2. Meanwhile, steady-state inactivation curves significantly shifted towards depolarization at week 2. The period between week 2 and week 3 is critical for the development of L-type Ca(2+) channels. Early sevoflurane exposure inhibits L-type Ca(2+) channel activity and rTMS can regulate L-type Ca(2+) channel activity inhibited by sevoflurane. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Barnidipine block of L-type Ca(2+) channel currents in rat ventricular cardiomyocytes.

    PubMed

    Wegener, J W; Meyrer, H; Rupp, J; Nawrath, H

    2000-08-01

    The effects of barnidipine and nifedipine on L-type Ca(2+) current (I(Ca(L))) were investigated in ventricular cardiomyocytes from rats. Both barnidipine and nifedipine reduced I(Ca(L)) in a concentration and voltage dependent manner; the EC(50) were 80 and 130 nM at a holding potential of -80 mV, respectively, and 18 and 6 nM at -40 mV, respectively. Both drugs induced a leftward shift of the steady-state inactivation curve of I(Ca(L)). Using a twin pulse protocol, the relationships between the amount of block of I(Ca(L)) by either drug, seen during the second pulse, and the length of the first pulse were described by monoexponential functions reflecting onset of block, dependent on drug concentration. The onset of block by barnidipine was three times faster than that by nifedipine. With both drugs, recovery of I(Ca(L)) was 50 times slower than under control conditions and described by monoexponential functions reflecting offset of block (independent of drug concentration). The offset of block with barnidipine was three times slower than that with nifedipine. The time constants of block and unblock of I(Ca(L)) by both drugs were used to calculate binding and unbinding and to predict their effects at two frequencies. It is suggested that barnidipine exhibits a higher affinity to the inactivated Ca(2+) channel state as compared to nifedipine.

  20. The molecular architecture of dihydropyrindine receptor/L-type Ca2+ channel complex

    PubMed Central

    Hu, Hongli; Wang, Zhao; Wei, Risheng; Fan, Guizhen; Wang, Qiongling; Zhang, Kaiming; Yin, Chang-Cheng

    2015-01-01

    Dihydropyridine receptor (DHPR), an L-type Ca2+ channel complex, plays an essential role in muscle contraction, secretion, integration of synaptic input in neurons and synaptic transmission. The molecular architecture of DHPR complex remains elusive. Here we present a 15-Å resolution cryo-electron microscopy structure of the skeletal DHPR/L-type Ca2+ channel complex. The DHPR has an asymmetrical main body joined by a hook-like extension. The main body is composed of a “trapezoid” and a “tetrahedroid”. Homologous crystal structure docking and site-specific antibody labelling revealed that the α1 and α2 subunits are located in the “trapezoid” and the β subunit is located in the “tetrahedroid”. This structure revealed the molecular architecture of a eukaryotic Ca2+ channel complex. Furthermore, this structure provides structural insights into the key elements of DHPR involved in physical coupling with the RyR/Ca2+ release channel and shed light onto the mechanism of excitation-contraction coupling. PMID:25667046

  1. Capturing single L-type Ca(2+) channel function with optics.

    PubMed

    Nystoriak, Matthew A; Nieves-Cintrón, Madeline; Navedo, Manuel F

    2013-07-01

    Advances in imaging technology have allowed optical analysis of Ca(2+)-permeable ion channel activity. Here, we briefly review novel developments in optical recording of L-type voltage-dependent Ca(2+) channel (LTCC) function with high spatial and temporal resolution. Underlying principles supporting the use of total internal reflection fluorescence (TIRF) microscopy for optical measurement of channel activity and new functional characteristics of LTCCs revealed by application of this approach are discussed. Visualization of Ca(2+) influx through single LTCCs ("LTCC sparklets") has demonstrated that channel activity is regionally heterogeneous and that clustered channels are capable of operating in a cooperative, or "coupled" manner. In light of these findings, we describe a current molecular model for the local control of LTCC activity and coupled gating in physiological and pathological contexts. This article is part of a Special Issue entitled: 12th European Symposium on Calcium. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes.

    PubMed

    Alvarez, Julio L; Petzhold, Daria; Pankonien, Ines; Behlke, Joachim; Kouno, Michiyoshi; Vassort, Guy; Morano, Ingo; Haase, Hannelore

    2010-09-01

    Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.

  3. Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels.

    PubMed Central

    Peterson, B Z; Lee, J S; Mulle, J G; Wang, Y; de Leon, M; Yue, D T

    2000-01-01

    L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation

  4. Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

    PubMed Central

    1993-01-01

    Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations

  5. Ca2+ influx through L-type Ca2+ channels and transient receptor potential channels activate pathological hypertrophy signaling

    PubMed Central

    Gao, Hui; Wang, Fang; Wang, Wei; Makarewich, Catherine A.; Zhang, Hongyu; Kubo, Hajime; Berretta, Remus M.; Barr, Larry A.; Molkentin, Jeffrey D.; Houser, Steven R.

    2012-01-01

    Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca2+]. These diseases induce cardiac hypertrophy and increases in [Ca2+] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of “hypertrophic” [Ca2+] is not known and is the topic of this study. The role of Ca2+ influx through L-type Ca2+ channels (LTCC), T-type Ca2+ channels (TTCC) and transient receptor potential (TRP) channels on the activation of Calcineurin (Cn) – Nuclear Factor of Activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat (NRVMs) and adult feline (AFVM) ventricular myocytes were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca2+ or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca2+-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca2+ induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca2+ transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca2+ influx through LTCCs is the primary source of Ca2+ to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca2+ entry via LTCCs. PMID:22921230

  6. Betaxolol, a beta1-adrenoceptor antagonist, has an affinity for L-type Ca2+ channels.

    PubMed

    Melena, J; Wood, J P; Osborne, N N

    1999-08-13

    The effect of betaxolol on the specific binding of [3H]diltiazem and [3H]nitrendipine to rat cortical membranes was examined. Betaxolol inhibited specific [3H]diltiazem and [3H]nitrendipine binding with IC50 values of 19.7 and 46.3 microM, respectively. The effect of betaxolol on L-type Ca2+ channels showed little stereospecificity, since similar inhibitions of radioligand binding were observed with both racemic betaxolol and L-betaxolol. The dissociation kinetics of [3H]diltiazem were unaffected by 30 microM betaxolol, whereas it increased the [3H]nitrendipine dissociation rate, thus suggesting that betaxolol directly interacts with the benzothiazepine binding site and allosterically modulates the dihydropyridine binding site. Carteolol, propranolol and timolol were also found to inhibit both specific [3H]diltiazem and [3H]nitrendipine binding to rat cortical membranes, but with less potency than betaxolol. The ability of betaxolol to interact with L-type Ca2+ channels may have a role in its therapeutic effects in the management of systemic hypertension and in reducing neuronal death as occurring in glaucoma.

  7. Plasticity of L-Type Ca2+ Channels After Cocaine Withdrawal

    PubMed Central

    Ford, Kerstin A.; Wolf, Marina E.; Hu, Xiu-Ti

    2010-01-01

    Increased reactivity of certain frontal cortical brain regions to cocaine re-exposure or drug-associated cues in cocaine-abstinent human addicts is linked to drug craving. Similarly, in rats tested after withdrawal from repeated cocaine exposure, cocaine or other strong excitatory stimuli produce greater activation of pyramidal neurons in the medial prefrontal cortex (mPFC). Our recent findings indicate that the increased mPFC neuronal activation depends primarily upon enhanced voltage-sensitive Ca2+ influx, most likely through high-voltage activated (HVA) L-type Ca2+ channels, but the mechanism underlying the enhanced Ca2+ currents is unknown. In this study, we used a protein crosslinking assay to show that repeated cocaine injections, resulting in behavioral sensitization, increased total protein levels and cell surface expression of HVA-Cav1.2 L-type channels in pyramidal neurons in deep layers of the mPFC. These changes in Cav1.2 L-channels were time-dependent and subtype-specific (i.e., differed from those observed for Cav1.3 L-channels). Furthermore, we found enhanced PKA activity in the mPFC of cocaine-sensitized rats that persisted for 21 days after withdrawal. PKA phosphorylation of L-channels increases their activity, so Ca2+ currents after cocaine withdrawal could be enhanced as a result of both increased activity and number of HVA-Cav1.2 L-channels on the cell surface. By increasing the supra-firing threshold excitability of mPFC pyramidal neurons, excessive upregulation of HVA L-channel activity and number may contribute to the cortical hyper-responsiveness that enhances vulnerability to cocaine craving and relapse. More generally, our results are the first to demonstrate that repeated cocaine exposure alters the membrane trafficking of a voltage-sensitive ion channel. PMID:19360908

  8. Influence of channel subunit composition on L-type Ca2+ current kinetics and cardiac wave stability.

    PubMed

    Gudzenko, Vadim; Shiferaw, Yohannes; Savalli, Nicoletta; Vyas, Roshni; Weiss, James N; Olcese, Riccardo

    2007-09-01

    Previous studies have demonstrated that the slope of the function relating the action potential duration (APD) and the diastolic interval, known as the APD restitution curve, plays an important role in the initiation and maintenance of ventricular fibrillation. Since the APD restitution slope critically depends on the kinetics of the L-type Ca(2+) current, we hypothesized that manipulation of the subunit composition of these channels may represent a powerful strategy to control cardiac arrhythmias. We studied the kinetic properties of the human L-type Ca(2+) channel (Ca(v)1.2) coexpressed with the alpha(2)delta-subunit alone (alpha(1C) + alpha(2)delta) or in combination with beta(2a), beta(2b), or beta(3) subunits (alpha(1C) + alpha(2)delta + beta), using Ca(2+) as the charge carrier. We then incorporated the kinetic properties observed experimentally into the L-type Ca(2+) current mathematical model of the cardiac action potential to demonstrate that the APD restitution slope can be selectively controlled by altering the subunit composition of the Ca(2+) channel. Assuming that beta(2b) most closely resembles the native cardiac L-type Ca(2+) current, the absence of beta, as well as the coexpression of beta(2a), was found to flatten restitution slope and stabilize spiral waves. These results imply that subunit modification of L-type Ca(2+) channels can potentially be used as an antifibrillatory strategy.

  9. Sarcolemmal Ca(2+)-entry through L-type Ca(2+) channels controls the profile of Ca(2+)-activated Cl(-) current in canine ventricular myocytes.

    PubMed

    Horváth, Balázs; Váczi, Krisztina; Hegyi, Bence; Gönczi, Mónika; Dienes, Beatrix; Kistamás, Kornél; Bányász, Tamás; Magyar, János; Baczkó, István; Varró, András; Seprényi, György; Csernoch, László; Nánási, Péter P; Szentandrássy, Norbert

    2016-08-01

    Ca(2+)-activated Cl(-) current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

  10. CALCINEURIN ENHANCES L-TYPE Ca2+ CHANNEL ACTIVITY IN HIPPOCAMPAL NEURONS: INCREASED EFFECT WITH AGE IN CULTURE

    PubMed Central

    NORRIS, C. M.; BLALOCK, E. M.; CHEN, K.-C.; PORTER, N. M.; LANDFIELD, P. W.

    2006-01-01

    The Ca2+/calmodulin-dependent protein phosphatase, calcineurin, modulates a number of key Ca2+ signaling pathways in neurons, and has been implicated in Ca2+-dependent negative feedback inactivation of N-methyl-d-aspartate receptors and voltage-sensitive Ca2+ channels. In contrast, we report here that three mechanistically disparate calcineurin inhibitors, FK-506, cyclosporin A, and the calcineurin autoinhibitory peptide, inhibited high-voltage-activated Ca2+ channel currents by up to 40% in cultured hippocampal neurons, suggesting that calcineurin acts to enhance Ca2+ currents. This effect occurred with Ba2+ or Ca2+ as charge carrier, and with or without intracellular Ca2+ buffered by EGTA. Ca2+-dependent inactivation of Ca2+ channels was not affected by FK-506. The immunosuppressant, rapamycin, and the protein phosphatase 1/2A inhibitor, okadaic acid, did not decrease Ca2+ channel current, showing specificity for effects on calcineurin. Blockade of L-type Ca2+ channels with nimodipine fully negated the effect of FK-506 on Ca2+ channel current, while blockade of N-, and P-/Q-type Ca2+ channels enhanced FK-506-mediated inhibition of the remaining L-type-enriched current. FK-506 also inhibited substantially more Ca2+ channel current in 4-week-old vs. 2-week-old cultures, an effect paralleled by an increase in calcineurin A mRNA levels. These studies provide the first evidence that calcineurin selectively enhances L-type Ca2+ channel activity in neurons. Moreover, this action appears to be increased concomitantly with the well-characterized increase in L-type Ca2+ channel availability in hippocampal neurons with age-in-culture. PMID:11958864

  11. A mathematical model of phase 2 reentry: role of L-type Ca current.

    PubMed

    Miyoshi, Shunichiro; Mitamura, Hideo; Fujikura, Kana; Fukuda, Yukiko; Tanimoto, Kojiro; Hagiwara, Yoko; Ita, Makoto; Ogawa, Satoshi

    2003-04-01

    Phase 2 reentry (P2R) is known to be one of the mechanisms of malignant ventricular arrhythmias, especially those associated with Brugada syndrome. However, little is known about the underlying mechanism for P2R. Our aim in this study was to simulate P2R in a mathematical model to enable us to understand its mechanism and identify a potential therapeutic target. A mathematical model of the L-type Ca current was composed according to whole cell current data from guinea pig ventricular myocytes recorded at 37 degrees C. Our mathematical model was incorporated into the modified Luo-Rudy phase 2 model. We set a dispersion in transient outward current (I(to)) density within the theoretical fiber, composed of 80 serially arranged epicardial cells with gap junctions and then observed the P2R. The dispersion in I(to) density within an only 0.8-cm epicardial theoretical fiber generated P2R with our Ca channel but not with the original model. When the P2R developed in the theoretical fiber, the calculated extracellular field potential showed coved-type ST segment elevation. We succeeded in generating P2R in our model for the first time. The local epicardial P2R may contribute the genesis of coved-type ST segment elevation in the Brugada syndrome.

  12. Barnidipine block of L-type Ca2+ channel currents in rat ventricular cardiomyocytes

    PubMed Central

    Wegener, Jörg W; Meyrer, Hans; Rupp, Johanna; Nawrath, Hermann

    2000-01-01

    The effects of barnidipine and nifedipine on L-type Ca2+ current (ICa(L)) were investigated in ventricular cardiomyocytes from rats.Both barnidipine and nifedipine reduced ICa(L) in a concentration and voltage dependent manner; the EC50 were 80 and 130 nM at a holding potential of −80 mV, respectively, and 18 and 6 nM at −40 mV, respectively.Both drugs induced a leftward shift of the steady-state inactivation curve of ICa(L).Using a twin pulse protocol, the relationships between the amount of block of ICa(L) by either drug, seen during the second pulse, and the length of the first pulse were described by monoexponential functions reflecting onset of block, dependent on drug concentration. The onset of block by barnidipine was three times faster than that by nifedipine.With both drugs, recovery of ICa(L) was 50 times slower than under control conditions and described by monoexponential functions reflecting offset of block (independent of drug concentration). The offset of block with barnidipine was three times slower than that with nifedipine.The time constants of block and unblock of ICa(L) by both drugs were used to calculate binding and unbinding and to predict their effects at two frequencies.It is suggested that barnidipine exhibits a higher affinity to the inactivated Ca2+ channel state as compared to nifedipine. PMID:10952695

  13. Effects of trimebutine maleate on colonic motility through Ca²+-activated K+ channels and L-type Ca²+ channels.

    PubMed

    Tan, Wei; Zhang, Hong; Luo, He-Sheng; Xia, Hong

    2011-06-01

    The effects of trimebutine maleate (TM) on spontaneous contractions of colonic longitudinal muscle were investigated in guinea pigs. The contractile responses of smooth muscle strips were recorded by an isometric force transducer. Membrane and action potentials were detected by an intracellular microelectrode technique. The whole-cell patch clamp recording technique was used to record the changes in large conductance Ca(2+)-activated K(+) (BK(ca)) and L-type Ca(2+) currents in colonic smooth muscle cells. At high concentrations (30, 100, and 300 μM), TM inhibited the amplitude of spontaneous contractions. At low concentrations (1 and 10 μM), TM attenuated the frequency and tone of smooth muscle strips, whereas TM had no influence on the amplitude of spontaneous contractions. TM depolarized the membrane potentials, but decreased the amplitude and frequency of action potentials at high concentrations. TM inhibited BK(ca) and L-type Ca(2+) currents in a dose-dependent manner. In the presence of the BK(ca) channel opener, NS1619, TM also inhibited BK(ca) currents. Bayk8644, a L-type Ca(2+) channel opener, increased L-type Ca(2+) currents. This augmentation was also attenuated by TM. These results suggest that TM attenuates intestinal motility through inhibition of L-type Ca(2+) currents, and depolarizes membrane potentials by reducing BK(ca) currents. Thus, TM may be a multiple-ion channel regulator in the gastrointestinal tract.

  14. Ryanodine modification of RyR1 retrogradely affects L-type Ca(2+) channel gating in skeletal muscle.

    PubMed

    Bannister, R A; Beam, K G

    2009-01-01

    In skeletal muscle, there is bidirectional signalling between the L-type Ca(2+) channel (1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine-sensitive Ca(2+) release channel (RyR1) of the sarcoplasmic reticulum (SR). In the case of "orthograde signalling" (i.e., excitation-contraction coupling), the conformation of RyR1 is controlled by depolarization-induced conformational changes of the DHPR resulting in Ca(2+) release from the SR. "Retrograde coupling" is manifested as enhanced L-type current. The nature of this retrograde signal, and its dependence on RyR1 conformation, are poorly understood. Here, we have examined L-type currents in normal myotubes after an exposure to ryanodine (200 microM, 1 h at 37 degrees C) sufficient to lock RyR1 in a non-conducting, inactivated, conformational state. This treatment caused an increase in L-type current at less depolarized test potentials in comparison to myotubes similarly exposed to vehicle as a result of a approximately 5 mV hyperpolarizing shift in the voltage-dependence of activation. Charge movements of ryanodine-treated myotubes were also shifted to more hyperpolarizing potentials (approximately 13 mV) relative to vehicle-treated myotubes. Enhancement of the L-type current by ryanodine was absent in dyspedic (RyR1 null) myotubes, indicating that ryanodine does not act directly on the DHPR. Our findings indicate that in retrograde signaling, the functional state of RyR1 influences conformational changes of the DHPR involved in activation of L-type current. This raises the possibility that physiological regulators of the conformational state of RyR1 (e.g., Ca(2+), CaM, CaMK, redox potential) may also affect DHPR gating.

  15. Block of single L-type Ca2+ channels in skeletal muscle fibers by aminoglycoside antibiotics

    PubMed Central

    1996-01-01

    The activity of single L-type Ca2+ channels was recorded from cell- attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were concerned with the mechanism by which aminoglycoside antibiotics inhibit ion flow through the channel. Aminoglycosides produced discrete fluctuations in the single-channel current when added to the external solution. The blocking kinetics could be described as a simple bimolecular reaction between an aminoglycoside molecule and the open channel. The blocking rate was found to be increased when either the membrane potential was made more negative or the concentration of external permeant ion was reduced. Both of these effects are consistent with a blocking site that is located within the channel pore. Other features of block, however, were incompatible with a simple pore blocking mechanism. Hyperpolarization enhanced the rate of unblocking, even though an aminoglycoside molecule must dissociate from its binding site in the channel toward the external solution against the membrane field. Raising the external permeant ion concentration also enhanced the rate of unblocking. This latter finding suggests that aminglycoside affinity is modified by repulsive interactions that arise when the pore is simultaneously occupied by a permeant ion and an aminoglycoside molecule. PMID:8868052

  16. Temperature-dependence of L-type Ca(2+) current in ventricular cardiomyocytes of the Alaska blackfish (Dallia pectoralis).

    PubMed

    Kubly, Kerry L; Stecyk, Jonathan A W

    2015-12-01

    To lend insight into the overwintering strategy of the Alaska blackfish (Dallia pectoralis), we acclimated fish to 15 or 5 °C and then utilized whole-cell patch clamp to characterize the effects of thermal acclimation and acute temperature change on the density and kinetics of ventricular L-type Ca(2+) current (I Ca). Peak I Ca density at 5 °C (-1.1 ± 0.1 pA pF(-1)) was 1/8th that at 15 °C (-8.8 ± 0.6 pA pF(-1)). However, alterations of the Ca(2+)- and voltage-dependent inactivation properties of L-type Ca(2+) channels partially compensated against the decrease. The time constant tau (τ) for the kinetics of inactivation of I Ca was ~4.5 times greater at 5 °C than at 15 °C, and the voltage for half-maximal inactivation was shifted from -23.3 ± 1.0 mV at 15 °C to -19.8 ± 1.2 mV at 5 °C. These modifications increase the open probability of the channel and culminate in an approximate doubling of the L-type Ca(2+) window current, which contributes to approximately 15% of the maximal Ca(2+) conductance at 5 °C. Consequently, the charge density of I Ca (Q Ca) and the total Ca(2+) transferred through the L-type Ca(2+) channels (Δ[Ca(2+)]) were not as severely reduced at 5 °C as compared to peak I Ca density. In combination, the results suggest that while the Alaska blackfish substantially down-regulates I Ca with acclimation to low temperature, there is sufficient compensation in the kinetics of the L-type Ca(2+) channel to support the level of cardiac performance required for the fish to remain active throughout the winter.

  17. Stac adaptor proteins regulate trafficking and function of muscle and neuronal L-type Ca2+ channels.

    PubMed

    Polster, Alexander; Perni, Stefano; Bichraoui, Hicham; Beam, Kurt G

    2015-01-13

    Excitation-contraction (EC) coupling in skeletal muscle depends upon trafficking of CaV1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca(2+) channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that CaV1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine CaV isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of CaV1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and CaV1.1 traffic together to the tsA201 plasma membrane, whereas CaV1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca(2+) channel function in tsA201 cells coexpressing Stac3 and CaV1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of CaV1.2, the principle subunit of the cardiac/brain L-type Ca(2+) channel, Stac3 does bind to CaV1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of CaV1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate CaV1.2 function.

  18. Rock Tea extract (Jasonia glutinosa) relaxes rat aortic smooth muscle by inhibition of L-type Ca(2+) channels.

    PubMed

    Valero, Marta Sofía; Oliván-Viguera, Aida; Garrido, Irene; Langa, Elisa; Berzosa, César; López, Víctor; Gómez-Rincón, Carlota; Murillo, María Divina; Köhler, Ralf

    2015-12-01

    In traditional herbal medicine, Rock Tea (Jasonia glutinosa) is known for its prophylactic and therapeutic value in various disorders including arterial hypertension. However, the mechanism by which Rock Tea exerts blood pressure-lowering actions has not been elucidated yet. Our aim was to demonstrate vasorelaxing effects of Rock Tea extract and to reveal its possible action mechanism. Isometric myography was conducted on high-K+-precontracted rings from rat thoracic aorta and tested extracts at concentrations of 0.5-5 mg/ml. Whole-cell patch-clamp experiments were performed in rat aortic vascular smooth muscle cells (line A7r5) to determine blocking effects on L-type Ca(2+) channels. Rock Tea extract relaxed the aorta contracted by high [K+] concentration dependently with an EC50 of ≈2.4 mg/ml and produced ≈75 % relaxation at the highest concentration tested. The L-type Ca(2+) channel blocker, verapamil (10(-6) M), had similar effects. Rock Tea extract had no effect in nominally Ca(2+)-free high-K(+) buffer but significantly inhibited contractions to re-addition of Ca(2+). Rock Tea extract inhibited the contractions induced by the L-type Ca(2+) channel activator Bay K 8644 (10(-5) M) and by phenylephrine (10(-6) M). Rock Tea extract and Y-27632 (10(-6) M), Rho-kinase inhibitor, had similar effects and the respective effects were not additive. Patch-clamp experiments demonstrated that Rock Tea extract (2.5 mg/ml) virtually abolished L-type Ca(2+) currents in A7r5. We conclude that Rock Tea extract produced vasorelaxation of rat aorta and that this relaxant effect is mediated by inhibition of L-type Ca(2+) channels. Rock Tea extracts may be of phytomedicinal value for prevention and adjuvant treatment of hypertension and other cardiovascular diseases.

  19. Postnatal development and activation of L-type Ca2+ currents in locus ceruleus neurons: implications for a role for Ca2+ in central chemosensitivity

    PubMed Central

    Imber, Ann N.

    2012-01-01

    Little is known about the role of Ca2+ in central chemosensitive signaling. We use electrophysiology to examine the chemosensitive responses of tetrodotoxin (TTX)-insensitive oscillations and spikes in neurons of the locus ceruleus (LC), a chemosensitive region involved in respiratory control. We show that both TTX-insensitive spikes and oscillations in LC neurons are sensitive to L-type Ca2+ channel inhibition and are activated by increased CO2/H+. Spikes appear to arise from L-type Ca2+ channels on the soma whereas oscillations arise from L-type Ca2+ channels that are distal to the soma. In HEPES-buffered solution (nominal absence of CO2/HCO3−), acidification does not activate either oscillations or spikes. When CO2 is increased while extracellular pH is held constant by elevated HCO3−, both oscillation and spike frequency increase. Furthermore, plots of both oscillation and spike frequency vs. intracellular [HCO3−]show a strong linear correlation. Increased frequency of TTX-insensitive spikes is associated with increases in intracellular Ca2+ concentrations. Finally, both the appearance and frequency of TTX-insensitive spikes and oscillations increase over postnatal ages day 3–16. Our data suggest that 1) L-type Ca2+ currents in LC neurons arise from channel populations that reside in different regions of the neuron, 2) these L-type Ca2+ currents undergo significant postnatal development, and 3) the activity of these L-type Ca2+ currents is activated by increased CO2 through a HCO3−-dependent mechanism. Thus the activity of L-type Ca2+ channels is likely to play a role in the chemosensitive response of LC neurons and may underlie significant changes in LC neuron chemosensitivity during neonatal development. PMID:22403350

  20. Role of L-Type Ca[superscript 2+] Channel Isoforms in the Extinction of Conditioned Fear

    ERIC Educational Resources Information Center

    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J.; Striessnig, Jorg; Singewald, Nicolas

    2008-01-01

    Dihydropyridine (DHP) L-type Ca[superscript 2+] channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the…

  1. Role of L-Type Ca[superscript 2+] Channel Isoforms in the Extinction of Conditioned Fear

    ERIC Educational Resources Information Center

    Busquet, Perrine; Hetzenauer, Alfred; Sinnegger-Brauns, Martina J.; Striessnig, Jorg; Singewald, Nicolas

    2008-01-01

    Dihydropyridine (DHP) L-type Ca[superscript 2+] channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the…

  2. Anthracene based compounds as new L-type Ca2+ channel blockers: design, synthesis, and full biological profile.

    PubMed

    Bova, Sergio; Saponara, Simona; Rampa, Angela; Gobbi, Silvia; Cima, Lorenzo; Fusi, Fabio; Sgaragli, Giampietro; Cavalli, Maurizio; de los Rios, Cristobal; Striessnig, Jörg; Bisi, Alessandra

    2009-03-12

    L-Type Ca(2+) channels (LTCCs) play a key role in the regulation of vascular smooth muscle contraction, and substances that interfere with their function (Ca(2+) channel blockers) are widely used in the therapy of hypertension. Here, we report anthracene-maleimide derivatives as new LTCC blockers. Among these, 3, lacking intracellular effects, was investigated in more detail. The results show that 3 binds preferentially to inactivated LTCCs, directly interacting with the pore-forming subunit of the channel.

  3. L-type Ca2+ channels in the enteric nervous system mediate oscillatory Cl- secretion in guinea pig colon.

    PubMed

    Nishikitani, Mariko; Yasuoka, Yukiko; Kawada, Hideaki; Kawahara, Katsumasa

    2007-02-01

    The enteric nervous system regulates epithelial ion and fluid secretion. Our previous study has shown that the low (0.2-1 mM) concentrations of Ba2+, a K+ channel inhibitor, evoke Ca2+-dependent oscillatory Cl- secretion via activation of submucosal cholinergic neurons in guinea pig distal colon. However, it is still unclear which types of Ca2+ channels are involved in the oscillation at the neuroepithelial junction. We investigated the inhibitory effects of organic and inorganic Ca2+ channel antagonists on the short circuit current (I(sc)) of colonic epithelia (mucosa-submucosa sheets) mounted in Ussing chambers. The amplitude (412 +/- 37 microA cm(-2)) and frequency (2.6 +/- 0.1 cycles min(-1)) of the Ba2+-induced I(sc) in normal (1.8 mM) Ca2+ solution (n = 26) significantly decreased by 37.6% and 38.5%, respectively, in the low (0.1 mM) Ca2+ solution (n = 14). The I(sc) amplitude was reversibly inhibited by either verapamil (an L-type Ca2+ channel antagonist) or divalent cations (Cd2+, Mn2+, Ni2+) in a concentration-dependent manner. The concentration of verapamil for half-maximum inhibition (IC50) was 4 and 2 microM in normal and low Ca2+ solution, respectively. The relative blocking potencies of metal ions were Cd2+ > Mn2+, Ni2+ in normal Ca2+ solution. In contrast, the frequency of I(sc) was unchanged over the range of concentrations of the Ca2+ channel antagonists used. Our results show that the oscillatory I(sc) evoked by Ba2+ involves L-type voltage-gated Ca2+ channels. We conclude that L-type Ca2+ channels play a key role in the oscillation at the neuroepithelial junctions of guinea pig colon.

  4. Glutathionylation of the L-type Ca2+ channel in oxidative stress-induced pathology of the heart.

    PubMed

    Johnstone, Victoria P A; Hool, Livia C

    2014-10-22

    There is mounting evidence to suggest that protein glutathionylation is a key process contributing to the development of pathology. Glutathionylation occurs as a result of posttranslational modification of a protein and involves the addition of a glutathione moiety at cysteine residues. Such modification can occur on a number of proteins, and exerts a variety of functional consequences. The L-type Ca2+ channel has been identified as a glutathionylation target that participates in the development of cardiac pathology. Ca2+ influx via the L-type Ca2+ channel increases production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes during periods of oxidative stress. This induces a persistent increase in channel open probability, and the resulting constitutive increase in Ca2+ influx amplifies the cross-talk between the mitochondria and the channel. Novel strategies utilising targeted peptide delivery to uncouple mitochondrial ROS and Ca2+ flux via the L-type Ca2+ channel following ischemia-reperfusion have delivered promising results, and have proven capable of restoring appropriate mitochondrial function in myocytes and in vivo.

  5. Glutathionylation of the L-type Ca2+ Channel in Oxidative Stress-Induced Pathology of the Heart

    PubMed Central

    Johnstone, Victoria P. A.; Hool, Livia C.

    2014-01-01

    There is mounting evidence to suggest that protein glutathionylation is a key process contributing to the development of pathology. Glutathionylation occurs as a result of posttranslational modification of a protein and involves the addition of a glutathione moiety at cysteine residues. Such modification can occur on a number of proteins, and exerts a variety of functional consequences. The L-type Ca2+ channel has been identified as a glutathionylation target that participates in the development of cardiac pathology. Ca2+ influx via the L-type Ca2+ channel increases production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes during periods of oxidative stress. This induces a persistent increase in channel open probability, and the resulting constitutive increase in Ca2+ influx amplifies the cross-talk between the mitochondria and the channel. Novel strategies utilising targeted peptide delivery to uncouple mitochondrial ROS and Ca2+ flux via the L-type Ca2+ channel following ischemia-reperfusion have delivered promising results, and have proven capable of restoring appropriate mitochondrial function in myocytes and in vivo. PMID:25340983

  6. Ca2+-binding protein-1 facilitates and forms a postsynaptic complex with Cav1.2 (L-type) Ca2+ channels.

    PubMed

    Zhou, Hong; Kim, Seong-Ah; Kirk, Elizabeth A; Tippens, Alyssa L; Sun, Hong; Haeseleer, Françoise; Lee, Amy

    2004-05-12

    Ca2+-binding protein-1 (CaBP1) is a Ca2+-binding protein that is closely related to calmodulin (CaM) and localized in somatodendritic regions of principal neurons throughout the brain, but how CaBP1 participates in postsynaptic Ca2+ signaling is not known. Here, we describe a novel role for CaBP1 in the regulation of Ca2+ influx through Ca(v)1.2 (L-type) Ca2+ channels. CaBP1 interacts directly with the alpha1 subunit of Ca(v)1.2 at sites that also bind CaM. CaBP1 binding to one of these sites, the IQ domain, is Ca2+ dependent and competitive with CaM binding. The physiological significance of this interaction is supported by the association of Ca(v)1.2 and CaBP1 in postsynaptic density fractions purified from rat brain. Moreover, in double-label immunofluorescence experiments, CaBP1 and Ca(v)1.2 colocalize in numerous cell bodies and dendrites of neurons, particularly in pyramidal cells in the CA3 region of the hippocampus and in the dorsal cortex. In electrophysiological recordings of cells transfected with Ca(v)1.2, CaBP1 greatly prolonged Ca2+ currents, prevented Ca2+-dependent inactivation, and caused Ca2+-dependent facilitation of currents evoked by step depolarizations and repetitive stimuli. These effects contrast with those of CaM, which promoted strong Ca2+-dependent inactivation of Ca(v)1.2 with these same voltage protocols. Our findings reveal how Ca2+-binding proteins, such as CaM and CaBP1, differentially adjust Ca2+ influx through Ca(v)1.2 channels, which may specify diverse modes of Ca2+ signaling in neurons.

  7. Two classes of gating current from L-type Ca channels in guinea pig ventricular myocytes

    PubMed Central

    1992-01-01

    that the same molecules that produce the charge movement in normally polarized cells also produce the slow component in depolarized cells. 100 microns D600 increased by 77% the slow charge movement after a 500-ms conditioning pulse. These results demonstrate two classes of charge movement associated with L-type Ca channels, with kinetics and voltage dependence similar to charge 1 and charge 2 of skeletal muscle.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1322450

  8. Temperature-dependence of L-type Ca2+ current in ventricular cardiomyocytes of the Alaska blackfish (Dallia pectoralis)

    PubMed Central

    Kubly, Kerry L.; Stecyk, Jonathan A.W.

    2016-01-01

    Summary To lend insight into the overwintering strategy of the Alaska blackfish (Dallia pectoralis), we acclimated fish to 15°C or 5°C and then utilized whole-cell patch-clamp to characterize the effects of thermal acclimation and acute temperature change on the density and kinetics of ventricular L-type Ca2+ current (ICa). Peak ICa density at 5°C (−1.1± 0.1 pA pF−1) was 1/8th that at 15°C (−8.8 ± 0.6 pA pF−1). However, alterations of the Ca2+- and voltage-dependent inactivation properties of L-type Ca2+ channels partially compensated against the decrease. The time constant tau (τ) for the kinetics of inactivation of ICa was ~4.5-times greater at 5°C than at 15°C, and the voltage for half-maximal inactivation was shifted from −23.3 ± 1.0 mV at 15°C to - 19.8 ± 1.2 mV at 5°C. These modifications increase the open probability of the channel and culminated in an approximate doubling of the L-type Ca2+ window current, which contributed to approximately 15% of the maximal Ca2+ conductance at 5°C. Consequently, the charge density of ICa (QCa) and the total Ca2+ transferred through the L-type Ca channels (Δ[Ca2+]) were not as severely reduced at 5°C as compared to peak ICa density. In combination, the results suggest that while the Alaska blackfish substantially down-regulates ICa with acclimation to low temperature, there is sufficient compensation in the kinetics of the L-type Ca2+ channel to support the level of cardiac performance required for the fish to remain active throughout the winter. PMID:26439127

  9. 1,25 (OH)2D3 enhances PTH-induced Ca2+ transients in preosteoblasts by activating L-type Ca2+ channels

    NASA Technical Reports Server (NTRS)

    Li, W.; Duncan, R. L.; Karin, N. J.; Farach-Carson, M. C.

    1997-01-01

    We previously demonstrated electrophysiologically that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] shifts the activation threshold of L-type Ca2+ channels in osteoblasts toward the resting potential and prolongs mean open time. Presently, we used single-cell Ca2+ imaging to study the combined effects of 1,25(OH)2D3 and parathyroid hormone (PTH) during generation of Ca2+ transients in fura 2-loaded MC3T3-E1 cells. Pretreatment with 1,25(OH)2D3 concentrations, which alone did not produce Ca2+ transients, consistently enhanced Ca2+ responses to PTH. Enhancement was dose dependent over the range of 1 to 10 nM and was blocked by pretreatment with 5 microM nitrendipine during pretreatment. A 1,25(OH)2D3 analog that activates L-type channels and shifts their activation threshold also enhanced PTH responses. In contrast, an analog devoid of membrane Ca2+ effects did not enhance PTH-induced Ca2+ transients. The PTH-induced Ca2+ transient involved activation of a dihydropyridine-insensitive cation channel that was inhibited by Gd3+. Together, these data suggest that 1,25(OH)2D3 increases osteoblast responsiveness to PTH through rapid modification of L-type Ca2+ channel gating properties, whose activation enhances Ca2+ entry through other channels such as the PTH-responsive, Gd(3+)-sensitive cation channel.

  10. 1,25 (OH)2D3 enhances PTH-induced Ca2+ transients in preosteoblasts by activating L-type Ca2+ channels

    NASA Technical Reports Server (NTRS)

    Li, W.; Duncan, R. L.; Karin, N. J.; Farach-Carson, M. C.

    1997-01-01

    We previously demonstrated electrophysiologically that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] shifts the activation threshold of L-type Ca2+ channels in osteoblasts toward the resting potential and prolongs mean open time. Presently, we used single-cell Ca2+ imaging to study the combined effects of 1,25(OH)2D3 and parathyroid hormone (PTH) during generation of Ca2+ transients in fura 2-loaded MC3T3-E1 cells. Pretreatment with 1,25(OH)2D3 concentrations, which alone did not produce Ca2+ transients, consistently enhanced Ca2+ responses to PTH. Enhancement was dose dependent over the range of 1 to 10 nM and was blocked by pretreatment with 5 microM nitrendipine during pretreatment. A 1,25(OH)2D3 analog that activates L-type channels and shifts their activation threshold also enhanced PTH responses. In contrast, an analog devoid of membrane Ca2+ effects did not enhance PTH-induced Ca2+ transients. The PTH-induced Ca2+ transient involved activation of a dihydropyridine-insensitive cation channel that was inhibited by Gd3+. Together, these data suggest that 1,25(OH)2D3 increases osteoblast responsiveness to PTH through rapid modification of L-type Ca2+ channel gating properties, whose activation enhances Ca2+ entry through other channels such as the PTH-responsive, Gd(3+)-sensitive cation channel.

  11. G protein-dependent inhibition of L-type Ca2+ currents by acetylcholine in mouse pancreatic B-cells.

    PubMed Central

    Gilon, P; Yakel, J; Gromada, J; Zhu, Y; Henquin, J C; Rorsman, P

    1997-01-01

    1. The effect of acetylcholine (ACh) on voltage-dependent Ca2+ currents in mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. 2. ACh (0.25-250 microM) reversibly and dose-dependently inhibited the Ca2+ current elicited by depolarizations from -80 mV to +10 mV. Maximal inhibition was observed at concentrations > 25 microM where it amounted to approximately 35%. The effect was voltage independent and prevented by atropine (10 microM) suggesting that it was mediated by muscarinic receptors. 3. The inhibitory action of ACh on the Ca2+ current was abolished when the cytoplasmic solution contained GDP beta S (2 mM) and became irreversible when the non-hydrolysable GTP analogue GTP gamma S (10 microM) was included in the pipette. This indicates the participation of G proteins in the inhibitory effect of ACh but pretreatment of the cells with either pertussis or cholera toxin failed to prevent the effect of ACh on the Ca2+ current. 4. ACh remained equally effective as an inhibitor of the whole-cell Ca2+ current in the presence of the L-type Ca2+ channel agonist (-)-Bay K 8644 and after partial inhibition of the current by nifedipine. Addition of omega-agatoxin IVA, omega-conotoxin GVIA or omega-conotoxin MVIIC neither affected the peak Ca2+ current amplitude nor the extent of inhibition produced by ACh. These pharmacological properties indicate that ACh acts by inhibiting L-type Ca2+ channels. 5. The inhibitory action of ACh on the B-cell Ca2+ current was not secondary to elevation of [Ca2+]i and ACh remained equally effective as an inhibitor when Ba2+ was used as the charge carrier, when [Ca2+]i was buffered to low concentrations using EGTA and under experimental conditions preventing the mobilization of Ca2+ from intracellular stores. 6. These results suggest that ACh reduces the whole-cell Ca2+ current in the B-cell through a G protein-regulated, voltage- and Ca(2+)-independent inhibition of L-type Ca2+ channels. PMID

  12. G protein-dependent inhibition of L-type Ca2+ currents by acetylcholine in mouse pancreatic B-cells.

    PubMed

    Gilon, P; Yakel, J; Gromada, J; Zhu, Y; Henquin, J C; Rorsman, P

    1997-02-15

    1. The effect of acetylcholine (ACh) on voltage-dependent Ca2+ currents in mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. 2. ACh (0.25-250 microM) reversibly and dose-dependently inhibited the Ca2+ current elicited by depolarizations from -80 mV to +10 mV. Maximal inhibition was observed at concentrations > 25 microM where it amounted to approximately 35%. The effect was voltage independent and prevented by atropine (10 microM) suggesting that it was mediated by muscarinic receptors. 3. The inhibitory action of ACh on the Ca2+ current was abolished when the cytoplasmic solution contained GDP beta S (2 mM) and became irreversible when the non-hydrolysable GTP analogue GTP gamma S (10 microM) was included in the pipette. This indicates the participation of G proteins in the inhibitory effect of ACh but pretreatment of the cells with either pertussis or cholera toxin failed to prevent the effect of ACh on the Ca2+ current. 4. ACh remained equally effective as an inhibitor of the whole-cell Ca2+ current in the presence of the L-type Ca2+ channel agonist (-)-Bay K 8644 and after partial inhibition of the current by nifedipine. Addition of omega-agatoxin IVA, omega-conotoxin GVIA or omega-conotoxin MVIIC neither affected the peak Ca2+ current amplitude nor the extent of inhibition produced by ACh. These pharmacological properties indicate that ACh acts by inhibiting L-type Ca2+ channels. 5. The inhibitory action of ACh on the B-cell Ca2+ current was not secondary to elevation of [Ca2+]i and ACh remained equally effective as an inhibitor when Ba2+ was used as the charge carrier, when [Ca2+]i was buffered to low concentrations using EGTA and under experimental conditions preventing the mobilization of Ca2+ from intracellular stores. 6. These results suggest that ACh reduces the whole-cell Ca2+ current in the B-cell through a G protein-regulated, voltage- and Ca(2+)-independent inhibition of L-type Ca2+ channels.

  13. Localization of Rod Bipolar Cells in the Mammalian Retina Using an Antibody Against the α1c L-type Ca(2+) Channel.

    PubMed

    Huh, Yu-Jin; Choi, Jae-Sik; Jeon, Chang-Jin

    2015-04-28

    Bipolar cells transmit stimuli via graded changes in membrane potential and neurotransmitter release is modulated by Ca(2+) influx through L-type Ca(2+) channels. The purpose of this study was to determine whether the α1c subunit of L-type voltage-gated Ca(2+) channel (α1c L-type Ca(2+) channel) colocalizes with protein kinase C alpha (PKC-α), which labels rod bipolar cells. Retinal whole mounts and vertical sections from mouse, hamster, rabbit, and dog were immunolabeled with antibodies against PKC-α and α1c L-type Ca(2+) channel, using fluorescein isothiocyanate (FITC) and Cy5 as visualizing agents. PKC-α-immunoreactive cells were morphologically identical to rod bipolar cells as previously reported. Their cell bodies were located within the inner nuclear layer, dendritic processes branched into the outer plexiform layer, and axons extended into the inner plexiform layer. Immunostaining showed that α1c L-type Ca(2+) channel colocalized with PKC-α in rod bipolar cells. The identical expression of PKC-α and α1c L-type Ca(2+) channel indicates that the α1c L-type Ca(2+) channel has a specific role in rod bipolar cells, and the antibody against the α1c L-type Ca(2+) channel may be a useful marker for studying the distribution of rod bipolar cells in mouse, hamster, rabbit, and dog retinas.

  14. Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

    PubMed

    Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K; Morales, Cyndi R; Contreras-Ferrat, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J; Somlo, Stefan; Rothermel, Beverly A; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

    2015-06-16

    L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. © 2015 American Heart Association, Inc.

  15. Spatial characteristics of sarcoplasmic reticulum Ca2+ release events triggered by L-type Ca2+ current and Na+ current in guinea-pig cardiac myocytes

    PubMed Central

    Lipp, Peter; Egger, Marcel; Niggli, Ernst

    2002-01-01

    Ca2+ signals in cardiac muscle cells are composed of spatially limited elementary events termed Ca2+ sparks. Several studies have also indicated that Ca2+ signals smaller than Ca2+ sparks can be elicited. These signals have been termed Ca2+ quarks and were proposed to result from the opening of a single Ca2+ release channel of the sarcoplasmic reticulum. We used laser-scanning confocal microscopy to examine the subcellular properties of Na+ current (INa)- and L-type Ca2+ current (ICa,L)-induced Ca2+ transients in voltage-clamped ventricular myocytes isolated from guinea-pigs. Both currents, INa and ICa,L, evoked substantial, global Ca2+ transients. To examine the spatiotemporal properties of such Ca2+ signals, we performed power spectral analysis of these Ca2+ transients and found that both lacked spatial frequency components characteristic for Ca2+ sparks. The application of 10 μm verapamil to partially block L-type Ca2+ current reduced the corresponding Ca2+ transients down to individual Ca2+ sparks. In contrast, INa-induced Ca2+ responses were still spatially homogeneous and lacked Ca2+ sparks even for small current amplitudes. By using high resistance patch pipettes (> 4 MΩ) to exaggerate the loss of voltage control during INa, Ca2+ sparks appeared superimposed on a homogeneous Ca2+ release component and were exclusively triggered during the flow of INa. In the presence of 10 μm ryanodine both ICa,L and INa elicited small, residual Ca2+ transients that were spatially homogeneous but displayed distinctively different temporal profiles. We conclude that INa is indeed able to cause Ca2+ release in guinea-pig ventricular myocytes. In contrast to ICa,L-induced Ca2+ transients, which are built up from the recruitment of individual Ca2+ sparks, the INa-evoked cellular responses were always homogeneous, indicating that their underlying elementary Ca2+ release event is distinct from the Ca2+ spark. Thus, INa-induced Ca2+ transients are composed of smaller Ca2

  16. Caldendrin, a neuron-specific modulator of Cav/1.2 (L-type) Ca2+ channels.

    PubMed

    Tippens, Alyssa L; Lee, Amy

    2007-03-16

    EF-hand Ca2+-binding proteins such as calmodulin and CaBP1 have emerged as important regulatory subunits of voltage-gated Ca2+ channels. Here, we show that caldendrin, a variant of CaBP1 enriched in the brain, interacts with and distinctly modulates Cav1.2 (L-type) voltage-gated Ca2+ channels relative to other Ca2+-binding proteins. Caldendrin binds to the C-terminal IQ-domain of the pore-forming alpha1-subunit of Cav1.2 (alpha(1)1.2) and competitively displaces calmodulin and CaBP1 from this site. Compared with CaBP1, caldendrin causes a more modest suppression of Ca2+-dependent inactivation of Cav1.2 through a different subset of molecular determinants. Caldendrin does not bind to the N-terminal domain of alpha11.2, a site that is critical for functional interactions of the channel with CaBP1. Deletion of the N-terminal domain inhibits CaBP1, but spares caldendrin modulation of Cav1.2 inactivation. In contrast, mutations of the IQ-domain abolish physical and functional interactions of caldendrin and Cav1.2, but do not prevent channel modulation by CaBP1. Using antibodies specific for caldendrin and Cav1.2, we show that caldendrin coimmunoprecipitates with Cav1.2 from the brain and colocalizes with Cav1.2 in somatodendritic puncta of cortical neurons in culture. Our findings reveal functional diversity within related Ca2+-binding proteins, which may enhance the specificity of Ca2+ signaling by Cav1.2 channels in different cellular contexts.

  17. Cooperative and acute inhibition by multiple C-terminal motifs of L-type Ca2+ channels

    PubMed Central

    Liu, Nan; Yang, Yaxiong; Ge, Lin; Liu, Min; Colecraft, Henry M; Liu, Xiaodong

    2017-01-01

    Inhibitions and antagonists of L-type Ca2+ channels are important to both research and therapeutics. Here, we report C-terminus mediated inhibition (CMI) for CaV1.3 that multiple motifs coordinate to tune down Ca2+ current and Ca2+ influx toward the lower limits determined by end-stage CDI (Ca2+-dependent inactivation). Among IQV (preIQ3-IQ domain), PCRD and DCRD (proximal or distal C-terminal regulatory domain), spatial closeness of any two modules, e.g., by constitutive fusion, facilitates the trio to form the complex, compete against calmodulin, and alter the gating. Acute CMI by rapamycin-inducible heterodimerization helps reconcile the concurrent activation/inactivation attenuations to ensure Ca2+ influx is reduced, in that Ca2+ current activated by depolarization is potently (~65%) inhibited at the peak (full activation), but not later on (end-stage inactivation, ~300 ms). Meanwhile, CMI provides a new paradigm to develop CaV1 inhibitors, the therapeutic potential of which is implied by computational modeling of CaV1.3 dysregulations related to Parkinson’s disease. DOI: http://dx.doi.org/10.7554/eLife.21989.001 PMID:28059704

  18. Methylene blue counteracts H2S toxicity-induced cardiac depression by restoring L-type Ca channel activity

    PubMed Central

    Zhang, Xue-Qian; Sonobe, Takashi; Song, Jianliang; Rannals, Matthew D.; Wang, JuFang; Tubbs, Nicole; Cheung, Joseph Y.; Haouzi, Philippe

    2016-01-01

    We have previously reported that methylene blue (MB) can counteract hydrogen sulfide (H2S) intoxication-induced circulatory failure. Because of the multifarious effects of high concentrations of H2S on cardiac function, as well as the numerous properties of MB, the nature of this interaction, if any, remains uncertain. The aim of this study was to clarify 1) the effects of MB on H2S-induced cardiac toxicity and 2) whether L-type Ca2+ channels, one of the targets of H2S, could transduce some of the counteracting effects of MB. In sedated rats, H2S infused at a rate that would be lethal within 5 min (24 μM·kg−1·min−1), produced a rapid fall in left ventricle ejection fraction, determined by echocardiography, leading to a pulseless electrical activity. Blood concentrations of gaseous H2S reached 7.09 ± 3.53 μM when cardiac contractility started to decrease. Two to three injections of MB (4 mg/kg) transiently restored cardiac contractility, blood pressure, and V̇o2, allowing the animals to stay alive until the end of H2S infusion. MB also delayed PEA by several minutes following H2S-induced coma and shock in unsedated rats. Applying a solution containing lethal levels of H2S (100 μM) on isolated mouse cardiomyocytes significantly reduced cell contractility, intracellular calcium concentration ([Ca2+]i) transient amplitudes, and L-type Ca2+ currents (ICa) within 3 min of exposure. MB (20 mg/l) restored the cardiomyocyte function, ([Ca2+]i) transient, and ICa. The present results offer a new approach for counteracting H2S toxicity and potentially other conditions associated with acute inhibition of L-type Ca2+ channels. PMID:26962024

  19. Methylene blue counteracts H2S toxicity-induced cardiac depression by restoring L-type Ca channel activity.

    PubMed

    Judenherc-Haouzi, Annick; Zhang, Xue-Qian; Sonobe, Takashi; Song, Jianliang; Rannals, Matthew D; Wang, JuFang; Tubbs, Nicole; Cheung, Joseph Y; Haouzi, Philippe

    2016-06-01

    We have previously reported that methylene blue (MB) can counteract hydrogen sulfide (H2S) intoxication-induced circulatory failure. Because of the multifarious effects of high concentrations of H2S on cardiac function, as well as the numerous properties of MB, the nature of this interaction, if any, remains uncertain. The aim of this study was to clarify 1) the effects of MB on H2S-induced cardiac toxicity and 2) whether L-type Ca(2+) channels, one of the targets of H2S, could transduce some of the counteracting effects of MB. In sedated rats, H2S infused at a rate that would be lethal within 5 min (24 μM·kg(-1)·min(-1)), produced a rapid fall in left ventricle ejection fraction, determined by echocardiography, leading to a pulseless electrical activity. Blood concentrations of gaseous H2S reached 7.09 ± 3.53 μM when cardiac contractility started to decrease. Two to three injections of MB (4 mg/kg) transiently restored cardiac contractility, blood pressure, and V̇o2, allowing the animals to stay alive until the end of H2S infusion. MB also delayed PEA by several minutes following H2S-induced coma and shock in unsedated rats. Applying a solution containing lethal levels of H2S (100 μM) on isolated mouse cardiomyocytes significantly reduced cell contractility, intracellular calcium concentration ([Ca(2+)]i) transient amplitudes, and L-type Ca(2+) currents (ICa) within 3 min of exposure. MB (20 mg/l) restored the cardiomyocyte function, ([Ca(2+)]i) transient, and ICa The present results offer a new approach for counteracting H2S toxicity and potentially other conditions associated with acute inhibition of L-type Ca(2+) channels. Copyright © 2016 the American Physiological Society.

  20. The L-type Ca2+ Channels Blocker Nifedipine Represses Mesodermal Fate Determination in Murine Embryonic Stem Cells

    PubMed Central

    Nguemo, Filomain; Fleischmann, Bernd K.; Gupta, Manoj K.; Šarić, Tomo; Malan, Daniela; Liang, Huamin; Pfannkuche, Kurt; Bloch, Wilhelm; Schunkert, Heribert; Hescheler, Jürgen; Reppel, Michael

    2013-01-01

    Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca2+ channels of different cell types. Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca2+ transient and reduction of LTCCs current density (ICaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca2+ channel α1C (CaV1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function. PMID:23320083

  1. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

    NASA Technical Reports Server (NTRS)

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

    2005-01-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

  2. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

    NASA Technical Reports Server (NTRS)

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

    2005-01-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

  3. Superoxide enhances Ca2+ entry through L-type channels in the renal afferent arteriole.

    PubMed

    Vogel, Paul A; Yang, Xi; Moss, Nicholas G; Arendshorst, William J

    2015-08-01

    Reactive oxygen species regulate cardiovascular and renal function in health and disease. Superoxide participates in acute calcium signaling in afferent arterioles and renal vasoconstriction produced by angiotensin II, endothelin, thromboxane, and pressure-induced myogenic tone. Known mechanisms by which superoxide acts include quenching of nitric oxide and increased ADP ribosyl cyclase/ryanodine-mediated calcium mobilization. The effect(s) of superoxide on other calcium signaling pathways in the renal microcirculation is poorly understood. The present experiments examined the acute effect of superoxide generated by paraquat on calcium entry pathways in isolated rat afferent arterioles. The peak increase in cytosolic calcium concentration caused by KCl (40 mmol/L) was 99±14 nmol/L. The response to this membrane depolarization was mediated exclusively by L-type channels because it was abolished by nifedipine but was unaffected by the T-type channel blocker mibefradil. Paraquat increased superoxide production (dihydroethidium fluorescence), tripled the peak response to KCl to 314±68 nmol/L (P<0.001) and doubled the plateau response. These effects were abolished by tempol and nitroblue tetrazolium, but not by catalase, confirming actions of superoxide and not of hydrogen peroxide. Unaffected by paraquat and superoxide was calcium entry through store-operated calcium channels activated by thapsigargin-induced calcium depletion of sarcoplasmic reticular stores. Also unresponsive to paraquat was ryanodine receptor-mediated calcium-induced calcium release from the sarcoplasmic reticulum. Our results provide new evidence that superoxide enhances calcium entry through L-type channels activated by membrane depolarization in rat cortical afferent arterioles, without affecting calcium entry through store-operated entry or ryanodine receptor-mediated calcium mobilization.

  4. Splice variants of the CaV1.3 L-type calcium channel regulate dendritic spine morphology

    PubMed Central

    Stanika, Ruslan; Campiglio, Marta; Pinggera, Alexandra; Lee, Amy; Striessnig, Jörg; Flucher, Bernhard E.; Obermair, Gerald J.

    2016-01-01

    Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson’s disease. The L-type calcium channel CaV1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length CaV1.3L and two C-terminally truncated splice variants (CaV1.342A and CaV1.343S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged CaV1.3L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in CaV1.3L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with CaV1.3L and correlated with increased CaV1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of CaV1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of CaV1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders. PMID:27708393

  5. Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca2+ current in cardiac myocytes.

    PubMed

    Wang, Honglan; Kohr, Mark J; Traynham, Christopher J; Ziolo, Mark T

    2009-08-01

    Endothelial nitric oxide synthase (NOS3) regulates the functional response to beta-adrenergic (beta-AR) stimulation via modulation of the L-type Ca(2+) current (I(Ca)). However, the NOS3 signaling pathway modulating I(Ca) is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I(Ca). Myocytes were isolated from NOS3 knockout (NOS3(-/-)) and wildtype (WT) mice. We measured I(Ca) (whole-cell voltage-clamp), and simultaneously measured Ca(2+) transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased beta-AR stimulated (isoproterenol, ISO)-I(Ca), and Ca(2+) transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I(Ca), but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3(-/-) myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I(Ca), or Ca(2+) transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I(Ca), and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I(Ca); YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca(2+) transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I(Ca) and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating beta-AR stimulated I(Ca), while limiting effects on cardiac contraction.

  6. Β-adrenergic-stimulated L-type channel Ca²+ entry mediates hypoxic Ca²+ overload in intact heart.

    PubMed

    Zhang, Huiliang; Shang, Wei; Zhang, Xing; Gu, Jingli; Wang, Xianhua; Zheng, Ming; Wang, Yanru; Zhou, Zhuan; Cao, Ji-Min; Ji, Guangju; Zhang, Rongli; Cheng, Heping

    2013-12-01

    Ca(2+) mishandling plays a key role in ischemia- and hypoxia-related cardiac dysfunction and injury. However, the cellular and molecular mechanisms underlying hypoxic intracellular Ca(2+) ([Ca(2+)]i) overload remain incompletely understood. This study aimed to investigate possible mechanisms of [Ca(2+)]i overload during hypoxia in the intact heart. In Langendorff-perfused heart expressing the Ca(2+) indicator GCaMP2, confocal microscopy was used to simultaneously visualize [Ca(2+)]i, mitochondrial membrane potential (ΔΨm, by tetramethylrhodamine methyl ester) and sarcolemmal integrity (by Evans blue). Upon hypoxia (pO2 ~20 mmHg in glucose-free perfusate), [Ca(2+)]i transients were initially enhanced and then became depressed, arrhythmic, and completely abolished within 12 min. At ~20 min, basal [Ca(2+)]i rose to its first peak at a supraphysiological level, coincident with loss of ΔΨm and onset of rigor. A greater [Ca(2+)]i rise occurred at ~2h and was linked to the loss of sarcolemmal integrity. Removal of extracellular Ca(2+) or blockade of the l-type Ca(2+) channel (LTCC) (10 μM diltiazem or nifedipine) prevented [Ca(2+)]i overload and markedly delayed the loss of ΔΨm; by contrast, depletion of the sarcoplasmic reticulum Ca(2+) store by thapsigargin did not have any significant effect. Importantly, β-adrenergic blockade or depletion of the sympathetic catecholamine store by reserpine slowed the Ca(2+) and mitochondrial responses to hypoxia in intact heart. This LTCC-mediated hypoxic [Ca(2+)]i overload was reproduced in isolated cardiomyocytes when β-adrenergic agonist was present. Taken together, we conclude that Ca(2+) entry through β-adrenergic-stimulated LTCC underlies hypoxia-induced [Ca(2+)]i overload and the ensuing loss of mitochondrial function in intact heart. © 2013. Published by Elsevier Ltd. All rights reserved.

  7. Ca2+/calcineurin-dependent inactivation of neuronal L-type Ca2+ channels requires priming by AKAP-anchored protein kinase A.

    PubMed

    Dittmer, Philip J; Dell'Acqua, Mark L; Sather, William A

    2014-06-12

    Within neurons, Ca2+-dependent inactivation (CDI) of voltage-gated L-type Ca2+ channels shapes cytoplasmic Ca2+ signals. CDI is initiated by Ca2+ binding to channel-associated calmodulin and subsequent Ca2+/calmodulin activation of the Ca2+-dependent phosphatase, calcineurin (CaN), which is targeted to L channels by the A-kinase-anchoring protein AKAP79/150. Here, we report that CDI of neuronal L channels was abolished by inhibition of PKA activity or PKA anchoring to AKAP79/150 and that CDI was also suppressed by stimulation of PKA activity. Although CDI was reduced by positive or negative manipulation of PKA, interference with PKA anchoring or activity lowered Ca2+ current density whereas stimulation of PKA activity elevated it. In contrast, inhibition of CaN reduced CDI but had no effect on current density. These results suggest a model wherein PKA-dependent phosphorylation enhances neuronal L current, thereby priming channels to undergo CDI, and Ca2+/calmodulin-activated CaN actuates CDI by reversing PKA-mediated enhancement of channel activity.

  8. Tetrodotoxin Blockade on Canine Cardiac L-Type Ca2+ Channels Depends on pH and Redox Potential

    PubMed Central

    Hegyi, Bence; Komáromi, István; Kistamás, Kornél; Ruzsnavszky, Ferenc; Váczi, Krisztina; Horváth, Balázs; Magyar, János; Bányász, Tamás; Nánási, Péter P.; Szentandrássy, Norbert

    2013-01-01

    Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na+ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca2+ current (ICa) in canine cardiac cells. In the present study, the TTX-sensitivity of ICa was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC50 value obtained under physiological conditions) caused 60% ± 2% inhibition of ICa in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 μM H2O2). Phosphorylation of the channel protein (induced by 3 μM forskolin) failed to modify the inhibiting potency of TTX; an IC50 value of 50 ± 4 μM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels. PMID:23771047

  9. Polycystin-1 is a Cardiomyocyte Mechanosensor That Governs L-type Ca2+ Channel Protein Stability

    PubMed Central

    Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K.; Morales, Cyndi R.; Contreras, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J.; Somlo, Stefan; Rothermel, Beverly A.; Gillette, Thomas G.; Lavandero, Sergio; Hill, Joseph A.

    2015-01-01

    Background L-type calcium channel (LTCC) activity is critical to afterload-induced hypertrophic growth of the heart. However, mechanisms governing mechanical stress-induced activation of LTCC activity are obscure. Polycystin-1 (PC-1) is a G-protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. Methods and Results We subjected neonatal rat ventricular myocytes (NRVMs) to mechanical stretch by exposing them to hypo-osmotic (HS) medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on LTCC activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Over-expression of a C-terminal fragment of PC-1 was sufficient to trigger NRVM hypertrophy. Exposing NRVMs to HS medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 KO) and subjected them to mechanical stress in vivo (transverse aortic constriction, TAC). At baseline, PC-1 KO mice manifested decreased cardiac function relative to littermate controls, and α1C LTCC protein levels were significantly lower in PC-1 KO hearts. Whereas control mice manifested robust TAC-induced increases in cardiac mass, PC-1 KO mice showed no significant growth. Likewise, TAC-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals Conclusions PC-1 is a cardiomyocyte mechanosensor and is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. PMID:25888683

  10. Effects of SEA0400 and KB-R7943 on Na+/Ca2+ exchange current and L-type Ca2+ current in canine ventricular cardiomyocytes.

    PubMed

    Birinyi, Péter; Acsai, Károly; Bányász, Tamás; Tóth, András; Horváth, Balázs; Virág, László; Szentandrássy, Norbert; Magyar, János; Varró, András; Fülöp, Ferenc; Nánási, Péter P

    2005-07-01

    SEA0400 and KB-R7943 are compounds synthesised to block transsarcolemmal Na+/Ca2+ exchange current (I(Na/Ca)); however, they have also been shown to inhibit L-type Ca2+ current (I(Ca)). The potential value of these compounds depends critically on their relative selectivity for I(Na/Ca) over I(Ca). In the present work, therefore, the concentration-dependent effects of SEA0400 and KB-R7943 on I(Na/Ca) and I(Ca) were studied and compared in canine ventricular cardiomyocytes using the whole-cell configuration of the patch clamp technique. SEA0400 and KB-R7943 decreased I(Na/Ca) in a concentration-dependent manner, having EC50 values of 111+/-43 nM and 3.35+/-0.82 microM, when suppressing inward currents, while the respective EC50 values were estimated at 108+/-18 nM and 4.74+/-0.69 microM in the case of outward current block. SEA0400 and KB-R7943 also blocked I(Ca), having comparable EC50 values (3.6 microM and 3.2 microM, respectively). At higher concentrations (10 microM) both drugs accelerated inactivation of I(Ca), retarded recovery from inactivation and shifted the voltage dependence of inactivation towards more negative voltages. The voltage dependence of activation was slightly modified by SEA0400, but not by KB-R7943. Based on the relatively good selectivity of submicromolar concentrations of SEA0400--but not KB-R7943--for I(Na/Ca) over I(Ca), SEA0400 appears to be a suitable tool to study the role of I(Na/Ca) in Ca2+ handling in canine cardiac cells. At concentrations higher than 1 microM, however, I(Ca) is progressively suppressed by the compound.

  11. The voltage-dependent L-type Ca2+ (CaV1.2) channel C-terminus fragment is a bi-modal vasodilator.

    PubMed

    Bannister, John P; Leo, Marie Dennis; Narayanan, Damodaran; Jangsangthong, Wanchana; Nair, Anitha; Evanson, Kirk W; Pachuau, Judith; Gabrick, Kyle S; Boop, Frederick A; Jaggar, Jonathan H

    2013-06-15

    Voltage-dependent L-type Ca(2+) channels (CaV1.2) are the primary Ca(2+) entry pathway in vascular smooth muscle cells (myocytes). CaV1.2 channels control systemic blood pressure and organ blood flow and are pathologically altered in vascular diseases, which modifies vessel contractility. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt). Previous studies in cardiac myocytes and neurons have identified CCt as both a transcription factor and CaV1.2 channel inhibitor, with different signalling mechanisms proposed to underlie some of these effects. CCt existence and physiological functions in arterial myocytes are unclear, but important to study given the functional significance of CaV1.2 channels. Here, we show that CCt exists in myocytes of both rat and human resistance-size cerebral arteries, where it locates to both the nucleus and plasma membrane. Recombinant CCt expression in arterial myocytes inhibited CaV1.2 transcription and reduced CaV1.2 protein. CCt induced a depolarizing shift in the voltage dependence of both CaV1.2 current activation and inactivation, and reduced non-inactivating current in myocytes. Recombinant truncated CCt lacking a putative nuclear localization sequence (92CCt) did not locate to the nucleus and had no effect on arterial CaV1.2 transcription or protein. However, 92CCt shifted the voltage dependence of CaV1.2 activation and inactivation similarly to CCt. CCt and 92CCt both inhibited pressure- and depolarization-induced vasoconstriction, although CCt was a far more effective vasodilator. These data demonstrate that endogenous CCt exists and reduces both CaV1.2 channel expression and voltage sensitivity in arterial myocytes. Thus, CCt is a bi-modal vasodilator.

  12. A model of the L-type Ca2+ channel in rat ventricular myocytes: ion selectivity and inactivation mechanisms

    PubMed Central

    Sun, Liang; Fan, Jing-Song; Clark, John W; Palade, Philip T

    2000-01-01

    We have developed a mathematical model of the L-type Ca2+ current, which is based on data from whole-cell voltage clamp experiments on rat ventricular myocytes. Ion substitution methods were employed to investigate the ionic selectivity of the channel. Experiments were configured with Na+, Ca2+ or Ba2+ as the majority current carrier. The amplitude of current through the channel is attenuated in the presence of extracellular Ca2+ or Ba2+. Our model accounts for channel selectivity by using a modified Goldman-Hodgkin-Katz (GHK) configuration that employs voltage-dependent channel binding functions for external divalent ions. Stronger binding functions were used for Ca2+ than for Ba2+. Decay of the ionic current during maintained depolarization was characterized by means of voltage- and Ca2+-dependent inactivation pathways embedded in a five-state dynamic channel model. Particularly, Ca2+ first binds to calmodulin and the Ca2+-calmodulin complex is the mediator of Ca2+ inactivation. Ba2+-dependent inactivation was characterized using the same scheme, but with a decreased binding to calmodulin. A reduced amount of steady-state inactivation, as evidenced by a U-shaped curve at higher depolarization levels (>40 mV) in the presence of [Ca2+]o, was observed in double-pulse protocols used to study channel inactivation. To characterize this phenomenon, a mechanism was incorporated into the model whereby Ca2+ or Ba2+ also inhibits the voltage-dependent inactivation pathway. The five-state dynamic channel model was also used to simulate single channel activity. Calculations of the open probability of the channel model are generally consistent with experimental data. A sixth state can be used to simulate modal activity by way of introducing long silent intervals. Our model has been tested extensively using experimental data from a wide variety of voltage clamp protocols and bathing solution manipulations. It provides: (a) biophysically based explanations of putative mechanisms

  13. [Metabolites of long-time preserved-acutely isolated rat cardiomyocytes affect L-type Ca(2+) channel current].

    PubMed

    Yue, Zhi-Jie; Sheng, Juan-Juan; Xie, Man-Jiang; Yu, Zhi-Bin

    2013-02-25

    The variability of peak current of L-type calcium channel (I(Ca,L)) shows an increase in cardiomyocytes after 6 h of preservation when the acutely isolated cardiomyocytes are preserved in a small volume buffer solution. The mechanism of the increased variability of I(Ca,L) is not clear. In order to obtain more accurately and stably experimental data of I(Ca,L), the aim of this study was to observe the pH changes of preservation buffer solution with acutely isolated rat cardiomyocytes, and the effects of pH changes on the shape of cardiomyocytes, the function of mitochondria and the gating property of L-type calcium channel. The results indicated that the pH was kept stable in 100 mL buffer solution, but was decreased from 7.20 to 6.95 in 20 mL buffer solution during 10 h of cardiomyocyte preservation. Therefore, 100 mL or 20 mL preservation solution was used as a normal control or acidotic group, respectively. The ratio of abnormal to normal rod-shaped cardiomyocytes increased in the acidotic group after 6 h of preservation. The acidosis induced a reduction in mitochondrial membrane potential indicated by JC-1 fluorescent probe after 8 h of cardiomyocyte preservation. The acidosis also shifted the autofluorescence of NADPH from blue to green after 8 h of cardiomyocyte preservation. The above changes in mitochondrial function induced a significant decrease in the peak I(Ca,L) and a shift in the clamped voltage at peak I(Ca,L) from +10 mV to 0 mV, after 10 h of cardiomyocyte preservation. These results suggest that the best way to preserve acutely isolated cardiomyocytes is to use a larger volume buffer system. In order to get stable peak I(Ca,L), we need to not only select a normal shape of cardiomyocyte at a bright field but also a blue fluorescent myocyte at an ultraviolet excitation.

  14. Inhibitory effects of purified antibody against α-1 repeat (117-137) on Na(+)-Ca(2+) exchange and L-type Ca(2+) currents in rat cardiomyocytes.

    PubMed

    Feng, Qi-Long; Wu, Dong-Mei; Cui, Xiang-Li; Zhao, Hua-Chen; Lin, Yuan-Yuan; Zhao, Lu-Ying; Wu, Bo-Wei

    2010-10-25

    Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).

  15. Reduced density and altered regulation of rat atrial L-type Ca2+ current in heart failure

    PubMed Central

    Bond, Richard C.; Bryant, Simon M.; Watson, Judy J.; Hancox, Jules C.; Orchard, Clive H.

    2017-01-01

    Constitutive regulation by PKA has recently been shown to contribute to L-type Ca2+ current (ICaL) at the ventricular t-tubule in heart failure. Conversely, reduction in constitutive regulation by PKA has been proposed to underlie the downregulation of atrial ICaL in heart failure. The hypothesis that downregulation of atrial ICaL in heart failure involves reduced channel phosphorylation was examined. Anesthetized adult male Wistar rats underwent surgical coronary artery ligation (CAL, N=10) or equivalent sham-operation (Sham, N=12). Left atrial myocytes were isolated ~18 wk postsurgery and whole cell currents recorded (holding potential=-80 mV). ICaL activated by depolarizing pulses to voltages from -40 to +50 mV were normalized to cell capacitance and current density-voltage relations plotted. CAL cell capacitances were ~1.67-fold greater than Sham (P ≤ 0.0001). Maximal ICaL conductance (Gmax) was downregulated more than 2-fold in CAL vs. Sham myocytes (P < 0.0001). Norepinephrine (1 μmol/l) increased Gmax >50% more effectively in CAL than in Sham so that differences in ICaL density were abolished. Differences between CAL and Sham Gmax were not abolished by calyculin A (100 nmol/l), suggesting that increased protein dephosphorylation did not account for ICaL downregulation. Treatment with either H-89 (10 μmol/l) or AIP (5 μmol/l) had no effect on basal currents in Sham or CAL myocytes, indicating that, in contrast to ventricular myocytes, neither PKA nor CaMKII regulated basal ICaL. Expression of the L-type α1C-subunit, protein phosphatases 1 and 2A, and inhibitor-1 proteins was unchanged. In conclusion, reduction in PKA-dependent regulation did not contribute to downregulation of atrial ICaL in heart failure. NEW & NOTEWORTHY Whole cell recording of L-type Ca2+ currents in atrial myocytes from rat hearts subjected to coronary artery ligation compared with those from sham-operated controls reveals marked reduction in current density in heart failure without

  16. Reduced density and altered regulation of rat atrial L-type Ca(2+) current in heart failure.

    PubMed

    Bond, Richard C; Bryant, Simon M; Watson, Judy J; Hancox, Jules C; Orchard, Clive H; James, Andrew F

    2017-03-01

    Constitutive regulation by PKA has recently been shown to contribute to L-type Ca(2+) current (ICaL) at the ventricular t-tubule in heart failure. Conversely, reduction in constitutive regulation by PKA has been proposed to underlie the downregulation of atrial ICaL in heart failure. The hypothesis that downregulation of atrial ICaL in heart failure involves reduced channel phosphorylation was examined. Anesthetized adult male Wistar rats underwent surgical coronary artery ligation (CAL, N=10) or equivalent sham-operation (Sham, N=12). Left atrial myocytes were isolated ~18 wk postsurgery and whole cell currents recorded (holding potential=-80 mV). ICaL activated by depolarizing pulses to voltages from -40 to +50 mV were normalized to cell capacitance and current density-voltage relations plotted. CAL cell capacitances were ~1.67-fold greater than Sham (P ≤ 0.0001). Maximal ICaL conductance (Gmax ) was downregulated more than 2-fold in CAL vs. Sham myocytes (P < 0.0001). Norepinephrine (1 μmol/l) increased Gmax >50% more effectively in CAL than in Sham so that differences in ICaL density were abolished. Differences between CAL and Sham Gmax were not abolished by calyculin A (100 nmol/l), suggesting that increased protein dephosphorylation did not account for ICaL downregulation. Treatment with either H-89 (10 μmol/l) or AIP (5 μmol/l) had no effect on basal currents in Sham or CAL myocytes, indicating that, in contrast to ventricular myocytes, neither PKA nor CaMKII regulated basal ICaL Expression of the L-type α1C-subunit, protein phosphatases 1 and 2A, and inhibitor-1 proteins was unchanged. In conclusion, reduction in PKA-dependent regulation did not contribute to downregulation of atrial ICaL in heart failure.NEW & NOTEWORTHY Whole cell recording of L-type Ca(2+) currents in atrial myocytes from rat hearts subjected to coronary artery ligation compared with those from sham-operated controls reveals marked reduction in current density in heart failure

  17. Age-related homeostatic mid-channel proteolysis of neuronal L-type voltage-gated Ca2+ channels

    PubMed Central

    Michailidis, Ioannis E.; Abele-Henckels, Kathryn; Zhang, Wei K.; Lin, Bochao; Yu, Yong; Geyman, Larry; Ehlers, Michael D.; Pnevmatikakis, Eftychios A.; Yang, Jian

    2014-01-01

    SUMMARY Neural circuitry and brain activity depend critically on proper function of voltage-gated calcium channels (VGCCs), whose activity must be tightly controlled. We show that the main body of the pore-forming α1 subunit of neuronal L-type VGCCs, Cav1.2, is proteolytically cleaved, resulting in Cav1.2 fragment-channels that separate but remain on the plasma membrane. This “gmid-channel” proteolysis is regulated by channel activity, involves the Ca2+-dependent protease calpain and the ubiquitin-proteasome system, and causes attenuation and biophysical alterations of VGCC currents. Recombinant Cav1.2 fragment-channels mimicking the products of mid-channel proteolysis do not form active channels on their own, but when properly paired, produce currents with distinct biophysical properties. Mid-channel proteolysis increases dramatically with age and can be attenuated with an L-type VGCC blocker in vivo. Mid-channel proteolysis represents a novel form of homeostatic negative-feedback processing of VGCCs that could profoundly affect neuronal excitability, neurotransmission, neuroprotection, and calcium signaling in physiological and disease states. PMID:24908485

  18. Analysis of factors affecting Ca(2+)-dependent inactivation dynamics of L-type Ca(2+) current of cardiac myocytes in pulmonary vein of rabbit.

    PubMed

    Ryu, Ju Seok; Kim, Won Tae; Lee, Jeong Hoon; Kwon, Jeong Hoon; Kim, Hyun A; Shim, Eun Bo; Youm, Jae Boum; Leem, Chae Hun

    2012-09-15

    L-type Ca(2+) channels (ICaLs) are inactivated by an increase in intracellular [Ca(2+)], known as Ca(2+)-dependent inactivation (CDI). CDI is also induced by Ca(2+) released from the sarcoplasmic reticulum (SR), known as release-dependent inhibition (RDI). As both CDI and RDI occur in the junctional subsarcolemmal nanospace (JSS), we investigated which factors are involved within the JSS using isolated cardiac myocytes from the main pulmonary vein of the rabbit. Using the whole-cell patch clamp technique, RDI was readily observed with the application of a pre-pulse followed by a test pulse, during which the ICaLs exhibited a decrease in peak current amplitude and a slower inactivation. A fast acting Ca(2+) chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), abolished this effect. As the time interval between the pre-pulse and test pulse increased, the ICaLs exhibited greater recovery and the RDI was relieved. Inhibition of the ryanodine receptor (RyR) or the SR Ca(2+)-ATPase (SERCA) greatly attenuated RDI and facilitated ICaL recovery. Removal of extracellular Na(+),which inhibits the Na(+)-Ca(2+) exchange (Incx), greatly enhanced RDI and slowed ICaL recovery, suggesting that Incx critically controls the [Ca(2+)] in the JSS. We incorporated the Ca(2+)-binding kinetics of the ICaL into a previously published computational model. By assuming two Ca(2+)-binding sites in the ICaL, of which one is of low-affinity with fast kinetics and the other is of high-affinity with slower kinetics, the new model was able to successfully reproduce RDI and its regulation by Incx. The model suggests that Incx accelerates Ca(2+) removal from the JSS to downregulate CDI and attenuates SR Ca(2+) refilling. The model may be useful to elucidate complex mechanisms involved in excitation–contraction coupling in myocytes.

  19. Reversal of age-related alterations in synaptic plasticity by blockade of L-type Ca2+ channels.

    PubMed

    Norris, C M; Halpain, S; Foster, T C

    1998-05-01

    The role of L-type Ca2+ channels in the induction of synaptic plasticity in hippocampal slices of aged (22-24 months) and young adult (4-6 months) male Fischer 344 rats was investigated. Prolonged 1 Hz stimulation (900 pulses) of Schaffer collaterals, which normally depresses CA3/CA1 synaptic strength in aged rat slices, failed to induce long-term depression (LTD) during bath application of the L-channel antagonist nifedipine (10 microM). When 5 Hz stimulation (900 pulses) was used to modify synaptic strength, nifedipine facilitated synaptic enhancement in slices from aged, but not young, adult rats. This enhancement was pathway-specific, reversible, and impaired by the NMDA receptor (NMDAR) antagonist DL-2-amino-5-phosphonopentanoic acid (AP5). Induction of long-term potentiation (LTP) in aged rats, using 100 Hz stimulation, occluded subsequent synaptic enhancement by 5 Hz stimulation, suggesting that nifedipine-facilitated enhancement shares mechanisms in common with conventional LTP. Facilitation of synaptic enhancement by nifedipine likely was attributable to a reduction ( approximately 30%) in the Ca2+-dependent K+-mediated afterhyperpolarization (AHP), because the K+ channel blocker apamin (1 microM) similarly reduced the AHP and promoted synaptic enhancement by 5 Hz stimulation. In contrast, apamin did not block LTD induction using 1 Hz stimulation, suggesting that, in aged rats, the AHP does not influence LTD and LTP induction in a similar way. The results indicate that, during aging, L-channels can (1) facilitate LTD induction during low rates of synaptic activity and (2) impair LTP induction during higher levels of synaptic activation via an increase in the Ca2+-dependent AHP.

  20. CO(2) and pH independently modulate L-type Ca(2+) current in rabbit carotid body glomus cells.

    PubMed

    Summers, Beth A; Overholt, Jeffrey L; Prabhakar, Nanduri R

    2002-08-01

    affect cAMP levels in rabbit carotid bodies. Further, nisoldipine, but not omega-conotoxin MVIIC, prevented augmentation of the Ca(2+) current by CO(2). In addition, when combined, hypercapnia and hypoxia augmented the Ca(2+) current by 26 +/- 4% (n = 7), which is greater than either stimulus alone, suggesting the effects are additive. Taken together, these results indicate that L-type Ca(2+) current is augmented by hypercapnia. The effect of CO(2) is not secondary to changes in pH and seems to be mediated by a protein kinase A-dependent mechanism. Furthermore, hypercapnia and hypoxia act additively in stimulating Ca(2+) current in glomus cells.

  1. Low concentrations of alcohol inhibit BDNF-dependent GABAergic plasticity via L-type Ca2+ channel inhibition in developing CA3 hippocampal pyramidal neurons.

    PubMed

    Zucca, Stefano; Valenzuela, C Fernando

    2010-05-12

    Fetal alcohol spectrum disorder (FASD) is associated with learning and memory alterations that could be, in part, a consequence of hippocampal damage. The CA3 hippocampal subfield is one of the regions affected by ethanol (EtOH), including exposure during the third trimester-equivalent (i.e., neonatal period in rats). However, the mechanism of action of EtOH is poorly understood. In CA3 pyramidal neurons from neonatal rats, dendritic BDNF release causes long-term potentiation of the frequency of GABAA receptor-mediated spontaneous postsynaptic currents (LTP-GABAA) and this mechanism is thought to play a role in GABAergic synapse maturation. Here, we show that short- and long-term exposure of neonatal male rats to low EtOH concentrations abolishes LTP-GABAA by inhibiting L-type voltage-gated Ca2+ channels. These findings support the recommendation that even light drinking should be avoided during pregnancy.

  2. Beta-adrenergic regulation of the L-type Ca2+ channel Ca(V)1.2 by PKA rekindles excitement.

    PubMed

    Hell, Johannes W

    2010-09-28

    When we are frightened, our hearts beat more rapidly and forcefully so we can fight more intensely or run away faster. This fight-or-flight response is triggered by the release of norepinephrine from neurons of the sympathetic nervous system. Norepinephrine activates the classic β-adrenergic receptor-heterotrimeric G(s) protein-adenylyl cyclase-adenosine 3',5'-monophosphate-protein kinase A (PKA) signaling cascade. One of the main PKA targets implicated in this response is the L-type Ca2+ channel Ca(V)1.2, which mediates Ca2+ influx into cardiomyocytes. Because of its central function in regulating heartbeat, and because the underlying molecular mechanism has remained elusive, understanding the regulation of Ca(V)1.2 has been considered the holy grail for the field of channel regulation. New evidence from the quest to solve the mystery of Ca(V)1.2 regulation has revealed that reproducible reconstitution of this regulation in heterologous cells requires a perfect balance of the ratio of Ca(V)1.2 to A-kinase anchor proteins (AKAPs). Proteolytic processing of the cytosolic C terminus of the central, pore-forming α₁1.2 subunit of Ca(V)1.2 contributed to its regulation by PKA, and Ser(1700) in the C terminus of the α₁1.2 subunit emerged as the relevant PKA phosphorylation site.

  3. Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells

    PubMed Central

    Villarroya, Mercedes; Olivares, Román; Ruíz, Ana; Cano-Abad, María F; de Pascual, Ricardo; Lomax, Richard B; López, Manuela G; Mayorgas, Inés; Gandía, Luis; García, Antonio G

    1999-01-01

    In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+-free solution containing 1·2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0·65 ± 0·02 fmol cell−1; in depolarized cells (bathed in nominally Ca2+-free solution containing 100 mM K+) the net Ca2+ uptake was 0·16 ± 0·01 fmol cell−1. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura-2-loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1·2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells. The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca2+ channels. This was strengthened by two additional findings: (i) nifedipine (3 μM), an L-type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L-type Ca2+ channel ‘activator’, dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. In voltage

  4. Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents.

    PubMed

    Luo, An-tao; Cao, Zhen-zhen; Xiang, Yu; Zhang, Shuo; Qian, Chun-ping; Fu, Chen; Zhang, Pei-hua; Ma, Ji-hua

    2015-11-01

    Intracellular Ca(2+) ([Ca(2+)]i) overload occurs in myocardial ischemia. An increase in the late sodium current (INaL) causes intracellular Na(+) overload and subsequently [Ca(2+)]i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca(2+)]i overload. The aim of this study was to investigate the effects of ketamine on Na(+)-dependent Ca(2+) overload in ventricular myocytes in vitro. Ventricular myocytes were enzymatically isolated from hearts of rabbits. INaL, NCX current (INCX) and L-type Ca(2+) current (ICaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca(2+)]i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system. Ketamine (20, 40, 80 μmol/L) inhibited INaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), INaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented INaL. Ketamine (40 μmol/L) also significantly suppressed hypoxia or H2O2-induced enhancement of INaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode INCX. In addition, ketamine (40 μmol/L) inhibited ICaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca(2+)]i, and the rate and amplitude of [Ca(2+)]i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 μmol/L) or by ketamine (40 μmol/L). Ketamine protects isolated rabbit ventricular myocytes against [Ca(2+)]i overload by inhibiting INaL and ICaL.

  5. Interactions between N and C termini of α1C subunit regulate inactivation of CaV1.2 L-type Ca2+ channel

    PubMed Central

    Benmocha Guggenheimer, Adva; Almagor, Lior; Tsemakhovich, Vladimir; Tripathy, Debi Ranjan; Hirsch, Joel A; Dascal, Nathan

    2016-01-01

    The modulation and regulation of voltage-gated Ca2+ channels is affected by the pore-forming segments, the cytosolic parts of the channel, and interacting intracellular proteins. In this study we demonstrate a direct physical interaction between the N terminus (NT) and C terminus (CT) of the main subunit of the L-type Ca2+ channel CaV1.2, α1C, and explore the importance of this interaction for the regulation of the channel. We used biochemistry to measure the strength of the interaction and to map the location of the interaction sites, and electrophysiology to investigate the functional impact of the interaction. We show that the full-length NT (amino acids 1-154) and the proximal (close to the plasma membrane) part of the CT, pCT (amino acids 1508-1669) interact with sub-micromolar to low-micromolar affinity. Calmodulin (CaM) is not essential for the binding. The results further suggest that the NT-CT interaction regulates the channel's inactivation, and that Ca2+, presumably through binding to calmodulin (CaM), reduces the strength of NT-CT interaction. We propose a molecular mechanism in which NT and CT of the channel serve as levers whose movements regulate inactivation by promoting changes in the transmembrane core of the channel via S1 (NT) or S6 (pCT) segments of domains I and IV, accordingly, and not as a kind of pore blocker. We hypothesize that Ca2+-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of CaV1.2 inactivation induced by Ca2+ entry into the cell. PMID:26577286

  6. Caveolae-specific activation loop between CaMKII and L-type Ca(2+) channel aggravates cardiac hypertrophy in α1-adrenergic stimulation.

    PubMed

    Tonegawa, Kota; Otsuka, Wataru; Kumagai, Shohei; Matsunami, Sachi; Hayamizu, Nao; Tanaka, Shota; Moriwaki, Kazumasa; Obana, Masanori; Maeda, Makiko; Asahi, Michio; Kiyonari, Hiroshi; Fujio, Yasushi; Nakayama, Hiroyuki

    2017-03-01

    Activation of CaMKII induces a myriad of biological processes and plays dominant roles in cardiac hypertrophy. Caveolar microdomain contains many calcium/calmodulin-dependent kinase II (CaMKII) targets, including L-type Ca(2+) channel (LTCC) complex, and serves as a signaling platform. The location of CaMKII activation is thought to be critical; however, the roles of CaMKII in caveolae are still elusive due to lack of methodology for the assessment of caveolae-specific activation. Our aim was to develop a novel tool for the specific analysis of CaMKII activation in caveolae and to determine the functional role of caveolar CaMKII in cardiac hypertrophy. To assess the caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin-3 (cPLN-Cav3) and GFP fusion protein with caveolin-binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP), which inhibits CaMKII activation specifically in caveolae. Caveolae-specific activation of CaMKII was detected using phosphospecific antibody for PLN (Thr(17)). Furthermore, adenoviral overexpression of LTCC β2a-subunit (β2a) in NRCMs showed its constitutive phosphorylation by CaMKII, which induces hypertrophy, and that both phosphorylation and hypertrophy are abolished by CBD-GFP-AIP expression, indicating that β2a phosphorylation occurs specifically in caveolae. Finally, β2a phosphorylation was observed after phenylephrine stimulation in β2a-overexpressing mice, and attenuation of cardiac hypertrophy after chronic phenylephrine stimulation was observed in nonphosphorylated mutant of β2a-overexpressing mice. We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated β2a dominantly localizes to caveolae and induces cardiac hypertrophy after α1-adrenergic stimulation in mice.NEW & NOTEWORTHY While signaling in caveolae is thought to be important in cardiac hypertrophy, direct evidence is missing

  7. Functional proteins involved in regulation of intracellular Ca(2+) for drug development: chronic nicotine treatment upregulates L-type high voltage-gated calcium channels.

    PubMed

    Katsura, Masashi; Ohkuma, Seitaro

    2005-03-01

    Neurochemical mechanisms underlying drug dependence and withdrawal syndrome remain unclear. In this review, we discuss how chronic nicotine exposure to neurons affects expression of diazepam binding inhibitor (DBI), an endogenous anxiogenic neuropeptide supposed to be a common substance participating drug dependence, and function of L-type high voltage-gated Ca(2+) channels (HVCCs). We also discuss the functional interaction between DBI and L-type HVCCs in nicotine dependence. Both DBI levels and [(45)Ca(2+)] influx significantly increased in the brain from mice treated with nicotine for long term, which was further enhanced after abrupt cessation of nicotine and was abolished by nicotinic acetylcholine receptor (nAChR) antagonists. Similar responses of DBI expression and L-type HVCC function were observed in cerebral cortical neurons after sustained exposure to nicotine. In addition, increased DBI expression was inhibited by antagonists of nAChR and L-type HVCCs. Sustained exposure of neurons to nicotine significantly enhanced expression of alpha(1) and alpha(2)/delta(1) subunits for L-type HVCCs and caused an increase in the B(max) value of [(3)H]verapamil binding to the particulate fractions. Therefore, it is concluded that the alterations in DBI expression is mediated via increased influx of Ca(2+) through upregulated L-type HVCCs and these neurochemical changes have a close relationship with development of nicotine dependence and/or its withdrawal syndrome.

  8. Impaired Fear Extinction Due to a Deficit in Ca(2+) Influx Through L-Type Voltage-Gated Ca(2+) Channels in Mice Deficient for Tenascin-C.

    PubMed

    Morellini, Fabio; Malyshev, Aleksey; Volgushev, Maxim; Chistiakova, Marina; Papashvili, Giorgi; Fellini, Laetitia; Kleene, Ralf; Schachner, Melitta; Dityatev, Alexander

    2017-01-01

    Mice deficient in the extracellular matrix glycoprotein tenascin-C (TNC(-/-)) express a deficit in specific forms of hippocampal synaptic plasticity, which involve the L-type voltage-gated Ca(2+) channels (L-VGCCs). The mechanisms underlying this deficit and its functional implications for learning and memory have not been investigated. In line with previous findings, we report on impairment in theta-burst stimulation (TBS)-induced long-term potentiation (LTP) in TNC(-/-) mice in the CA1 hippocampal region and its rescue by the L-VGCC activator Bay K-8644. We further found that the overall pattern of L-VGCC expression in the hippocampus in TNC(-/-) mice was normal, but Western blot analysis results uncovered upregulated expression of the Cav1.2 and Cav1.3 α-subunits of L-VGCCs. However, these L-VGCCs were not fully functional in TNC(-/-) mice, as demonstrated by Ca(2+) imaging, which revealed a reduction of nifedipine-sensitive Ca(2+) transients in CA1 pyramidal neurons. TNC(-/-) mice showed normal learning and memory in the contextual fear conditioning paradigm but impaired extinction of conditioned fear responses. Systemic injection of the L-VGCC blockers nifedipine and diltiazem into wild-type mice mimicked the impairment of fear extinction observed in TNC(-/-) mice. The deficiency in TNC(-/-) mice substantially occluded the effects of these drugs. Our results suggest that TNC-mediated modulation of L-VGCC activity is essential for fear extinction.

  9. Profile of L-type Ca2+ current and Na+/Ca2+ exchange current during cardiac action potential in ventricular myocytes

    PubMed Central

    Banyasz, Tamas; Horvath, Balazs; Jian, Zhong; Izu, Leighton T.; Chen-Izu, Ye

    2011-01-01

    Objective The L-type Ca2+ current (ICa,L) and the Na+/Ca2+ exchange current (INCX) are major inward currents that shape the cardiac action potential (AP). Previously, the profile of these currents during AP was determined from voltage-clamp experiments that used Ca2+ buffer. In this study, we aimed to obtain direct experimental measurement of these currents during cardiac AP with Ca2+ cycling. Method A newly developed AP-clamp sequential dissection method was used to record ionic currents in guinea pig ventricular myocytes under a triad of conditions: using the cell’s own AP as the voltage command, using internal and external solutions that mimic the cell’s ionic composition and, importantly, no exogenous Ca2+ buffer was used. Results The nifedipine-sensitive current (INIFE), which is composed of ICa,L and INCX, revealed hitherto unreported features during AP with Ca2+ cycling in the cell. We identified two peaks in the current profile followed by a long residual current extending beyond the AP, coinciding with a residual depolarization. The second peak and the residual current become apparent only when Ca2+ is not buffered. Pharmacological dissection of INIFE using SEA0400 shows that ICa,L is dominant during phase-1&2 whereas INCX contributes significantly to the inward current at phase-3&4 of AP. Conclusion These data provide the first direct experimental visualization of ICa,L and INCX during cardiac AP and Ca2+ cycle. The residual current reported here can serve as a potential substrate for afterdepolarizations when increased under pathologic conditions. PMID:21884673

  10. Rab27a GTPase modulates L-type Ca2+ channel function via interaction with the II-III linker of CaV1.3 subunit.

    PubMed

    Reichhart, Nadine; Markowski, Magdalena; Ishiyama, Shimpei; Wagner, Andrea; Crespo-Garcia, Sergio; Schorb, Talitha; Ramalho, José S; Milenkovic, Vladimir M; Föckler, Renate; Seabra, Miguel C; Strauß, Olaf

    2015-11-01

    In a variety of cells, secretory processes require the activation of both Rab27a and L-type channels of the Ca(V)1.3 subtype. In the retinal pigment epithelium (RPE), Rab27a and Ca(V)1.3 channels regulate growth-factor secretion towards its basolateral side. Analysis of murine retina sections revealed a co-localization of both Rab27a and Ca(V)1.3 at the basolateral membrane of the RPE. Heterologously expressed Ca(V)1.3/β3/α2δ1 channels showed negatively shifted voltage-dependence and decreased current density of about 70% when co-expressed with Rab27a. However, co-localization analysis using α(5)β(1) integrin as a membrane marker revealed that Rab27a co-expression reduced the surface expression of Ca(V)1.3 only about 10%. Physical binding of heterologously expressed Rab27a with Ca(V)1.3 channels was shown by co-localization in immunocytochemistry as well as co-immunoprecipitation which was abolished after deletion of a MyRIP-homologous amino acid sequence at the II-III linker of the Ca(V)1.3 subunit. Rab27a over-expression in ARPE-19 cells positively shifted the voltage dependence, decreased current density of endogenous Ca(V)1.3 channels and reduced VEGF-A secretion. We show the first evidence of a direct functional modulation of an ion channel by Rab27a suggesting a new mechanism of Rab and ion channel interaction in the control of VEGF-A secretion in the RPE.

  11. Trypsin increases availability and open probability of cardiac L-type Ca2+ channels without affecting inactivation induced by Ca2+.

    PubMed Central

    Schmid, R; Seydl, K; Baumgartner, W; Groschner, K; Romanin, C

    1995-01-01

    The patch-clamp technique was employed to investigate the response of single L-type Ca2+ channels to the protease trypsin applied to the intracellular face of excised membrane patches from guinea pig ventricular myocytes. Calpastatin and ATP were used to prevent run-down of Ca2+ channel activity monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)-BAYK 8644. Upon application of trypsin (100 micrograms/ml) channel activity was enhanced fourfold and remained elevated upon removal of trypsin, as expected of a proteolytic, irreversible modification. The trypsin effect was not mediated by a proteolytic activation of protein kinases, as evidenced by the insensitivity of this effect to protein kinase inhibitors. Trypsin-modified Ca2+ channels exhibited the usual run-down phanomenon upon removal of calpastatin and ATP. In ensemble average currents trypsin-induced changes of channel function are apparent as a threefold increase in peak current and a reduction in current inactivation. At the single channel level these effects were based on about a twofold increase in both Ca2+ channels' availability and open probability. Neither the actual number of channels in the patch nor their unitary conductance as well as reversal potential was changed by trypsin. The Ca(2+)-induced inactivation was not impaired, as judged by a comparable sensitivity of trypsin-modified Ca2+ channels to intracellular Ca2+. Similarly, trypsin treatment did not affect the sensitivity of Ca2+ channels to phenylalkylmine inhibition. The observed alterations in channel function are discussed in terms of possible structural correlates. PMID:8580328

  12. Nitric Oxide Is Required for L-Type Ca2+ Channel-Dependent Long-Term Potentiation in the Hippocampus

    PubMed Central

    Pigott, Beatrice M.; Garthwaite, John

    2016-01-01

    Nitric oxide (NO) has long been implicated in the generation of long-term potentiation (LTP) and other types of synaptic plasticity, a role for which the intimate coupling between NMDA receptors (NMDARs) and the neuronal isoform of NO synthase (nNOS) is likely to be instrumental in many instances. While several types of synaptic plasticity depend on NMDARs, others do not, an example of which is LTP triggered by opening of L-type voltage-gated Ca2+ channels (L-VGCCs) in postsynaptic neurons. In CA3-CA1 synapses in the hippocampus, NMDAR-dependent LTP (LTPNMDAR) appears to be primarily expressed postsynaptically whereas L-VGCC-dependent LTP (LTPL−VGCC), which often coexists with LTPNMDAR, appears mainly to reflect enhanced presynaptic transmitter release. Since NO is an excellent candidate as a retrograde messenger mediating post-to-presynaptic signaling, we sought to determine if NO functions in LTPL−VGCC in mouse CA3-CA1 synapses. When elicited by a burst type of stimulation with NMDARs and the associated NO release blocked, LTPL−VGCC was curtailed by inhibition of NO synthase or of the NO-receptor guanylyl cyclase to the same extent as occurred with inhibition of L-VGCCs. Unlike LTPNMDAR at these synapses, LTPL−VGCC was unaffected in mice lacking endothelial NO synthase, implying that the major source of the NO is neuronal. Transient delivery of exogenous NO paired with tetanic synaptic stimulation under conditions of NMDAR blockade resulted in a long-lasting potentiation that was sensitive to inhibition of NO-receptor guanylyl cyclase but was unaffected by inhibition of L-VGCCs. The results indicate that NO, acting through its second messenger cGMP, plays an unexpectedly important role in L-VGCC-dependent, NMDAR-independent LTP, possibly as a retrograde messenger generated in response to opening of postsynaptic L-VGCCs and/or as a signal acting postsynaptically, perhaps to facilitate changes in gene expression. PMID:27445786

  13. Testosterone-induced relaxation involves L-type and store-operated Ca2+ channels blockade, and PGE 2 in guinea pig airway smooth muscle.

    PubMed

    Perusquía, Mercedes; Flores-Soto, Edgar; Sommer, Bettina; Campuzano-González, Elias; Martínez-Villa, Inocencio; Martínez-Banderas, Aldo I; Montaño, Luis M

    2015-04-01

    In vascular smooth muscle, it has been described that testosterone (TES) produces relaxation by blocking L-type Ca(2+) channels. Recently, we found that L-type Ca(2+) and store-operated Ca(2+) (SOC) channels are the main membranal structures that provide extracellular Ca(2+) for carbachol (CCh)-induced contraction in airway smooth muscle (ASM). We studied the possible interactions between L-type and SOC channels in TES-induced relaxation in guinea pig ASM. TES (10, 32, 100, and 178 μM) induced a complete relaxation of CCh-precontracted tracheal smooth muscle, and indomethacin partially inhibited this response. In single myocytes, the KCl-induced intracellular Ca(2+) increase ([Ca(2+)]i) was decreased by 32 and completely blocked by 100 nM TES. This androgen (32 and 100 μM) significantly diminished (~25 and 49 %, respectively) the capacitative Ca(2+) entry. Myocytes stimulated with CCh produced a transient Ca(2+) peak followed by a sustained plateau. D-600 was added during the plateau phase, and a partial diminution (~35 %) was observed. A greater decrease (~78 %) was seen when 2-aminoethyl diphenylborinate (2-APB, SOC antagonist) was used. The combination of both drugs completely abolished the Ca(2+) plateau induced by CCh. TES (100 μM) also completely abolished the CCh-induced Ca(2+) plateau. Indomethacin significantly diminished this effect of TES. PGE2 and butaprost proportionally decreased the Ca(2+) plateau as indomethacin blocked it. Sarcoplasmic reticulum refilling was partially, dependently, and significantly diminished by TES. We concluded that TES-induced relaxation involves blockade of L-type Ca(2+) channels at nanomolar and SOC channels at micromolar concentration and PGE2 seems to be also involved in this phenomenon.

  14. Block of the L-type Ca2+ channel pore by external and internal Mg2+ in rat phaeochromocytoma cells.

    PubMed Central

    Kuo, C C; Hess, P

    1993-01-01

    1. Three to eight micromolar external Mg2+ produces discrete block of the unitary inward currents through the L-type Ca2+ channel carried by 300 mM external Li+. Like the Ca2+ block, increasing Li+ concentration decreases the Mg2+ on-rate and increases the Mg2+ off-rate. 2. These kinetic changes are saturating and the apparent dissociation constant (Kd) for the on-rates in 75 mM Li+ (in activity), the same as that in the case of Ca2+ block. This suggests that Mg2+ and Ca2+ produce the discrete block at the same site. The apparent Kd for the off-rates is 300 mM, much smaller than that in the case of Ca2+ block. This indicates that Mg2+ exerts much less repulsion on the Li+ ion in the neighbouring (enhancement) site than Ca2+, although Mg2+ and Ca2+ both have two charges. The theoretical fits to the off-rates also suggest that Mg2+ can exit the blocking sites at a rate of several hundred per second in the absence of any enhancement effect. 3. Seventeen to forty-eight micromolar internal Mg2+ produces discrete block of the outward unitary currents carried by 300 mM internal Li+. The off-rates are in general approximately 20 times faster as compared to the Mg2+ off-rates in the inward currents. This finding suggests that Mg2+ in the high-affinity sites can much more easily exit to the outside than to the inside, implying significantly higher energy barriers on the inner side of the high-affinity sites for Mg2+. 4. At least 5-10 mM internal Mg2+ is needed to produce discrete block of the inward unitary currents carried by 215 mM external Na+. The off-rates in such experiments are generally the same as those in the case of external Mg2+ block of inward currents. This suggests that internal and external Mg2+ both reach the same site, namely the high-affinity Ca2+ binding sites in the pore, to produce the discrete block. 5. Other than discrete block, 5-10 mM internal Mg2+ also decreases the size of the inward unitary current. This is most probably due to a fast block at

  15. Proteolytic processing of the L-type Ca (2+) channel alpha 11.2 subunit in neurons.

    PubMed

    Buonarati, Olivia R; Henderson, Peter B; Murphy, Geoffrey G; Horne, Mary C; Hell, Johannes W

    2017-01-01

    Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α 11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α 11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (M R)of the α 11.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α 11.2 expressed in HEK293 cells was conducted using six different region-specific antibodies against α 11.2. Results: The full length form of α 11.2 migrated, as expected, with an apparent M R of ~250 kDa. A shorter form of comparable prevalence with an apparent M R of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α 11.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α 11.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.

  16. Mifepristone is a Vasodilator Due to the Inhibition of Smooth Muscle Cells L-Type Ca2+ Channels.

    PubMed

    Mariana, Melissa; Feiteiro, Joana; Cairrao, Elisa; Verde, Ignacio

    2016-06-01

    Derived from the estrane progestins, mifepristone was the first synthetic steroid of this class employed as abortifacient in the first months of pregnancy. Mifepristone reduces high potassium-induced contraction and prevents calcium-induced contraction. At the vascular level, mifepristone induces direct relaxation in rat and human arteries, and this effect seems to be endothelium- and NO independent, suggesting that the vascular smooth muscle is its target. Moreover, mifepristone's effect could involve the modulation of different calcium channels. The aim of the present study is to analyze the involvement of calcium channels in the relaxation induced by mifepristone on vascular smooth muscle cells (VSMCs). Planar cell surface area (PCSA) technique was used to analyze the effect of mifepristone on the VSMC contractility, and the whole cell configuration of patch-clamp technique to measure the activity of L-type Ca(2+) channels (LTCC) in A7r5 cells. Regarding the PCSA technique, mifepristone induced relaxation of the VSMC previously contracted by different agents. Also, a rapid inhibitory effect on basal and BAY K8644-stimulated calcium current was observed, which indicates that this drug has the ability to block LTCC. These results suggest that mifepristone induces relaxation on the VSMCs due to the inhibition of the calcium channels.

  17. Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity

    PubMed Central

    Mangoni, Matteo E.; Couette, Brigitte; Bourinet, Emmanuel; Platzer, Josef; Reimer, Daniel; Striessnig, Jörg; Nargeot, Joël

    2003-01-01

    The spontaneous activity of pacemaker cells in the sino-atrial node (SAN) controls the heart rhythm and rate under physiological conditions. Pacemaker activity in SAN cells is due to the presence of the diastolic depolarization, a slow depolarization phase that drives the membrane voltage from the end of an action potential to the threshold of a new action potential. SAN cells express a wide array of ionic channels, but we have limited knowledge about their functional role in pacemaker activity and we still do not know which channels play a prominent role in the generation of the diastolic depolarization. It is thus important to provide genetic evidence linking the activity of genes coding for ionic channels to specific alterations of pacemaker activity of SAN cells. Here, we show that target inactivation of the gene coding for α1D (Cav1.3) Ca2+ channels in the mouse not only significantly slows pacemaker activity but also promotes spontaneous arrhythmia in SAN pacemaker cells. These alterations of pacemaker activity are linked to abolition of the major component of the L-type current (ICa,L) activating at negative voltages. Pharmacological analysis of ICa,L demonstrates that Cav1.3 gene inactivation specifically abolishes ICa,L in the voltage range corresponding to the diastolic depolarization. Taken together, our data demonstrate that Cav1.3 channels play a major role in the generation of cardiac pacemaker activity by contributing to diastolic depolarization in SAN pacemaker cells. PMID:12700358

  18. Arrhythmogenic effects of mutated L-type Ca 2+-channels on an optogenetically paced muscular pump in Caenorhabditis elegans.

    PubMed

    Schüler, Christina; Fischer, Elisabeth; Shaltiel, Lior; Steuer Costa, Wagner; Gottschalk, Alexander

    2015-09-24

    Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.

  19. Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes

    PubMed Central

    Nystoriak, Matthew A.; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R.; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C.; Scott, John D.; Ward, Sean M.; Hell, Johannes W.; Navedo, Manuel F.

    2017-01-01

    Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. PMID:28119464

  20. Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for β2-adrenergic regulation

    PubMed Central

    Balijepalli, Ravi C.; Foell, Jason D.; Hall, Duane D.; Hell, Johannes W.; Kamp, Timothy J.

    2006-01-01

    L-type Ca2+ channels play a critical role in regulating Ca2+-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca2+ channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca2+ channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for β2-adrenergic receptor (AR) regulation of ICa,L. Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Cav1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Cav1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Cav1.2, β2-AR (not β1-AR), G protein αs, adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized β2-AR/Cav1.2 signaling complex, β2-AR stimulation (salbutamol plus atenolol) of ICa,L was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of ICa,L in response to β2-AR activation was eliminated by disruption of caveolae with 10 mM methyl β-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas β1-AR stimulation (norepinephrine plus prazosin) of ICa,L was not altered. These findings demonstrate that subcellular localization of L-type Ca2+ channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways. PMID:16648270

  1. Impaired Fear Extinction Due to a Deficit in Ca2+ Influx Through L-Type Voltage-Gated Ca2+ Channels in Mice Deficient for Tenascin-C

    PubMed Central

    Morellini, Fabio; Malyshev, Aleksey; Volgushev, Maxim; Chistiakova, Marina; Papashvili, Giorgi; Fellini, Laetitia; Kleene, Ralf; Schachner, Melitta; Dityatev, Alexander

    2017-01-01

    Mice deficient in the extracellular matrix glycoprotein tenascin-C (TNC−/−) express a deficit in specific forms of hippocampal synaptic plasticity, which involve the L-type voltage-gated Ca2+ channels (L-VGCCs). The mechanisms underlying this deficit and its functional implications for learning and memory have not been investigated. In line with previous findings, we report on impairment in theta-burst stimulation (TBS)-induced long-term potentiation (LTP) in TNC−/− mice in the CA1 hippocampal region and its rescue by the L-VGCC activator Bay K-8644. We further found that the overall pattern of L-VGCC expression in the hippocampus in TNC−/− mice was normal, but Western blot analysis results uncovered upregulated expression of the Cav1.2 and Cav1.3 α-subunits of L-VGCCs. However, these L-VGCCs were not fully functional in TNC−/− mice, as demonstrated by Ca2+ imaging, which revealed a reduction of nifedipine-sensitive Ca2+ transients in CA1 pyramidal neurons. TNC−/− mice showed normal learning and memory in the contextual fear conditioning paradigm but impaired extinction of conditioned fear responses. Systemic injection of the L-VGCC blockers nifedipine and diltiazem into wild-type mice mimicked the impairment of fear extinction observed in TNC−/− mice. The deficiency in TNC−/− mice substantially occluded the effects of these drugs. Our results suggest that TNC-mediated modulation of L-VGCC activity is essential for fear extinction. PMID:28824389

  2. The hypotensive agent dodoneine inhibits L-type Ca2+ current with negative inotropic effect on rat heart.

    PubMed

    Carré, Grégoire; Carreyre, Hélène; Ouedraogo, Maurice; Becq, Frédéric; Bois, Patrick; Thibaudeau, Sébastien; Vandebrouck, Clarisse; Bescond, Jocelyn

    2014-04-05

    Agelanthus dodoneifolius is one of the medicinal plants used in African pharmacopeia and traditional medicine for the treatment of cardiovascular diseases. A chemical analysis has identified one of the active principles: Dodoneine (Ddn). It is a new dihydropyranone which exerts hypotensive and vasorelaxant effects on rat. Since the mechanism of the hypotensive effect is unknown, we performed a variety of preclinical and mechanistic studies to characterize the specific cardiac effect of Ddn at tissue (ex-vivo) and cellular levels (in-vitro) in order to determine a molecular target. Ddn effects were evaluated in an isolated rat heart preparation using Langendorff retrograde perfusion and then, the effects of Ddn were characterized in freshly dissociated cardiac ventricular myocytes using the whole-cell patch-clamp configuration. Ex-vivo, Ddn produced a dose-dependent negative inotropic effect with an IC50 value of 10 µM without changed heart rate. 100 µM Ddn decreased left ventricular developed pressure of about 40%. In isolated cardiac myocytes, Ddn reduced I(Ca),L density of about 30% with an IC50 value estimated at 3 µM. Ddn did not change current-voltage relation but it shifted the inactivation curve toward negative potentials and modified the half inactivation potentials. Furthermore, Ddn induced a phasic-dependent blocking on ICa,L. This study demonstrates that the hypotensive property of dodoneine is likely associated with a negative inotropic effect and the blockade of the L-type calcium channels. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Adenylyl Cyclase Subtype-Specific Compartmentalization: Differential Regulation of L-type Ca2+ Current in Ventricular Myocytes

    PubMed Central

    Timofeyev, Valeriy; Myers, Richard E.; Kim, Hyo Jeong; Woltz, Ryan L.; Sirish, Padmini; Heiserman, James P.; Li, Ning; Singapuri, Anil; Tang, Tong; Yarov-Yarovoy, Vladimir; Yamoah, Ebenezer N.; Hammond, H. Kirk; Chiamvimonvat, Nipavan

    2013-01-01

    Rationale Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor (βAR) signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC type 5 (ACV) and 6 (ACVI) are the two main isoforms in the heart. While highly homologous in sequence, these two proteins nevertheless play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is predominantly located along invaginations of the cell membrane known as t-tubules. Objective We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these two isoforms in ventricular myocytes. Methods and Results We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. ACVI isoform is localized in the plasma membrane outside of the t-tubular region, and is responsible for β1AR signaling-mediated enhancement of the L-type Ca2+ current (ICa,L) in ventricular myocytes. In contrast, ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase (PDE). We further demonstrate that the interaction between caveolin-3 with ACV and PDE is responsible for the compartmentalization of ACV signaling. Conclusions Our results provide new insights into the compartmentalization of the two AC isoforms in the regulation of ICa,L in ventricular myocytes. Since caveolae are found in most mammalian cells, the mechanism of βAR and AC compartmentalization may also be important for βAR signaling in other cell types. PMID:23609114

  4. Knockout of Na+/Ca2+ exchanger in smooth muscle attenuates vasoconstriction and L-type Ca2+ channel current and lowers blood pressure

    PubMed Central

    Ren, Chongyu; Chen, Ling; Navedo, Manuel F.; Antos, Laura K.; Kinsey, Stephen P.; Iwamoto, Takahiro; Philipson, Kenneth D.; Kotlikoff, Michael I.; Santana, Luis F.; Wier, W. Gil; Matteson, Donald R.; Blaustein, Mordecai P.

    2010-01-01

    Mice with smooth muscle (SM)-specific knockout of Na+/Ca2+ exchanger type-1 (NCX1SM−/−) and the NCX inhibitor, SEA0400, were used to study the physiological role of NCX1 in mouse mesenteric arteries. NCX1 protein expression was greatly reduced in arteries from NCX1SM−/− mice generated with Cre recombinase. Mean blood pressure (BP) was 6–10 mmHg lower in NCX1SM−/− mice than in wild-type (WT) controls. Vasoconstriction was studied in isolated, pressurized mesenteric small arteries from WT and NCX1SM−/− mice and in heterozygotes with a global null mutation (NCX1Fx/−). Reduced NCX1 activity was manifested by a marked attenuation of responses to low extracellular Na+ concentration, nanomolar ouabain, and SEA0400. Myogenic tone (MT, 70 mmHg) was reduced by ∼15% in NCX1SM−/− arteries and, to a similar extent, by SEA0400 in WT arteries. MT was normal in arteries from NCX1Fx/− mice, which had normal BP. Vasoconstrictions to phenylephrine and elevated extracellular K+ concentration were significantly reduced in NCX1SM−/− arteries. Because a high extracellular K+ concentration-induced vasoconstriction involves the activation of L-type voltage-gated Ca2+ channels (LVGCs), we measured LVGC-mediated currents and Ca2+ sparklets in isolated mesenteric artery myocytes. Both the currents and the sparklets were significantly reduced in NCX1SM−/− (vs. WT or NCX1Fx/−) myocytes, but the voltage-dependent inactivation of LVGCs was not augmented. An acute application of SEA0400 in WT myocytes had no effect on LVGC current. The LVGC agonist, Bay K 8644, eliminated the differences in LVGC currents and Ca2+ sparklets between NCX1SM−/− and control myocytes, suggesting that LVGC expression was normal in NCX1SM−/− myocytes. Bay K 8644 did not, however, eliminate the difference in myogenic constriction between WT and NCX1SM−/− arteries. We conclude that, under physiological conditions, NCX1-mediated Ca2+ entry contributes significantly to the

  5. Single-Channel Monitoring of Reversible L-Type Ca2+ Channel CaVα1-CaVβ Subunit Interaction

    PubMed Central

    Jangsangthong, Wanchana; Kuzmenkina, Elza; Böhnke, Ann Kristin; Herzig, Stefan

    2011-01-01

    Voltage-dependent Ca2+ channels are heteromultimers of CaVα1 (pore), CaVβ- and CaVα2δ-subunits. The stoichiometry of this complex, and whether it is dynamically regulated in intact cells, remains controversial. Fortunately, CaVβ-isoforms affect gating differentially, and we chose two extremes (CaVβ1a and CaVβ2b) regarding single-channel open probability to address this question. HEK293α1C cells expressing the CaV1.2 subunit were transiently transfected with CaVα2δ1 alone or with CaVβ1a, CaVβ2b, or (2:1 or 1:1 plasmid ratio) combinations. Both CaVβ-subunits increased whole-cell current and shifted the voltage dependence of activation and inactivation to hyperpolarization. Time-dependent inactivation was accelerated by CaVβ1a-subunits but not by CaVβ2b-subunits. Mixtures induced intermediate phenotypes. Single channels sometimes switched between periods of low and high open probability. To validate such slow gating behavior, data were segmented in clusters of statistically similar open probability. With CaVβ1a-subunits alone, channels mostly stayed in clusters (or regimes of alike clusters) of low open probability. Increasing CaVβ2b-subunits (co-)expressed (1:2, 1:1 ratio or alone) progressively enhanced the frequency and total duration of high open probability clusters and regimes. Our analysis was validated by the inactivation behavior of segmented ensemble averages. Hence, a phenotype consistent with mutually exclusive and dynamically competing binding of different CaVβ-subunits is demonstrated in intact cells. PMID:22261054

  6. Adrenal fasciculata cells express T-type and rapidly and slowly activating L-type Ca2+ channels that regulate cortisol secretion

    PubMed Central

    Enyeart, Judith A.

    2015-01-01

    In whole cell patch-clamp recordings, we characterized the L-type Ca2+ currents in bovine adrenal zona fasciculata (AZF) cells and explored their role, along with the role of T-type channels, in ACTH- and angiotensin II (ANG II)-stimulated cortisol secretion. Two distinct dihydropyridine-sensitive L-type currents were identified, both of which were activated at relatively hyperpolarized potentials. One activated with rapid kinetics and, in conjunction with Northern blotting and PCR, was determined to be Cav1.3. The other, expressed in approximately one-half of AZF cells, activated with extremely slow voltage-dependent kinetics and combined properties not previously reported for an L-type Ca2+ channel. The T-type Ca2+ channel antagonist 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2) inhibited Cav3.2 current in these cells, as well as ACTH- and ANG II-stimulated cortisol secretion, at concentrations that did not affect L-type currents. In contrast, nifedipine specifically inhibited L-type currents and cortisol secretion, but less effectively than TTA-P2. Diphenylbutylpiperidine Ca2+ antagonists, including pimozide, penfluridol, and fluspirilene, and the dihydropyridine niguldipine blocked Cav3.2 and L-type currents and inhibited ACTH-stimulated cortisol secretion with similar potency. This study shows that bovine AZF cells express three Ca2+ channels, the voltage-dependent gating and kinetics of which could orchestrate complex mechanisms linking peptide hormone receptors to cortisol secretion through action potentials or sustained depolarization. The function of the novel, slowly activating L-type channel is of particular interest in this respect. Regardless, the well-correlated selective inhibition of T- and L-type currents and ACTH- and ANG II-stimulated cortisol secretion by TTA-P2 and nifedipine establish the critical importance of these channels in AZF cell physiology. PMID:25788571

  7. A light stabilizer (Tinuvin 770) that elutes from polypropylene plastic tubes is a potent L-type Ca(2+)-channel blocker.

    PubMed Central

    Glossmann, H; Hering, S; Savchenko, A; Berger, W; Friedrich, K; Garcia, M L; Goetz, M A; Liesch, J M; Zink, D L; Kaczorowski, G J

    1993-01-01

    A pharmacologically active agent was easily extracted by aqueous or organic solvents from laboratory plastic tubes (Falcon Blue Max) and has been chemically identified as bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate. This compound (approximately 12 micrograms per tube approximately 25 nmol) blocked 1,4-dihydropyridine-sensitive 45Ca2+ uptake into GH3 cells with an IC50 value of 3.6 microM, inhibited Sr2+ currents through L-type Ca2+ channels in A7r5 smooth-muscle cells in whole-cell patch-clamp experiments after extracellular application, and affected the high-affinity binding of Ca2+ entry-blocker ligands to a variety of preparations. Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate is a highly potent (IC50 values < 10 nM) inhibitor at the phenylalkylamine- and benzothiazepine-selective drug-binding domains of the alpha 1 subunit of L-type Ca2+ channels. This compound behaves as a heterotropic allosteric regulator for the 1,4-dihydropyridine-selective domain in purified Ca(2+)-channel preparations from rabbit skeletal muscle. (+)-Tetrandrine stimulation of 1,4-dihydropyridine binding to the membrane-bound L-type Ca2+ channel is inhibited by the compound in a competitive manner (Ki value = 6.8 nM). Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate is therefore classified as the prototype of another class of L-type Ca(2+)-channel blockers that binds to the alpha 1 subunit at the drug-binding domains selective for (+)-tetrandrine or (+)-cis-diltiazem. This compound is identical to Tinuvin 770, which is used worldwide as a light stabilizer for polyolefins. PMID:8415734

  8. Antagonism of 4-substituted 1,4-dihydropyridine-3,5-dicarboxylates toward voltage-dependent L-type Ca2+ channels Ca V 1.3 and Ca V 1.2.

    PubMed

    Chang, Che-Chien; Cao, Song; Kang, Soosung; Kai, Li; Tian, Xinyong; Pandey, Prativa; Dunne, Sara Fernandez; Luan, Chi-Hao; Surmeier, D James; Silverman, Richard B

    2010-05-01

    L-type Ca(2+) channels in mammalian brain neurons have either a Ca(V)1.2 or Ca(V)1.3 pore-forming subunit. Recently, it was shown that Ca(V)1.3 Ca(2+) channels underlie autonomous pacemaking in adult dopaminergic neurons in the substantia nigra pars compacta, and this reliance renders them sensitive to toxins used to create animal models of Parkinson's disease. Antagonism of these channels with the dihydropyridine antihypertensive drug isradipine diminishes the reliance on Ca(2+) and the sensitivity of these neurons to toxins, pointing to a potential neuroprotective strategy. However, for neuroprotection without an antihypertensive side effect, selective Ca(V)1.3 channel antagonists are required. In an attempt to identify potent and selective antagonists of Ca(V)1.3 channels, 124 dihydropyridines (4-substituted-1,4-dihydropyridine-3,5-dicarboxylic diesters) were synthesized. The antagonism of heterologously expressed Ca(V)1.2 and Ca(V)1.3 channels was then tested using electrophysiological approaches and the FLIPR Calcium 4 assay. Despite the large diversity in substitution on the dihydropyridine scaffold, the most Ca(V)1.3 selectivity was only about twofold. These results support a highly similar dihydropyridine binding site at both Ca(V)1.2 and Ca(V)1.3 channels and suggests that other classes of compounds need to be identified for Ca(V)1.3 selectivity.

  9. Hippocampal sharp waves and ripples: Effects of aging and modulation by NMDA receptors and L-type Ca2+ channels.

    PubMed

    Kouvaros, S; Kotzadimitriou, D; Papatheodoropoulos, C

    2015-07-09

    Aging is accompanied by a complicated pattern of changes in the brain organization and often by alterations in specific memory functions. One of the brain activities with important role in the process of memory consolidation is thought to be the hippocampus activity of sharp waves and ripple oscillation (SWRs). Using field recordings from the CA1 area of hippocampal slices we compared SWRs as well as single pyramidal cell activity between adult (3-6-month old) and old (24-34-month old) Wistar rats. The slices from old rats displayed ripple oscillation with a significantly less number of ripples and lower frequency compared with those from adult animals. However, the hippocampus from old rats had significantly higher propensity to organized SWRs in long sequences. Furthermore, the bursts recorded from complex spike cells in slices from old compared with adult rats displayed higher number of spikes and longer mean inter-spike interval. Blockade of N-methyl-D-aspartic acid (NMDA) receptors by 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) increased the amplitude of both sharp waves and ripples and increased the interval between events of SWRs in both age groups. On the contrary, CPP reduced the probability of occurrence of sequences of SWRs more strongly in slices from adult than old rats. Blockade of L-type voltage-dependent calcium channels by nifedipine only enhanced the amplitude of sharp waves in slices from adult rats. CPP increased the postsynaptic excitability and the paired-pulse inhibition in slices from both adult and old rats similarly while nifedipine increased the postsynaptic excitability only in slices from adult rats. We propose that the tendency of the aged hippocampus to generate long sequences of SWR events might represent the consequence of homeostatic mechanisms that adaptively try to compensate the impairment in the ripple oscillation in order to maintain the behavioral outcome efficient in the old individuals. The age

  10. Epigenetic regulation of L-type voltage-gated Ca(2+) channels in mesenteric arteries of aging hypertensive rats.

    PubMed

    Liao, Jingwen; Zhang, Yanyan; Ye, Fang; Zhang, Lin; Chen, Yu; Zeng, Fanxing; Shi, Lijun

    2016-11-24

    Accumulating evidence has shown that epigenetic regulation is involved in hypertension and aging. L-type voltage-gated Ca(2+) channels (LTCCs), the dominant channels in vascular myocytes, greatly contribute to arteriole contraction and blood pressure (BP) control. We investigated the dynamic changes and epigenetic regulation of LTCC in the mesenteric arteries of aging hypertensive rats. LTCC function was evaluated by using microvascular rings and whole-cell patch-clamp in the mesenteric arteries of male Wistar-Kyoto rats and spontaneously hypertensive rats at established hypertension (3 month old) and an aging stage (16 month old), respectively. The expression of the LTCC α1C subunit was determined in the rat mesenteric microcirculation. The expression of miR-328, which targets α1C mRNA, and the DNA methylation status at the promoter region of the α1C gene (CACNA1C) were also determined. In vitro experiments were performed to assess α1C expression after transfection of the miR-328 mimic into cultured vascular smooth muscle cells (VSMCs). The results showed that hypertension superimposed with aging aggravated BP and vascular remodeling. Both LTCC function and expression were significantly increased in hypertensive arteries and downregulated with aging. miR-328 expression was inhibited in hypertension, but increased with aging. There was no significant difference in the mean DNA methylation of CACNA1C among groups, whereas methylation was enhanced in the hypertensive group at specific sites on a CpG island located upstream of the gene promoter. Overexpression of miR-328 inhibited the α1C level of cultured VSMCs within 48 h. The results of the present study indicate that the dysfunction of LTCCs may exert an epigenetic influence at both pre- and post-transcriptional levels during hypertension pathogenesis and aging progression. miR-328 negatively regulated LTCC expression in both aging and hypertension.Hypertension Research advance online publication, 24

  11. L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca2+ release during sino-atrial node pacemaker activity.

    PubMed

    Torrente, Angelo Giovanni; Mesirca, Pietro; Neco, Patricia; Rizzetto, Riccardo; Dubel, Stefan; Barrere, Christian; Sinegger-Brauns, Martina; Striessnig, Joerg; Richard, Sylvain; Nargeot, Joël; Gomez, Ana Maria; Mangoni, Matteo Elia

    2016-03-01

    Sino-atrial node (SAN) automaticity is an essential mechanism of heart rate generation that is still not completely understood. Recent studies highlighted the importance of intracellular Ca(2+) ([Ca(2+)]i) dynamics during SAN pacemaker activity. Nevertheless, the functional role of voltage-dependent L-type Ca(2+) channels in controlling SAN [Ca(2+)]i release is largely unexplored. Since Cav1.3 is the predominant L-type Ca(2+) channel isoform in SAN cells, we studied [Ca(2+)]i dynamics in isolated cells and ex vivo SAN preparations explanted from wild-type (WT) and Cav1.3 knockout (KO) mice (Cav1.3(-/-)). We found that Cav1.3 deficiency strongly impaired [Ca(2+)]i dynamics, reducing the frequency of local [Ca(2+)]i release events and preventing their synchronization. This impairment inhibited the generation of Ca(2+) transients and delayed spontaneous activity. We also used action potentials recorded in WT SAN cells as voltage-clamp commands for Cav1.3(-/-) cells. Although these experiments showed abolished Ca(2+) entry through L-type Ca(2+) channels in the diastolic depolarization range of KO SAN cells, their sarcoplasmic reticulum Ca(2+) load remained normal. β-Adrenergic stimulation enhanced pacemaking of both genotypes, though, Cav1.3(-/-) SAN cells remained slower than WT. Conversely, we rescued pacemaker activity in Cav1.3(-/-) SAN cells and intact tissues through caffeine-mediated stimulation of Ca(2+)-induced Ca(2+) release. Cav1.3 channels play a critical role in the regulation of [Ca(2+)]i dynamics, providing an unanticipated mechanism for triggering local [Ca(2+)]i releases and thereby controlling pacemaker activity. Our study also provides an additional pathophysiological mechanism for congenital SAN dysfunction and heart block linked to Cav1.3 loss of function in humans. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  12. Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor

    PubMed Central

    Eltit, Jose Miguel; Bannister, Roger A.; Moua, Ong; Altamirano, Francisco; Hopkins, Philip M.; Pessah, Isaac N.; Molinski, Tadeusz F.; López, Jose R.; Beam, Kurt G.; Allen, Paul D.

    2012-01-01

    Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca2+ release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation–contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and CaV1.1, the principal subunit of the L-type Ca2+ channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca2+ entry. The CaV1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all CaV channels [Carpenter D, et al. (2009) BMC Med Genet 10:104–115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (CaV1.1 null) myotubes. Unlike previously described MH-linked mutations in CaV1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca2+ release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in CaV1.1 R174W-expressing myotubes, resting myoplasmic Ca2+ levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type CaV1.1. Our results indicate that CaV1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca2+ leak from the SR, and that perturbation of CaV1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers. PMID:22547813

  13. Nanoparticle-mediated dual delivery of an antioxidant and a peptide against the L-Type Ca2+ channel enables simultaneous reduction of cardiac ischemia-reperfusion injury.

    PubMed

    Hardy, Naviin; Viola, Helena M; Johnstone, Victoria P A; Clemons, Tristan D; Cserne Szappanos, Henrietta; Singh, Ruhani; Smith, Nicole M; Iyer, K Swaminathan; Hool, Livia C

    2015-01-27

    Increased reactive oxygen species (ROS) production and elevated intracellular Ca(2+) following cardiac ischemia-reperfusion injury are key mediators of cell death and the development of cardiac hypertrophy. The L-type Ca(2+) channel is the main route for calcium influx in cardiac myocytes. Activation of the L-type Ca(2+) channel leads to a further increase in mitochondrial ROS production and metabolism. We have previously shown that the application of a peptide derived against the alpha-interacting domain of the L-type Ca(2+) channel (AID) decreases myocardial injury post reperfusion. Herein, we examine the efficacy of simultaneous delivery of the AID peptide in combination with the potent antioxidants curcumin or resveratrol using multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles. We highlight that drug loading and dissolution are important parameters that have to be taken into account when designing novel combinatorial therapies following cardiac ischemia-reperfusion injury. In the case of resveratrol low loading capacity and fast release rates hinder its applicability as an effective candidate for simultaneous therapy. However, in the case of curcumin, high loading capacity and sustained release rates enable its effective simultaneous delivery in combination with the AID peptide. Simultaneous delivery of the AID peptide with curcumin allowed for effective attenuation of the L-type Ca(2+) channel-activated increases in superoxide (assessed as changes in DHE fluorescence; Empty NP = 53.1 ± 7.6%; NP-C-AID = 7.32 ± 3.57%) and mitochondrial membrane potential (assessed as changes in JC-1 fluorescence; Empty NP = 19.8 ± 2.8%; NP-C-AID=13.05 ± 1.78%). We demonstrate in isolated rat hearts exposed to ischemia followed by reperfusion, that curcumin and the AID peptide in combination effectively reduce muscle damage, decrease oxidative stress and superoxide production in cardiac myocytes.

  14. Adrenaline Stimulates Glucagon Secretion in Pancreatic A-Cells by Increasing the Ca2+ Current and the Number of Granules Close to the L-Type Ca2+ Channels

    PubMed Central

    Gromada, Jesper; Bokvist, Krister; Ding, Wei-Guang; Barg, Sebastian; Buschard, Karsten; Renström, Erik; Rorsman, Patrik

    1997-01-01

    We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through ω-conotoxin-GVIA–sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via β-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels. PMID:9276750

  15. Enhanced Ca²+ influx through cardiac L-type Ca²+ channels maintains the systolic Ca²+ transient in early cardiac atrophy induced by mechanical unloading.

    PubMed

    Schwoerer, A P; Neef, S; Broichhausen, I; Jacubeit, J; Tiburcy, M; Wagner, M; Biermann, D; Didié, M; Vettel, C; Maier, L S; Zimmermann, W H; Carrier, L; Eschenhagen, T; Volk, T; El-Armouche, A; Ehmke, H

    2013-12-01

    Cardiac atrophy as a consequence of mechanical unloading develops following exposure to microgravity or prolonged bed rest. It also plays a central role in the reverse remodelling induced by left ventricular unloading in patients with heart failure. Surprisingly, the intracellular Ca(2+) transients which are pivotal to electromechanical coupling and to cardiac plasticity were repeatedly found to remain unaffected in early cardiac atrophy. To elucidate the mechanisms underlying the preservation of the Ca(2+) transients, we investigated Ca(2+) cycling in cardiomyocytes from mechanically unloaded (heterotopic abdominal heart transplantation) and control (orthotopic) hearts in syngeneic Lewis rats. Following 2 weeks of unloading, sarcoplasmic reticulum (SR) Ca(2+) content was reduced by ~55 %. Atrophic cardiac myocytes also showed a much lower frequency of spontaneous diastolic Ca(2+) sparks and a diminished systolic Ca(2+) release, even though the expression of ryanodine receptors was increased by ~30 %. In contrast, current clamp recordings revealed prolonged action potentials in endocardial as well as epicardial myocytes which were associated with a two to fourfold higher sarcolemmal Ca(2+) influx under action potential clamp. In addition, Cav1.2 subunits which form the pore of L-type Ca(2+) channels (LTCC) were upregulated in atrophic myocardium. These data suggest that in early cardiac atrophy induced by mechanical unloading, an augmented sarcolemmal Ca(2+) influx through LTCC fully compensates for a reduced systolic SR Ca(2+) release to preserve the Ca(2+) transient. This interplay involves an electrophysiological remodelling as well as changes in the expression of cardiac ion channels.

  16. On the top of ARB N/L type Ca channel blocker leads to less elevation of aldosterone

    PubMed Central

    Konoshita, Tadashi; Kaeriyama, Saori; Urabe, Machi; Nakaya, Takahiro; Yamada, Mika; Ichikawa, Mai; Yamamoto, Katsushi; Sato, Satsuki; Imagawa, Michiko; Fujii, Miki; Makino, Yasukazu; Zenimaru, Yasuo; Wakahara, Shigeyuki; Suzuki, Jinya; Ishizuka, Tamotsu; Nakamura, Hiroyuki

    2016-01-01

    The activation of the renin–angiotensin system (RAS) is one of the unfavourable characteristics of calcium channel blocker (CCB). N type calcium channel is thought to be involved in renin gene transcription and adrenal aldosterone release. Accordingly, N/L type CCB has a possibility of less elevation of plasma aldosterone concentrations (PAC) among CCBs. In a monotherapy study, we had already demonstrated that N/L type CCB leads to less activation of the RAS compared with L type CCB. The objective of this study is to substantiate the hypothesis that at the condition of additive administration on the top of an angiotensin receptor blocker (ARB), still N/L type CCB leads to less elevation of PAC compared with L type one. Subjects were 60 hypertensives administered with valsartan. As an open label study, amlodipine (L type) or cilnidipine (N/L type) were administered on the top of valsartan (ARB) in a cross-over manner. Results were as follows (valsartan+amlodipine compared with valsartan+cilnidipine): systolic blood pressure (SBP)/diastolic blood pressure (DBP) (mmHg): 132±10/76±10 compared with 131±10/77±9, P=0.95/0.48, plasma renin activity (PRA) (ng/ml·h): 2.41±2.67 compared with 2.00±1.50 P=0.20, PAC (pg/ml): 77.3±31.0 compared with 67.4±24.8, P<0.05, urinary albumin excretion (UAE) (mg/gCr): 105.9±216.1 compared with 73.9±122.2, P<0.05. Thus, PAC at cilnidipine was significantly lower than those at amlodipine in spite of the comparable BP reductions. Besides, UAE was significantly lower at cilnidipine. In conclusion, on the top of the ARB, it is suggested that cilnidipine administration might lead to less elevation of PAC and reduction in UAE compared with amlodipine. PMID:27515419

  17. Gentamicin Blocks the ACh-Induced BK Current in Guinea Pig Type II Vestibular Hair Cells by Competing with Ca2+ at the l-Type Calcium Channel

    PubMed Central

    Yu, Hong; Guo, Chang-Kai; Wang, Yi; Zhou, Tao; Kong, Wei-Jia

    2014-01-01

    Type II vestibular hair cells (VHCs II) contain big-conductance Ca2+-dependent K+ channels (BK) and l-type calcium channels. Our previous studies in guinea pig VHCs II indicated that acetylcholine (ACh) evoked the BK current by triggering the influx of Ca2+ ions through l-type Ca2+ channels, which was mediated by M2 muscarinic ACh receptor (mAChRs). Aminoglycoside antibiotics, such as gentamicin (GM), are known to have vestibulotoxicity, including damaging effects on the efferent nerve endings on VHCs II. This study used the whole-cell patch clamp technique to determine whether GM affects the vestibular efferent system at postsynaptic M2-mAChRs or the membrane ion channels. We found that GM could block the ACh-induced BK current and that inhibition was reversible, voltage-independent, and dose-dependent with an IC50 value of 36.3 ± 7.8 μM. Increasing the ACh concentration had little influence on GM blocking effect, but increasing the extracellular Ca2+ concentration ([Ca2+]o) could antagonize it. Moreover, 50 μM GM potently blocked Ca2+ currents activated by (−)-Bay-K8644, but did not block BK currents induced by NS1619. These observations indicate that GM most likely blocks the M2 mAChR-mediated response by competing with Ca2+ at the l-type calcium channel. These results provide insights into the vestibulotoxicity of aminoglycoside antibiotics on mammalian VHCs II. PMID:24758923

  18. Apo calmodulin binding to the L-type voltage-gated calcium channel Ca{sub v}1.2 IQ peptide

    SciTech Connect

    Lian Luyun . E-mail: lu-yun.lian@liverpool.ac.uk; Myatt, Daniel; Kitmitto, Ashraf . E-mail: ashraf.kitmitto@manchester.ac.uk

    2007-02-16

    The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic recticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645-1668) housed within the cytoplasmic domain of the LTCC Ca{sub v}1.2 subunit has been shown to bind both calcium-loaded (Ca{sup 2+}CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction of apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca{sup 2+}CaM can bind to the intact channel.

  19. Regulation of Postsynaptic Stability by the L-type Calcium Channel CaV1.3 and its Interaction with PDZ Proteins

    PubMed Central

    Stanika, Ruslan I.; Flucher, Bernhard E.; Obermair, Gerald J.

    2015-01-01

    Alterations in dendritic spine morphology and postsynaptic structure are a hallmark of neurological disorders. Particularly spine pruning of striatal medium spiny neurons and aberrant rewiring of corticostriatal synapses have been associated with the pathology of Parkinson’s disease and L-DOPA induced dyskinesia, respectively. Owing to its low activation threshold the neuronal L-type calcium channel CaV1.3 is particularly critical in the control of neuronal excitability and thus in the calcium-dependent regulation of neuronal functions. CaV1.3 channels are located in dendritic spines and contain a C-terminal class 1 PDZ domain-binding sequence. Until today the postsynaptic PDZ domain proteins shank, densin-180, and erbin have been shown to interact with CaV1.3 channels and to modulate their current properties. Interestingly experimental evidence suggests an involvement of all three PDZ proteins as well as CaV1.3 itself in regulating dendritic and postsynaptic morphology. Here we briefly review the importance of CaV1.3 and its proposed interactions with PDZ proteins for the stability of dendritic spines. With a special focus on the pathology associated with Parkinson’s disease, we discuss the hypothesis that CaV1.3 L-type calcium channels may be critical modulators of dendritic spine stability. PMID:25966696

  20. Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats.

    PubMed

    Zhang, Man; Zang, Kai-Hong; Luo, Jia-Lie; Leung, Fung-Ping; Huang, Yu; Lin, Cheng-Yuan; Yang, Zhi-Jun; Lu, Ai-Ping; Tang, Xu-Dong; Xu, Hong-Xi; Sung, Joseph Jao-yiu; Bian, Zhao-Xiang

    2013-11-15

    This study aimed to investigate the effect of magnolol (5,5'-diallyl-2,2'-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca(2+) currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3-100 μM). In the presence of Bay K8644 (100 nM), magnolol (10-100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and Nώ-nitro-L-arginine methyl ester (L-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3-100 μM) inhibited the L-type Ca(2+) currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca(2+) channel activity.

  1. CaV1.3 L-type channels, maxiK Ca(2+)-dependent K(+) channels and bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells.

    PubMed

    Müller, Claudia; Más Gómez, Néstor; Ruth, Peter; Strauss, Olaf

    2014-05-01

    Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca(2+) regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Cav1.3 L-type Ca(2+) channels (Ca1.3(-/-)) and maxiK Ca(2+)-dependent K(+) channels (BK(-/-)). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca(2+) homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (-)BayK8644 had no impact. The expression rate of Cav1.3, the predominant L-type Ca(2+) channel in RPE cells, varied at different times of day. CaV1.3(-/-) RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK(-/-) RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK(-/-) mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca(2+) channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca(2+) channels.

    PubMed

    Luo, Fujun; Liu, Xinran; Südhof, Thomas C; Acuna, Claudio

    2017-09-05

    Fast neurotransmitter release from ribbon synapses via Ca(2+)-triggered exocytosis requires tight coupling of L-type Ca(2+) channels to release-ready synaptic vesicles at the presynaptic active zone, which is localized at the base of the ribbon. Here, we used genetic, electrophysiological, and ultrastructural analyses to probe the architecture of ribbon synapses by perturbing the function of RIM-binding proteins (RBPs) as central active-zone scaffolding molecules. We found that genetic deletion of RBP1 and RBP2 did not impair synapse ultrastructure of ribbon-type synapses formed between rod bipolar cells (RBCs) and amacrine type-2 (AII) cells in the mouse retina but dramatically reduced the density of presynaptic Ca(2+) channels, decreased and desynchronized evoked neurotransmitter release, and rendered evoked and spontaneous neurotransmitter release sensitive to the slow Ca(2+) buffer EGTA. These findings suggest that RBPs tether L-type Ca(2+) channels to the active zones of ribbon synapses, thereby synchronizing vesicle exocytosis and promoting high-fidelity information transfer in retinal circuits.

  3. Neurochemical characterization of the striatum and the nucleus accumbens in L-type Ca(v)1.3 channels knockout mice.

    PubMed

    Sagala, Ferry S P; Harnack, Daniel; Bobrov, Evgeny; Sohr, Reinhard; Gertler, Christoph; James Surmeier, D; Kupsch, Andreas

    2012-02-01

    L-type Ca(v)1.3 channels control the autonomous pacemaking of the substantia nigra (SN) dopamine (DA) neurons, which maintains the sustained release of DA in the striatum, its target structure. The persistent engagement of L-type channels during pacemaking might lead to increased vulnerability to environmental stressors or degenerative processes, providing a mechanism for the development of Parkinson's disease (PD). Interestingly, L-type channels are not necessary for pacemaking, opening the possible use of calcium channel antagonists as neuroprotective agents for PD without disturbing normal DA function. In this study we aimed to evaluate the consequences of Ca(v)1.3 channels deletion at the neurochemical level. For this purpose, tissue concentrations of DA and their respective metabolites were measured using high performance liquid chromatography (HPLC) in the striatum and the nucleus accumbens (NAcc) of mice lacking the gene for the Ca(v)1.3 channel subunit (CACNA1D) and compared to those in wild-type mice. Striatal DA level did not differ between the two groups. In contrast, the level of serotonin, glutamate, GABA, and taurine were increased by more than 50% in the striatum of Ca(v)1.3 null mice. Neurotransmitters levels in the NAcc did not differ between the different groups. In conclusion, our results neurochemically corroborate the robustness of the nigrostriatal DA neurons in the absence of Ca(v)1.3 channels, but suggest that complete deletion of this channel affected a variety of other transmitter systems.

  4. Differential zinc permeation and blockade of L-type Ca2+ channel isoforms Cav1.2 and Cav1.3.

    PubMed

    Park, So-Jung; Min, Se-Hong; Kang, Ho-Won; Lee, Jung-Ha

    2015-10-01

    Certain voltage-activated Ca2+ channels have been reported to act as potential zinc entry routes. However, it remains to be determined whether zinc can permeate individual Ca2+ channel isoforms. We expressed recombinant Ca2+ channel isoforms in Xenopus oocytes and attempted to record zinc currents from them using a two-electrode voltage clamp method. We found that, in an extracellular zinc solution, inward currents arising from zinc permeation could be recorded from Xenopus oocytes expressing L-type Cav1.2 or Cav1.3 isoforms, but not from oocytes expressing Cav2.2, Cav2.3, Cav3.1, or Cav3.2. Zinc currents through Cav1.2 and Cav1.3 were blocked by nimodipine, but enhanced by (±)Bay K8644, supporting the finding that zinc can permeate both L-type Cav1.2 and Cav1.3 channel isoforms. We also examined the blocking effects of low concentrations of zinc on Ca2+ currents through the L-type channel isoforms. Low micro-molar zinc potently blocked Ca2+ currents through Cav1.2 and Cav1.3 with different sensitivities (IC50 for Cav1.2 and Cav1.3=18.4 and 34.1 μM) and de-accelerated the activation and inactivation kinetics in a concentration-dependent manner. Notably, mild acidifications of the external zinc solution increased zinc currents through Cav1.2 and Cav1.3, with the increment level for Cav1.3 being greater than that for Cav1.2. In overall, we provide evidence that Cav1.2 and Cav1.3 isoforms are capable of potentially functioning as zinc permeation routes, through which zinc entry can be differentially augmented by mild acidifications.

  5. Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway.

    PubMed

    Sandoval, Alejandro; Duran, Paz; Gandini, María A; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

    2017-09-01

    cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca(2+) currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated CaV1.3L-type Ca(2+) channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca(2+) macroscopic currents and impair insulin release stimulated with high K(+). In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for CaV1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Sustained Contraction in Vascular Smooth Muscle by Activation of L-type Ca2+ Channels Does Not Involve Ca2+ Sensitization or Caldesmon

    PubMed Central

    Ets, Hillevi K.; Seow, Chun Y.; Moreland, Robert S.

    2016-01-01

    Vascular smooth muscle (VSM) is unique in its ability to maintain an intrinsic level of contractile force, known as tone. Vascular tone is believed to arise from the constitutive activity of membrane-bound L-type Ca2+ channels (LTCC). This study used a pharmacological agonist of LTCC, Bay K8644, to elicit a sustained, sub-maximal contraction in VSM that mimics tone. Downstream signaling was investigated in order to determine what molecules are responsible for tone. Medial strips of swine carotid artery were stimulated with 100 nM Bay K8644 to induce a sustained level of force. Force and phosphorylation levels of myosin light chain (MLC), MAP kinase, MYPT1, CPI-17, and caldesmon were measured during Bay K8644 stimulation in the presence and absence of nifedipine, ML-7, U0126, bisindolylmaleimide (Bis), and H-1152. Nifedipine and ML-7 inhibited force and MLC phosphorylation in response to Bay K8644. Inhibition of Rho kinase (H-1152) but not PKC (Bis) inhibited Bay K8644 induced force. U0126 significantly increased Bay K8644-dependent force with no effect on MLC phosphorylation. Neither CPI-17 nor caldesmon phosphorylation were increased during the maintenance of sustained force. Our results suggest that force due to the influx of calcium through LTCCs is partially MLC phosphorylation-dependent but does not involve PKC or caldesmon. Interestingly, inhibition of MLC kinase (MLCK) and PKC significantly increased MAP kinase phosphorylation suggesting that MLCK and PKC may directly or indirectly inhibit MAP kinase activity during prolonged contractions induced by Bay K8544. PMID:28082901

  7. L-type Ca2+ currents at CA1 synapses, but not CA3 or dentate granule neuron synapses, are increased in 3xTgAD mice in an age-dependent manner

    PubMed Central

    Wang, Yue; Mattson, Mark P.

    2013-01-01

    Abnormal neuronal excitability and impaired synaptic plasticity might occur before the degeneration and death of neurons in Alzheimer’s disease (AD). To elucidate potential biophysical alterations underlying aberrant neuronal network activity in AD, we performed whole-cell patch clamp analyses of L-type (nifedipine-sensitive) Ca2+ currents (L-VGCC), 4–aminopyridine-sensitive K+ currents, and AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) and NMDA (N-methyl-D-aspartate) currents in CA1, CA3, and dentate granule neurons in hippocampal slices from young, middle-age, and old 3xTgAD mice and age-matched wild type mice. 3xTgAD mice develop progressive widespread accumulation of amyloid b-peptide, and selective hyperphosphorylated tau pathology in hippocampal CA1 neurons, which are associated with cognitive deficits, but independent of overt neuronal degeneration. An age-related elevation of L-type Ca2+ channel current density occurred in CA1 neurons in 3xTgAD mice, but not in wild type mice, with the magnitude being significantly greater in older 3xTgAD mice. The NMDA current was also significantly elevated in CA1 neurons of old 3xTgAD mice compared with in old wild type mice. There were no differences in the amplitude of K+ or AMPA currents in CA1 neurons of 3xTgAD mice compared with wild type mice at any age. There were no significant differences in Ca2+, K+, AMPA, or NMDA currents in CA3 and dentate neurons from 3xTgAD mice compared with wild type mice at any age. Our results reveal an age-related increase of L-VGCC density in CA1 neurons, but not in CA3 or dentate granule neurons, of 3xTgAD mice. These findings suggest a potential contribution of altered L-VGCC to the selective vulnerability of CA1 neurons to tau pathology in the 3xTgAD mice and to their degeneration in AD patients. PMID:23932880

  8. Chronic treatment with otilonium bromide induces changes in L-type Ca²⁺ channel, tachykinins, and nitric oxide synthase expression in rat colon muscle coat.

    PubMed

    Traini, C; Cipriani, G; Evangelista, S; Santicioli, P; Faussone-Pellegrini, M-S; Vannucchi, M-G

    2013-11-01

    Otilonium bromide (OB) is a quaternary ammonium derivative used for the treatment of intestinal hypermotility and is endowed with neurokinin2 receptor (NK2r) antagonist and Ca²⁺ channel blocker properties. Therefore, the possibility that OB might play a role in the neurokinin receptor/Substance-P/nitric oxide (NKr/SP/NO) circuit was investigated after chronic exposition to the drug. Rats were treated with OB 2-20 mg kg⁻¹ for 10 and 30 days. In the proximal colon, the expression and distribution of muscle NOsynthase 1 (NOS1), NK1r, NK2r, SP and Cav 1.2 subunit (for L-type Ca²⁺ channel) and the spontaneous activity and stimulated responses to NK1r and NK2r agonists were investigated. Immunohistochemistry showed a redistribution of NK1r and L-type Ca²⁺ channel in muscle cells with no change of NK2r at 30 days, a significant increase in muscle NOS1 expression at 10 days and a significant decrease in the SP content early in the ganglia and later in the intramuscular nerve fibers. Functional studies showed no change in spontaneous activity but a significant increase in maximal contraction induced by NK1r agonist. Chronic exposition to OB significantly affects the NKr/SP/NO circuit. The progressive decrease in SP-expression might be the consequence of the persistent presence of OB, the increase of NOS1 expression in muscle cells at 10 days in an attempt to guarantee an adequate NO production, and, at 30 days, the redistribution of the L-type Ca²⁺ channel and NK1r as a sign to compensate the drug channel block by re-cycling both of them. The physiological data suggest NK1r hypersensitivity. © 2013 John Wiley & Sons Ltd.

  9. The L-type Ca2+ Channel Blocker Nifedipine Inhibits Mycelial Growth, Sporulation, and Virulence of Phytophthora capsici

    PubMed Central

    Liu, Peiqing; Gong, Jie; Ding, Xueling; Jiang, Yue; Chen, Guoliang; Li, Benjin; Weng, Qiyong; Chen, Qinghe

    2016-01-01

    The oomycete vegetable pathogen Phytophthora capsici causes significant losses of important vegetable crops worldwide. Calcium and other plant nutrients have been used in disease management of oomycete pathogens. Calcium homeostasis and signaling is essential for numerous biological processes, and Ca2+ channel blockers prevent excessive Ca2+ influx into the fungal cell. However, it is not known whether voltage-gated Ca2+ channel blockers improve control over oomycete pathogens. In the present study, we compared the inhibitory effects of CaCl2 and the extracellular Ca2+ chelator EDTA on mycelial growth and found that calcium assimilation plays a key role in P. capsici mycelial growth. Next, we involved the voltage-gated Ca2+ channel blockers verapamil (VP) and nifedipine (NFD) to analyze the effect of Ca2+ channel blockers on mycelial growth and sporulation; the results suggested that NFD, but not VP, caused significant inhibition. Ion rescue in an NFD-induced inhibition assay suggested that NFD-induced inhibition is calcium-dependent. In addition, NFD increased P. capsici sensitivity to H2O2 in a calcium-dependent manner, and extracellular calcium rescued it. Furthermore, NFD inhibited the virulence and gene expression related to its pathogenicity. These results suggest that NFD inhibits mycelial growth, sporulation, and virulence of P. capsici. PMID:27540377

  10. Ultrastructural evidence for pre- and postsynaptic localization of Cav1.2 L-type Ca2+ channels in the rat hippocampus.

    PubMed

    Tippens, Alyssa L; Pare, Jean-Francois; Langwieser, Nicole; Moosmang, Sven; Milner, Teresa A; Smith, Yoland; Lee, Amy

    2008-02-01

    In the hippocampal formation, Ca(v)1.2 (L-type) voltage-gated Ca(2+) channels mediate Ca(2+) signals that can trigger long-term alterations in synaptic efficacy underlying learning and memory. Immunocytochemical studies indicate that Ca(v)1.2 channels are localized mainly in the soma and proximal dendrites of hippocampal pyramidal neurons, but electrophysiological data suggest a broader distribution of these channels. To define the subcellular substrates underlying Ca(v)1.2 Ca(2+) signals, we analyzed the localization of Ca(v)1.2 in the hippocampal formation by using antibodies against the pore-forming alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2). By light microscopy, alpha(1)1.2-like immunoreactivity (alpha(1)1.2-IR) was detected in pyramidal cell soma and dendritic fields of areas CA1-CA3 and in granule cell soma and fibers in the dentate gyrus. At the electron microscopic level, alpha(1)1.2-IR was localized in dendrites, but also in axons, axon terminals, and glial processes in all hippocampal subfields. Plasmalemmal immunogold particles representing alpha(1)1.2-IR were more significant for small- than large-caliber dendrites and were largely associated with extrasynaptic regions in dendritic spines and axon terminals. These findings provide the first detailed ultrastructural analysis of Ca(v)1.2 localization in the brain and support functionally diverse roles of these channels in the hippocampal formation.

  11. Participation of IP3R, RyR and L-type Ca2+ channel in the nuclear maturation of Rhinella arenarum oocytes.

    PubMed

    Toranzo, G Sánchez; Bühler, M C Gramajo; Bühler, M I

    2014-05-01

    During meiosis resumption, oocytes undergo a series of nuclear and cytosolic changes that prepare them for fertilization and that are referred to as oocyte maturation. These events are characterized by germinal vesicle breakdown (GVBD), chromatin condensation and spindle formation and, among cytosolic changes, organelle redistribution and maturation of Ca2+-release mechanisms. The progression of the meiotic cell cycle is regulated by M phase/maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Changes in the levels of intracellular free Ca2+ ion have also been implicated strongly in the triggering of the initiation of the M phase. Ca2+ signals can be generated by Ca2+ release from intracellular Ca2+ stores (endoplasmic reticulum; ER) or by Ca2+ influx from the extracellular space. In this sense, the L-type Ca2+ channel plays an important role in the incorporation of Ca2+ from the extracellular space. Two types of intracellular Ca2+ receptor/channels are known to mediate the intracellular Ca2+ release from the ER lumen. The most abundant, the inositol 1,4,5-trisphosphate receptor (IP3R), and the other Ca2+ channel, the ryanodine receptor (RyR), have also been reported to mediate Ca2+ release in several oocytes. In amphibians, MPF and MAPK play a central role during oocyte maturation, controlling several events. However, no definitive relationships have been identified between Ca2+ and MPF or MAPK. We investigated the participation of Ca2+ in the spontaneous and progesterone-induced nuclear maturation in Rhinella arenarum oocytes and the effect of different pharmacological agents known to produce modifications in the Ca2+ channels. We demonstrated that loading competent and incompetent oocytes with the intracellular calcium chelator BAPTA/AM produced suppression of spontaneous and progesterone-induced GVBD. In our results, the capacity of progesterone to trigger meiosis reinitiation in Rhinella in the presence of L-type Ca2+ channel blockers

  12. Leptin increases L-type Ca2+ channel expression and GnRH-stimulated LH release in LβT2 gonadotropes

    PubMed Central

    Avelino-Cruz, José E.; Flores, Amira; Cebada, Jorge; Mellon, Pamela L.; Felix, Ricardo; Monjaraz, Eduardo

    2009-01-01

    Leptin, a mediator of long-term regulation of energy balance, has been implicated in the release of adenohypophyseal gonadotropins by regulating gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. However, a direct effect of leptin on hormone release from gonadotropes remains virtually unexplored. In the current report, we assessed the long-term (48 h) actions of leptin on voltage-gated channel activity and luteinizing hormone (LH) production in mouse pituitary gonadotrope LβT2 cells. Electrophysiological recordings showed that leptin treatment significantly increased whole-cell patch clamp Ba2+ current through L-type Ca2+ channels. Quantitative RT-PCR analysis revealed increased levels of L-type (α1D) Ca2+ channel mRNA. Likewise, radioimmunoassays using specific antibodies provided evidence that leptin alone had no effect on LH release but did enhance GnRH-induced secretion of the hormone. Leptin had no apparent effects on LH gene transcription in absence of GnRH, as measured by transient transfection assays using a LH promoter-reporter gene and real time RT-PCR. These observations suggest that leptin might affect LH release by acting directly on the gonadotropes, favoring hormone production by enhancing responsiveness to GnRH as a result of increased Ca2+ channel expression. PMID:18834922

  13. KMUP-1 inhibits L-type Ca²⁺ channels involved the protein kinase C in rat basilar artery myocytes.

    PubMed

    Chen, Jun-Yih; Jiang, Min-Chi; Chu, Li-Wen; Hsieh, Su-Ling; Chen, Ing-Jun; Wu, Bin-Nan

    2011-12-01

    This study investigated whether KMUP-1, a xanthine-based derivative, inhibits L-type Ca(2+) currents (I(Ca,L)) in rat basilar artery smooth muscle cells (RBASMCs). We used whole cell patch-clamp recording to monitor Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels (LTCCs). Under voltage-clamp conditions, holding at -40 mV, KMUP-1 (1, 3, 10 μM) inhibited I(Ba) in a concentration-dependent manner and its IC(50) value was 2.27 ± 0.45 μM. A high concentration of KMUP-1 (10 μM) showed without modifying the I(Ba) current-voltage relationship. On the other hand, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM) increase I(Ba) was inhibited by KMUP-1. Pretreatment with the PKC inhibitor chelerythrine (5 μM) intensified KMUP-1-inhibited I(Ba). However, the Rho kinase inhibitor Y-27632 (30 μM) failed to affect the I(Ba) inhibition by KMUP-1. In light of these results, we suggest that KMUP-1 inhibition of LTCCs in concentration- and voltage-dependent manners in RBASMCs may be due, at least in part, to its modulation of the PKC pathway. Copyright © 2011 Elsevier Taiwan LLC. All rights reserved.

  14. Modulation of L-type Ca²⁺ channel activity by neuronal nitric oxide synthase and myofilament Ca²⁺ sensitivity in cardiac myocytes from hypertensive rat.

    PubMed

    Wang, Yue; Youm, Jae Boum; Jin, Chun Zi; Shin, Dong Hoon; Zhao, Zai Hao; Seo, Eun Yeong; Jang, Ji Hyun; Kim, Sung Joon; Jin, Zhe Hu; Zhang, Yin Hua

    2015-09-01

    Neuronal nitric oxide synthase (nNOS) is important in cardiac protection in diseased heart. Recently, we have reported that nNOS is associated with myofilament Ca(2+) desensitization in cardiac myocytes from hypertensive rats. So far, the effect of myofilament Ca(2+) desensitization or nNOS on L-type Ca(2+) channel activity (I(Ca)) in cardiac myocyte is unclear. Here, we examined nNOS regulation of I(Ca) in left ventricular (LV) myocytes from sham and angiotensin II (Ang II)-induced hypertensive rats. Our results showed that basal I(Ca) was not different between sham and hypertension (from -60 to +40 mV, 0.1 Hz). S-methyl-L-thiocitrulline (SMTC), a selective nNOS inhibitor, increased peak I(Ca) similarly in both groups. However, chelation of intracellular Ca(2+) [Ca(2+)]i with BAPTA increased I(Ca) and abolished SMTC-augmentation of I(Ca) only in hypertension. Myofilament Ca(2+) desensitization with butanedione monoxime (BDM), a myosin ATPase inhibitor, decreased I(Ca) in both groups but to a greater extent in hypertension. Intracellular BAPTA or nNOS inhibition reinstated I(Ca) in the presence of BDM to the basal level, suggesting Ca(2+)-dependent inactivation of I(Ca) by nNOS and greater vulnerability in hypertension. Increasing stimulation frequencies (2, 4 and 8 Hz) attenuated myofilament Ca(2+) sensitivity in sham and reduced peak ICa in both groups. Nevertheless, SMTC or BAPTA exerted no effect on I(Ca) at high frequencies in either group. These results suggest that nNOS attenuates I(Ca) via Ca(2+)-dependent mechanism and the vulnerability is greater in hypertension subject to myofilament Ca(2+) desensitization. nNOS or [Ca(2+)]i does not affect I(Ca) at high stimulation frequencies. The results were recapitulated with computer simulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons

    PubMed Central

    Qian, Hai; Patriarchi, Tommaso; Price, Jennifer L.; Matt, Lucas; Lee, Boram; Nieves-Cintrón, Madeline; Buonarati, Olivia R.; Chowdhury, Dhrubajyoti; Nanou, Evanthia; Nystoriak, Matthew A.; Catterall, William A.; Poomvanicha, Montatip; Hofmann, Franz; Navedo, Manuel F.; Hell, Johannes W.

    2017-01-01

    The L-type Ca2+ channel Cav1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser1928 in Cav1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser1928 is not required for regulation of cardiac Cav1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Cav1.2 and enhancement of its activity by the β2-adrenergic receptor (β2AR)–cAMP–PKA cascade. Thus, there is an unexpected dichotomy in the control of Cav1.2 by PKA in cardiomyocytes and hippocampal neurons. PMID:28119465

  16. Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons.

    PubMed

    Qian, Hai; Patriarchi, Tommaso; Price, Jennifer L; Matt, Lucas; Lee, Boram; Nieves-Cintrón, Madeline; Buonarati, Olivia R; Chowdhury, Dhrubajyoti; Nanou, Evanthia; Nystoriak, Matthew A; Catterall, William A; Poomvanicha, Montatip; Hofmann, Franz; Navedo, Manuel F; Hell, Johannes W

    2017-01-24

    The L-type Ca(2+) channel Cav1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser(1928) in Cav1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser(1928) is not required for regulation of cardiac Cav1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Cav1.2 and enhancement of its activity by the β2-adrenergic receptor (β2AR)-cAMP-PKA cascade. Thus, there is an unexpected dichotomy in the control of Cav1.2 by PKA in cardiomyocytes and hippocampal neurons. Copyright © 2017, American Association for the Advancement of Science.

  17. Roscovitine, a cyclin-dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L-type (Ca(V)1.2) calcium channels.

    PubMed

    Yarotskyy, V; Elmslie, K S

    2007-10-01

    L-type calcium channels (Ca((V))1.2) play an important role in cardiac contraction. Roscovitine, a cyclin-dependent kinase inhibitor and promising anticancer drug, has been shown to affect Ca((V))1.2 by inhibiting current amplitude and slowing activation. This research investigates the mechanism by which roscovitine inhibits Ca((V))1.2 channels. Ca((V))1.2 channels were transfected into HEK 293 cells, using the calcium phosphate precipitation method, and currents were measured using the whole-cell patch clamp technique. Roscovitine slows activation at all voltages, which precludes one previously proposed mechanism. In addition, roscovitine enhances voltage-dependent, but not calcium-dependent inactivation. This enhancement resulted from both an acceleration of inactivation and a slowing of the recovery from inactivation. Internally applied roscovitine failed to affect Ca((V))1.2 currents, which supports a kinase-independent mechanism and extracellular binding site. Unlike the dihydropyridines, closed state inactivation was not affected by roscovitine. Inactivation was enhanced in a dose-dependent manner with an IC(50)=29.5+/-12 microM, which is close to that for slow activation and inhibition. We conclude that roscovitine binds to an extracellular site on Ca((V))1.2 channels to inhibit current by both slowing activation and enhancing inactivation. Purine-based drugs could become a new option for treatment of diseases that benefit from L-channel inhibition such as cardiac arrhythmias and hypertension.

  18. Is comparative cardiotoxicity of S(-) and R(+) bupivacaine related to enantiomer-selective inhibition of L-type Ca(2+) channels?

    PubMed

    Zapata-Sudo, G; Trachez, M M; Sudo, R T; Nelson, T E

    2001-02-01

    Cardiac toxicity can occur after accidental intravascular injection of bupivacaine. Racemic bupivacaine can inhibit both cardiac Na(+) and Ca(2+) channels, but the contribution of these actions to cardiac depression is not totally understood. We tested whether the effect of R(+) bupivacaine on cardiac electrical activity in isolated hearts and on L-type Ca(2+) channels (I(Ca-L)) in isolated cardiac myocytes could be responsible for its increased cardiotoxicity compared with S(-) bupivacaine. Cardiac electrical activity of spontaneously beating isolated hearts was recorded before and after exposure to increasing concentrations of R(+) and S(-) bupivacaine. An increase of the PR interval (80%) and the QRS duration (370%) by 10microM R(+) bupivacaine (80% and 370%) was significantly higher than for S(-) bupivacaine (25% and 200%, respectively). R(+) but not S(-) bupivacaine produced severe arrhythmias at concentrations larger than 2.5microM. The intensity of I(Ca-L) inhibition did not differ between bupivacaine isomers. At 0 mV, I(Ca-L) was irreversibly reduced by 40.2% +/- 8.8% and 51.4% +/- 3.8% in the presence of 10microM R(+) and S(-) bupivacaine, respectively. The arrhythmogenic effect of R(+) bupivacaine could not be explained by stereoselectivity on the I(Ca-L) inhibition. Thus, other mechanisms could contribute to the cardiac electrical and contractile dysfunction induced by R(+) bupivacaine.

  19. Tetrodotoxin blockade on canine cardiac L-type Ca²⁺ channels depends on pH and redox potential.

    PubMed

    Hegyi, Bence; Komáromi, István; Kistamás, Kornél; Ruzsnavszky, Ferenc; Váczi, Krisztina; Horváth, Balázs; Magyar, János; Bányász, Tamás; Nánási, Péter P; Szentandrássy, Norbert

    2013-06-14

    Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na⁺ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca²⁺ current (I(Ca)) in canine cardiac cells. In the present study, the TTX-sensitivity of I(Ca) was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC₅₀ value obtained under physiological conditions) caused 60% ± 2% inhibition of I(Ca) in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 μM H₂O₂). Phosphorylation of the channel protein (induced by 3 μM forskolin) failed to modify the inhibiting potency of TTX; an IC₅₀ value of 50 ± 4 μM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels.

  20. CaLecRK-S.5, a pepper L-type lectin receptor kinase gene, confers broad-spectrum resistance by activating priming

    PubMed Central

    Woo, Joo Yong; Jeong, Kwang Ju; Kim, Young Jin; Paek, Kyung-Hee

    2016-01-01

    In Arabidopsis, several L-type lectin receptor kinases (LecRKs) have been identified as putative immune receptors. However, to date, there have been few analyses of LecRKs in crop plants. Virus-induced gene silencing of CaLecRK-S.5 verified the role of CaLecRK-S.5 in broad-spectrum resistance. Compared with control plants, CaLecRK-S.5-silenced plants showed reduced hypersensitive response, reactive oxygen species burst, secondary metabolite production, mitogen-activated protein kinase activation, and defense-related gene expression in response to Tobacco mosaic virus pathotype P0 (TMV-P0) infection. Suppression of CaLecRK-S.5 expression significantly enhanced the susceptibility to Pepper mild mottle virus pathotype P1,2,3, Xanthomonas campestris pv. vesicatoria, Phytophthora capsici, as well as TMV-P0. Additionally, β-aminobutyric acid treatment and a systemic acquired resistance assay revealed that CaLecRK-S.5 is involved in priming of plant immunity. Pre-treatment with β-aminobutyric acid before viral infection restored the reduced disease resistance phenotypes shown in CaLecRK-S.5-silenced plants. Systemic acquired resistance was also abolished in CaLecRK-S.5-silenced plants. Finally, RNA sequencing analysis indicated that CaLecRK-S.5 positively regulates plant immunity at the transcriptional level. Altogether, these results suggest that CaLecRK-S.5-mediated broad-spectrum resistance is associated with the regulation of priming. PMID:27647723

  1. CaLecRK-S.5, a pepper L-type lectin receptor kinase gene, confers broad-spectrum resistance by activating priming.

    PubMed

    Woo, Joo Yong; Jeong, Kwang Ju; Kim, Young Jin; Paek, Kyung-Hee

    2016-10-01

    In Arabidopsis, several L-type lectin receptor kinases (LecRKs) have been identified as putative immune receptors. However, to date, there have been few analyses of LecRKs in crop plants. Virus-induced gene silencing of CaLecRK-S.5 verified the role of CaLecRK-S.5 in broad-spectrum resistance. Compared with control plants, CaLecRK-S.5-silenced plants showed reduced hypersensitive response, reactive oxygen species burst, secondary metabolite production, mitogen-activated protein kinase activation, and defense-related gene expression in response to Tobacco mosaic virus pathotype P0 (TMV-P0) infection. Suppression of CaLecRK-S.5 expression significantly enhanced the susceptibility to Pepper mild mottle virus pathotype P1,2,3, Xanthomonas campestris pv. vesicatoria, Phytophthora capsici, as well as TMV-P0 Additionally, β-aminobutyric acid treatment and a systemic acquired resistance assay revealed that CaLecRK-S.5 is involved in priming of plant immunity. Pre-treatment with β-aminobutyric acid before viral infection restored the reduced disease resistance phenotypes shown in CaLecRK-S.5-silenced plants. Systemic acquired resistance was also abolished in CaLecRK-S.5-silenced plants. Finally, RNA sequencing analysis indicated that CaLecRK-S.5 positively regulates plant immunity at the transcriptional level. Altogether, these results suggest that CaLecRK-S.5-mediated broad-spectrum resistance is associated with the regulation of priming.

  2. Effects of L-type Ca2+ channel antagonists on in vitro excystment of Paragonimus ohirai metacercariae induced by sodium cholate.

    PubMed

    Ikeda, Teruaki

    2006-09-01

    The inhibitory effects of L-type Ca2+ channel antagonists on Na cholate-induced in vitro excystment (CIIE) of Paragonimus ohirai metacercariae were studied. At concentrations of 10 microM, nicardipine and nimodipine inhibited CIIE completely and by approximately 92%, respectively. Nitrendipine and (+/-)-verapamil inhibited CIIE by about one half and one third, respectively. Nifedipine and diltiazem did not inhibit CIIE significantly. At higher concentrations, nitrendipine at 20 microM completely inhibited CIIE, and (+/-)-verapamil at 40 microM inhibited CIIE by 93%. Nifedipine and diltiazem inhibited CIIE only slightly and little, respectively, even at 40 microM. Complete inhibition by nicardipine at 10 microM required preincubation of metacercariae with the antagonist for 15 min. The inhibitory effects of nicardipine and nimodipine were reversible, and most of the nimodipine-treated metacercariae could excyst within 1 h after being washed, but the nicardipine-treated ones started to excyst 1 h after washing. Nicardipine suppressed the active movement of encysted juveniles evoked by Na cholate, whereas nimodipine did not suppress this significantly. These results suggested that L-type Ca2+ channels appeared to be involved in CIIE of P. ohirai metacercariae and that the inhibitory effect of the channels was due primarily to factors other than the inhibition of muscular activity, probably involving the secretion and release of enzymes lytic against the metacercarial cyst wall.

  3. Involvement of L-type Ca²⁺ channel and toll-like receptor-4 in nickel-induced interleukin-8 gene expression.

    PubMed

    Lin, Chia-Hsien; Chung, Chih-Ang; Wong, Jhen-Hong; Chen, Ben-Kuen; Chiu, Siou-Jin; Klahan, Sukhontip; Lee, Yi-Chao; Chang, Wei-Chiao

    2016-01-01

    The metal nickel (Ni(2+)) is found everywhere in our daily lives, including coins, costume jewelry, and even nuts and chocolates. Nickel poisoning can cause inflammatory reactions, respiratory diseases, and allergic contact dermatitis. To clarify the mechanism by which nickel induces mediators of inflammation, we used the human acute monocytic leukemia THP-1 cell line as a model. Interleukin (IL)-8 promoter activity as well as gene expression were tested by luciferase assay and real-time polymerase chain reaction. The underlying mechanisms of nickel-induced IL-8 were investigated. We found that nickel induced IL-8 gene expression via the L-type Ca(2+) channel, Toll-like receptor-4 (TRL-4) and nuclear factor NF-κB signal transduction pathways. Nickel activated NF-κB expression through extracellular signal-regulated kinase 1/2 phosphorylation and then increased IL-8 expression. Thus, the L-type Ca(2+) channel and TRL-4 play important roles in nickel-induced inflammatory gene expressions.

  4. Forgetting of long-term memory requires activation of NMDA receptors, L-type voltage-dependent Ca2+ channels, and calcineurin

    PubMed Central

    Sachser, Ricardo Marcelo; Santana, Fabiana; Crestani, Ana Paula; Lunardi, Paula; Pedraza, Lizeth Katherine; Quillfeldt, Jorge Alberto; Hardt, Oliver; de Oliveira Alvares, Lucas

    2016-01-01

    In the past decades, the cellular and molecular mechanisms underlying memory consolidation, reconsolidation, and extinction have been well characterized. However, the neurobiological underpinnings of forgetting processes remain to be elucidated. Here we used behavioral, pharmacological and electrophysiological approaches to explore mechanisms controlling forgetting. We found that post-acquisition chronic inhibition of the N-methyl-D-aspartate receptor (NMDAR), L-type voltage-dependent Ca2+ channel (LVDCC), and protein phosphatase calcineurin (CaN), maintains long-term object location memory that otherwise would have been forgotten. We further show that NMDAR activation is necessary to induce forgetting of object recognition memory. Studying the role of NMDAR activation in the decay of the early phase of long-term potentiation (E-LTP) in the hippocampus, we found that ifenprodil infused 30 min after LTP induction in vivo blocks the decay of CA1-evoked postsynaptic plasticity, suggesting that GluN2B-containing NMDARs activation are critical to promote LTP decay. Taken together, these findings indicate that a well-regulated forgetting process, initiated by Ca2+ influx through LVDCCs and GluN2B-NMDARs followed by CaN activation, controls the maintenance of hippocampal LTP and long-term memories over time. PMID:26947131

  5. Forgetting of long-term memory requires activation of NMDA receptors, L-type voltage-dependent Ca2+ channels, and calcineurin.

    PubMed

    Sachser, Ricardo Marcelo; Santana, Fabiana; Crestani, Ana Paula; Lunardi, Paula; Pedraza, Lizeth Katherine; Quillfeldt, Jorge Alberto; Hardt, Oliver; Alvares, Lucas de Oliveira

    2016-03-07

    In the past decades, the cellular and molecular mechanisms underlying memory consolidation, reconsolidation, and extinction have been well characterized. However, the neurobiological underpinnings of forgetting processes remain to be elucidated. Here we used behavioral, pharmacological and electrophysiological approaches to explore mechanisms controlling forgetting. We found that post-acquisition chronic inhibition of the N-methyl-D-aspartate receptor (NMDAR), L-type voltage-dependent Ca(2+) channel (LVDCC), and protein phosphatase calcineurin (CaN), maintains long-term object location memory that otherwise would have been forgotten. We further show that NMDAR activation is necessary to induce forgetting of object recognition memory. Studying the role of NMDAR activation in the decay of the early phase of long-term potentiation (E-LTP) in the hippocampus, we found that ifenprodil infused 30 min after LTP induction in vivo blocks the decay of CA1-evoked postsynaptic plasticity, suggesting that GluN2B-containing NMDARs activation are critical to promote LTP decay. Taken together, these findings indicate that a well-regulated forgetting process, initiated by Ca(2+) influx through LVDCCs and GluN2B-NMDARs followed by CaN activation, controls the maintenance of hippocampal LTP and long-term memories over time.

  6. Putative resolution of the EEEE selectivity paradox in L-type Ca2+ and bacterial Na+ biological ion channels

    NASA Astrophysics Data System (ADS)

    Kaufman, I. Kh; Luchinsky, D. G.; Gibby, W. A. T.; McClintock, P. V. E.; Eisenberg, R. S.

    2016-05-01

    The highly selective permeation of ions through biological ion channels can be described and explained in terms of fluctuational dynamics under the influence of powerful electrostatic forces. Hence valence selectivity, e.g. between Ca2+ and Na+ in calcium and sodium channels, can be described in terms of ionic Coulomb blockade, which gives rise to distinct conduction bands and stop-bands as the fixed negative charge Q f at the selectivity filter of the channel is varied. This picture accounts successfully for a wide range of conduction phenomena in a diversity of ion channels. A disturbing anomaly, however, is that what appears to be the same electrostatic charge and structure (the so-called EEEE motif) seems to select Na+ conduction in bacterial channels but Ca2+ conduction in mammalian channels. As a possible resolution of this paradox it is hypothesised that an additional charged protein residue on the permeation path of the mammalian channel increases |{{Q}f}| by e, thereby altering the selectivity from Na+ to Ca2+. Experiments are proposed that will enable the hypothesis to be tested.

  7. Tetanic depression is overcome by tonic adenosine A2A receptor facilitation of L-type Ca2+ influx into rat motor nerve terminals

    PubMed Central

    Oliveira, Laura; Timóteo, M Alexandrina; Correia-de-Sá, Paulo

    2004-01-01

    Motor nerve terminals possess multiple voltage-sensitive calcium channels operating acetylcholine (ACh) release. In this study, we investigated whether facilitation of neuromuscular transmission by adenosine generated during neuronal firing was operated by Ca2+ influx via ‘prevalent’ P-type or via the recruitment of ‘silent’ L-type channels. The release of [3H]ACh from rat phrenic nerve endings decreased upon increasing the stimulation frequency of the trains (750 pulses) from 5 Hz (83 ± 4 × 103 disintegrations per minute per gram (d.p.m. g−1); n = 11) to 50 Hz (30 ± 3 × 103 d.p.m. g−1; n = 5). The P-type Ca2+ channel blocker, ω-agatoxin IVA (100 nm) reduced (by 40 ± 10%; n = 6) the release of [3H]ACh evoked by 50-Hz trains, while nifedipine (1 μm, an L-type blocker) was inactive. Tetanic depression was overcome (88 ± 6 × 103 d.p.m. g−1; n = 12) by stimulating the phrenic nerve with 50-Hz bursts (five bursts of 150 pulses, 20 s interburst interval). In these conditions, ω-agatoxin IVA (100 nm) failed to affect transmitter release, but nifedipine (1 μm) decreased [3H]ACh release by 21 ± 7% (n = 4). Inactivation of endogenous adenosine with adenosine deaminase (ADA, 0.5 U ml−1) reduced (by 54 ± 8%, n = 5) the release of [3H]ACh evoked with 50-Hz bursts. This effect was opposite to the excitatory actions of adenosine (0.5 mm), S-(p-nitrobenzyl)-6-thioinosine (5 μm, an adenosine uptake blocker) and CGS 21680C (3 nm, a selective A2A receptor agonist); as the A1 receptor agonist R-N6-phenylisopropyl adenosine (R-PIA, 300 nm) failed to affect the release of [3H]ACh, the results indicate that adenosine generated during 50-Hz bursts exerts an A2A-receptor-mediated tonus. The effects of ADA (0.5 U ml−1) and CGS 21680C (3 nm) were prevented by nifedipine (1 μm). Blocking tonic A2A receptor activation, with ADA (0.5 U ml−1) or 3,7-dimethyl-1-propargyl xanthine (10 μm, an A2A antagonist), recovered ω-agatoxin IVA (100 nm) inhibition and

  8. An aqueous extract of the marine sponge Ectyoplasia ferox stimulates L-type Ca2+-current by direct interaction with the Cav1.2 subunit.

    PubMed

    Christ, Torsten; Wüst, Melinda; Matthes, Jan; Jänchen, Michael; Jürgens, Susanne; Herzig, Stefan; Wettwer, Erich; Dobrev, Dobromir; Matschke, Klaus; Mebs, Dietrich; Ravens, Ursula

    2004-12-01

    Marine organisms have attracted much attention as a source of pharmacological tools or potential drugs. We have produced and screened a library of sponge extracts in search of biologically active compounds that may contain useful pharmaceutical lead structures. Sponges were collected from various locations and their aqueous extracts were freeze dried. Murine right and left atria were used to screen 75 extracts for putative cardiac effects. Among seven extracts with a positive inotropic and chronotropic effect the extract C47 from Ectyoplasia ferox proved to be the most active and was chosen for further analysis. C47 also produced a beta-adrenoceptor-independent, propranolol-resistant positive inotropic effect in human atrial trabeculae. To elucidate one possible mode of action the effects of C47 on L-type Ca(2+) current (I(Ca,L)) were measured with a standard patch-clamp technique. In isolated human atrial myocytes exposure to C47 increased peak amplitude of I(Ca,L) in a concentration-dependent manner. The threshold concentration was 15 microg/ml. In addition, voltage dependency of activation and steady-state inactivation were shifted to more negative potentials. C47 slowed the initial phase of time-dependent current inactivation and the recovery from inactivation. In cell-attached patches of HEK 293 cells expressing human Ca(v)1.2 addition of C47 to the bath solution did not affect gating properties, whereas inclusion of the extract into the pipette solution strongly increased single-channel activity, suggesting a direct effect on the pore-forming channel subunit. Despite its robust effect on I(Ca,L) C47 enhanced cardiac force of contraction by only a fraction of the maximum increase caused by high extracellular concentrations of Ca(2+) and failed to increase vascular tone. These findings suggest that the effect of C47 is restricted to the Ca(2+) channel.

  9. Deletion of the L-type Calcium Channel CaV1.3 but not CaV1.2 Results in a Diminished sAHP in Mouse CA1 Pyramidal Neurons

    PubMed Central

    Gamelli, Amy E.; McKinney, Brandon C.; White, Jessica A.; Murphy, Geoffrey G.

    2009-01-01

    Trains of action potentials in CA1 pyramidal neurons are followed by a prolonged calcium-dependent post-burst afterhyperpolarization (AHP) that serves to limit further firing to a sustained depolarizing input. A reduction in the AHP accompanies acquisition of several types of learning and increases in the AHP are correlated with age-related cognitive impairment. The AHP develops primarily as the result of activation of outward calcium-activated potassium currents; however the precise source of calcium for activation of the AHP remains unclear. There is substantial experimental evidence suggesting that calcium influx via voltage-gated L-type calcium channels (L-VGCCs) contributes to the generation of the AHP. Two L-VGCC subtypes are predominately expressed in the hippocampus, CaV1.2 and CaV1.3, however it is not known which L-VGCC subtype is involved in generation of the AHP. This ambiguity is due in large part to the fact that at present there are no subunit-specific agonists or antagonists. Therefore, using mice in which the gene encoding CaV1.2 or CaV1.3 was deleted, we sought to determine the impact of alterations in levels of these two L-VCGG subtypes on neuronal excitability. No differences in any AHP measure were seen between neurons from CaV1.2 knockout mice and controls. However, the total area of the AHP was significantly smaller in neurons from CaV1.3 knockout mice as compared to neurons from wildtype controls. A significant reduction in the amplitude of the AHP was also seen at the 1 sec time point in neurons from CaV1.3 knockout mice as compared to those from controls. Reductions in both the area and 1 sec amplitude suggest the involvement of calcium influx via CaV1.3 in the slow AHP (sAHP). Thus, the results of our study demonstrate that deletion of CaV1.3, but not CaV1.2, significantly impacts the generation of the sAHP. PMID:20014384

  10. Low-dose combination of Rho kinase and L-type Ca(2+) channel antagonists for selective inhibition of depolarization-induced sustained arterial contraction.

    PubMed

    Porras-González, Cristina; González-Rodríguez, Patricia; Calderón-Sánchez, Eva; López-Barneo, José; Ureña, Juan

    2014-06-05

    L-type Ca(2+) channels (LTCCs) are involved in the maintenance of tonic arterial contractions and regulate the RhoA/Rho-associated kinase (ROCK) sensitization cascade. We have tested effects of individual and combined low concentrations of LTCCs and ROCK inhibitors to produce arterial relaxation without the adverse side effects of LTCCs antagonists. We have also studied whether this pharmacological strategy alters Ca(2+)-dependent electrical properties of isolated arterial and cardiac myocytes as well as cardiac contractility. Rat basilar, human carotid and coronary arterial rings were mounted on a small-vessel myograph to measure isometric tension and cardiac contractility was measured in Langendorff-perfused rat heart. Simultaneous cytosolic Ca(2+) concentration and arterial diameter were measured in intact pressurized arteries loaded with Fura-2. Patch-clamp techniques were used to measure electrical properties in isolated cardiac and arterial myocytes. Low concentrations of LTCCs and ROCK inhibitors reduced the tonic component of moderate depolarization-evoked contraction, leaving the phasic component practically unaltered. This selective vasorelaxant effect was more marked when the LTCCs and ROCK inhibitors were applied together. In the concentration range used (nM), Ca(2+) currents in arterial myocytes, cardiac action potentials and heart contractility were unaffected by this pharmacological approach. In conclusion, low doses of LTCCs and ROCK inhibitors could be used to selectively relax precontracted arteries in pathologic conditions such as hypertension, and cerebral or coronary spasms with minor side effects on physiological contractile properties of vascular and cardiac myocytes.

  11. Densin-180 controls the trafficking and signaling of L-type voltage-gated Cav1.2 Ca(2+) channels at excitatory synapses.

    PubMed

    Wang, Shiyi; Stanika, Ruslan I; Wang, Xiaohan; Hagen, Jussara; Kennedy, Mary B; Obermair, Gerald J; Colbran, Roger J; Lee, Amy

    2017-03-31

    Voltage-gated Cav1.2 and Cav1.3 (L-type) Ca(2+) channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca(2+)-dependent facilitation of voltage-gated Cav1.3 Ca(2+) channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors --phenotypes that more closely match those in mice lacking Cav1.2 than Cav1.3. Thus, we investigated the functional impact of densin on Cav1.2. We report that densin is an essential regulator of Cav1.2 in neurons, but has distinct modulatory effects compared to its regulation of Cav1.3. Densin binds to the N-terminal domain of Cav1.2 but not Cav1.3, and increases Cav1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Cav1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Cav1.2 channels, overexpression of densin increases the clustering of Cav1.2 in dendrites of hippocampal neurons in culture. Compared to wild-type mice, the cell-surface levels of Cav1.2 in the brain as well as Cav1.2 current density and signaling to the nucleus are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Cav1 channels and ensures efficient Cav1.2 Ca(2+) signaling at excitatory synapses.SIGNIFICANCE STATEMENTThe number and localization of voltage-gated Cav Ca(2+) channels are crucial determinants of neuronal excitability and synaptic transmission. We report that a protein that is highly enriched at excitatory synapses in the brain, densin-180, enhances the cell-surface trafficking and postsynaptic localization of Cav1.2 L-type Ca(2+) channels in neurons. This interaction promotes coupling of Cav1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that

  12. The reduction of EPSC amplitude in CA1 pyramidal neurons by the peroxynitrite donor SIN-1 requires Ca2+ influx via postsynaptic non-L-type voltage gated calcium channels.

    PubMed

    Zhaowei, Liu; Yongling, Xie; Jiajia, Yang; Zhuo, Yang

    2014-02-01

    The peroxynitrite free radical (ONOO(-)) modulation of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) was investigated in rat CA1 pyramidal neurons using the whole-cell patch clamp technique. SIN-1(3-morpholino-sydnonimine), which can lead the simultaneous generation of superoxide anion and nitric oxide, and then form the highly reactive species ONOO(-), induced dose-dependent inhibition in amplitudes of both mEPSCs and sEPSCs. The SIN-1 action on mEPSC amplitude was completely blocked by U0126, a selective MEK inhibitor, suggesting that MEK contributed to the action of ONOO(-) on mEPSCs. The effect of SIN-1 was completely occluded either in the presence of the calcium chelator EGTA or the non-selective calcium channel antagonist Cd(2+). Furthermore, the application of nifedipine (20 μM), the L-type calcium channel blocker, had no effect on the ONOO(-)-induced decrease in mEPSC amplitude, excluding a role for L-type voltage-gated Ca(2+) channels in this process. SIN-1 inhibited the frequency of sEPSCs but had no effect on mEPSC frequency, which suggested a presynaptic action potential-dependent the action of ONOO(-) at CA1 pyramidal neuron synapses. The best-known glutamatergic input to CA1 pyramidal neurons is via Schaffer collaterals from CA3 area. However, no changes were observed in slices treated with SIN-1 on the spontaneous firing rates of CA3 pyramidal neurons. These findings suggested that SIN-1 inhibited glutamatergic synaptic transmission of CA1 pyramidal neurons by a postsynaptic non-L-type voltage gated calcium channel-dependent mechanism.

  13. Significance of KATP channels, L-type Ca2+ channels and CYP450-4A enzymes in oxygen sensing in mouse cremaster muscle arterioles In vivo

    PubMed Central

    2013-01-01

    Background ATP-sensitive K+ channels (KATP channels), NO, prostaglandins, 20-HETE and L-type Ca2+ channels have all been suggested to be involved in oxygen sensing in skeletal muscle arterioles, but the role of the individual mechanisms remain controversial. We aimed to establish the importance of these mechanisms for oxygen sensing in arterioles in an in vivo model of metabolically active skeletal muscle. For this purpose we utilized the exteriorized cremaster muscle of anesthetized mice, in which the cremaster muscle was exposed to controlled perturbation of tissue PO2. Results Change from “high” oxygen tension (PO2 = 153.4 ± 3.4 mmHg) to “low” oxygen tension (PO2 = 13.8 ± 1.3 mmHg) dilated cremaster muscle arterioles from 11.0 ± 0.4 μm to 32.9 ± 0.9 μm (n = 28, P < 0.05). Glibenclamide (KATP channel blocker) caused maximal vasoconstriction, and abolished the dilation to low oxygen, whereas the KATP channel opener cromakalim caused maximal dilation and prevented the constriction to high oxygen. When adding cromakalim on top of glibenclamide or vice versa, the reactivity to oxygen was gradually restored. Inhibition of L-type Ca2+ channels using 3 μM nifedipine did not fully block basal tone in the arterioles, but rendered them unresponsive to changes in PO2. Inhibition of the CYP450-4A enzyme using DDMS blocked vasoconstriction to an increase in PO2, but had no effect on dilation to low PO2. Conclusions We conclude that: 1) L-type Ca2+ channels are central to oxygen sensing, 2) KATP channels are permissive for the arteriolar response to oxygen, but are not directly involved in the oxygen sensing mechanism and 3) CYP450-4A mediated 20-HETE production is involved in vasoconstriction to high PO2. PMID:23663730

  14. Roscovitine, a cyclin-dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L-type (Ca(V)1.2) calcium channels

    PubMed Central

    Yarotskyy, V; Elmslie, K S

    2007-01-01

    Background and purpose: L-type calcium channels (Ca (V)1.2) play an important role in cardiac contraction. Roscovitine, a cyclin-dependent kinase inhibitor and promising anticancer drug, has been shown to affect Ca (V)1.2 by inhibiting current amplitude and slowing activation. This research investigates the mechanism by which roscovitine inhibits Ca (V)1.2 channels. Experimental approach: Ca (V)1.2 channels were transfected into HEK 293 cells, using the calcium phosphate precipitation method, and currents were measured using the whole-cell patch clamp technique. Key results: Roscovitine slows activation at all voltages, which precludes one previously proposed mechanism. In addition, roscovitine enhances voltage-dependent, but not calcium-dependent inactivation. This enhancement resulted from both an acceleration of inactivation and a slowing of the recovery from inactivation. Internally applied roscovitine failed to affect Ca (V)1.2 currents, which supports a kinase-independent mechanism and extracellular binding site. Unlike the dihydropyridines, closed state inactivation was not affected by roscovitine. Inactivation was enhanced in a dose-dependent manner with an IC50=29.5±12 μM, which is close to that for slow activation and inhibition. Conclusions and implications: We conclude that roscovitine binds to an extracellular site on Ca (V)1.2 channels to inhibit current by both slowing activation and enhancing inactivation. Purine-based drugs could become a new option for treatment of diseases that benefit from L-channel inhibition such as cardiac arrhythmias and hypertension. PMID:17700718

  15. T-type and L-type Ca2+ conductances define and encode the bimodal firing pattern of vestibulocerebellar unipolar brush cells.

    PubMed

    Diana, Marco A; Otsu, Yo; Maton, Gilliane; Collin, Thibault; Chat, Mireille; Dieudonné, Stéphane

    2007-04-04

    Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons that receive direct input from vestibular afferents in the form of a unique excitatory synapse on their dendritic brush. UBCs constitute independent relay lines for vestibular signals, and their inherent properties most likely determine how vestibular activity is encoded by the cerebellar cortex. We now demonstrate that UBCs are bimodal cells; they can either fire high-frequency bursts of action potentials when stimulated from hyperpolarized potentials or discharge tonically during sustained depolarizations. The two functional states can be triggered by physiological-like activity of the excitatory input and are encoded by distinct Ca2+-signaling systems. By combining complementary strategies, consisting of molecular and electrophysiological analysis and of ultrafast acousto-optical deflector-based two-photon imaging, we unraveled the identity and the subcellular localization of the Ca2+ conductances activating in each mode. Fast inactivating T-type Ca2+ channels produce low-threshold spikes, which trigger the high-frequency bursts and generate powerful Ca2+ transients in the brush and, to a much lesser extent, in the soma. The tonic firing mode is encoded by a signalization system principally composed of L-type channels. Ca2+ influx during tonic firing produces a linear representation of the spike rate of the cell in the form of a widespread and sustained Ca2+ concentration increase and regulates cellular excitability via BK potassium channels. The bimodal firing pattern of UBCs may underlie different coding strategies of the vestibular input by the cerebellum, thus likely increasing the computational power of this structure.

  16. Calcium dynamics during NMDA-induced membrane potential oscillations in lamprey spinal neurons--contribution of L-type calcium channels (CaV1.3).

    PubMed

    Wang, Di; Grillner, Sten; Wallén, Peter

    2013-05-15

      NMDA receptor-dependent, intrinsic membrane potential oscillations are an important element in the operation of the lamprey locomotor network. They involve a cyclic influx of calcium, leading to an activation of calcium-activated potassium (KCa) channels that in turn contributes to the termination of the depolarized plateau and membrane repolarization. In this study, we have investigated the calcium dynamics in different regions of lamprey spinal neurons during membrane potential oscillations, using confocal calcium imaging in combination with intracellular recordings. Calcium fluctuations were observed in both soma and dendrites, timed to the oscillations. The calcium level increased sharply at the onset of membrane depolarization, to reach its maximum by the end of the plateau. The calcium peak in distal dendrites typically occurred earlier than in the soma during the oscillatory cycle. The L-type calcium channel blocker nimodipine increased the duration of the depolarized plateau phase in most cells tested, whereas the agonist Bay K 8644 decreased plateau duration. Bay K 8644 increased the amplitude of calcium fluctuations, particularly in distal dendrites, whereas nimodipine caused a decrease, suggesting that L-type low-voltage-activated calcium channels are mainly localized in these regions. Our results thus indicate that dendritic CaV1.3-like calcium channels are activated during NMDA-mediated membrane potential oscillations. This calcium influx activates KCa channels involved in plateau termination.

  17. Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels

    PubMed Central

    Tosetti, Patrizia; Parente, Valeria; Taglietti, Vanni; Dunlap, Kathleen; Toselli, Mauro

    2003-01-01

    In neuronal cells, the influx of Ca2+ ions through voltage-dependent L-type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G-protein-coupled receptors. Intracellular molecules that modify G-protein activity – such as regulator of G-protein-signalling (RGS) proteins – are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)-mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)-sensitive (Gi/o-coupled) and the PTX-insensitive (presumably Gq/11-coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration. A low concentration of recombinant chick RGS2L (10 nm) preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas a 100-fold higher concentration attenuates both PTX-sensitive- and PTX-insensitive-mediated components equally. Our data suggest that factors promoting RGS2L gene induction may regulate Ca2+ influx through L channels by recruiting low-affinity interactions with Gi/o that are absent at basal RGS2L levels. PMID:12651916

  18. Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl- cotransport, intracellular Cl- and L-type Ca2+ channels.

    PubMed

    Anfinogenova, Yana J; Baskakov, Mikhail B; Kovalev, Igor V; Kilin, Alexander A; Dulin, Nickolai O; Orlov, Sergei N

    2004-10-01

    This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca2+ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca2+ o. Thirty minutes preincubation with bumetanide, a potent Na+, K+, CI- cotransport (NKCC) inhibitor, decreased Cl(-)i content, nifedipine-sensitive 45Ca uptake and contractions triggered by modest depolarization ([K+]o = 36 mM). Elevation of [K+]o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl(-)i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.

  19. Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels.

    PubMed

    Tosetti, Patrizia; Parente, Valeria; Taglietti, Vanni; Dunlap, Kathleen; Toselli, Mauro

    2003-05-15

    In neuronal cells, the influx of Ca2+ ions through voltage-dependent L-type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G-protein-coupled receptors. Intracellular molecules that modify G-protein activity - such as regulator of G-protein-signalling (RGS) proteins - are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)-mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)-sensitive (Gi/o-coupled) and the PTX-insensitive (presumably Gq/11-coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration. A low concentration of recombinant chick RGS2L (10 nM) preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas a 100-fold higher concentration attenuates both PTX-sensitive- and PTX-insensitive-mediated components equally. Our data suggest that factors promoting RGS2L gene induction may regulate Ca2+ influx through L channels by recruiting low-affinity interactions with Gi/o that are absent at basal RGS2L levels.

  20. 20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca2+ channel via a PKC-dependent mechanism in cardiomyocytes

    PubMed Central

    Han, Yong; Bao, Yuyan; Li, Wei; Li, Xingting; Shen, Xin; Wang, Xu; Yao, Fanrong; O'Rourke, Stephen T.; Sun, Chengwen

    2010-01-01

    The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type Ca2+ currents (ICa,L) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10–100 nM) resulted in a concentration-dependent increase in ICa,L, and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 μM), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on ICa,L. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 μM), attenuated the 20-HETE-induced increases in ICa,L and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca2+ channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases. PMID:20675568

  1. Downregulation of L-type Ca2+ channel in rat mesenteric arteries leads to loss of smooth muscle contractile phenotype and inward hypertrophic remodeling.

    PubMed

    Kudryavtseva, Olga; Herum, Kate Møller; Dam, Vibeke Secher; Straarup, Marthe Simonsen; Kamaev, Dmitry; Briggs Boedtkjer, Donna M; Matchkov, Vladimir V; Aalkjær, Christian

    2014-05-01

    L-type Ca(2+) channels (LTCCs) are important for vascular smooth muscle cell (VSMC) contraction, as well as VSMC differentiation, as indicated by loss of LTCCs during VSMC dedifferentiation. However, it is not clear whether loss of LTCCs is a primary event underlying phenotypic modulation or whether loss of LTCCs has significance for vascular structure. We used small interference RNA (siRNA) transfection in vivo to investigate the role of LTCCs in VSMC phenotypic expression and structure of rat mesenteric arteries. siRNA reduced LTCC mRNA and protein expression in rat mesenteric arteries 3 days after siRNA transfection to 12.7 ± 0.7% and 47.3 ± 13%, respectively: this was associated with an increased resting intracellular Ca(2+) concentration ([Ca(2+)]i). Despite the high [Ca(2+)]i, the contractility was reduced (tension development to norepinephrine was 3.5 ± 0.2 N/m and 0.8 ± 0.2 N/m for sham-transfected and downregulated arteries respectively; P < 0.05). Expression of contractile phenotype marker genes was reduced in arteries downregulated for LTCCs. Phenotypic changes were associated with a 45% increase in number of VSMCs and a consequent increase of media thickness and media area. Ten days after siRNA transfection arterial structure was again normalized. The contractile responses of LTCC-siRNA transfected arteries were elevated in comparison with matched controls 10 days after transfection. The study provides strong evidence for causal relationships between LTCC expression and VSMC contractile phenotype, as well as novel data addressing the complex relationship between VSMC contractility, phenotype, and vascular structure. These findings are relevant for understanding diseases, associated with phenotype changes of VSMC and vascular remodeling, such as atherosclerosis and hypertension.

  2. L-type Ca(2+) channels in mood, cognition and addiction: integrating human and rodent studies with a focus on behavioural endophenotypes.

    PubMed

    Kabir, Z D; Lee, A S; Rajadhyaksha, A M

    2016-10-15

    Brain Cav 1.2 and Cav 1.3 L-type Ca(2+) channels play key physiological roles in various neuronal processes that contribute to brain function. Genetic studies have recently identified CACNA1C as a candidate risk gene for bipolar disorder (BD), schizophrenia (SCZ), major depressive disorder (MDD) and autism spectrum disorder (ASD), and CACNA1D for BD and ASD, suggesting a contribution of Cav 1.2 and Cav 1.3 Ca(2+) signalling to the pathophysiology of neuropsychiatric disorders. Once considered sole clinical entities, it is now clear that BD, SCZ, MDD and ASD share common phenotypic features, most likely due to overlapping neurocircuitry and common molecular mechanisms. A major future challenge lies in translating the human genetic findings to pathological mechanisms that are translatable back to the patient. One approach for tackling such a daunting scientific endeavour for complex behaviour-based neuropsychiatric disorders is to examine intermediate biological phenotypes in the context of endophenotypes within distinct behavioural domains. This will better allow us to integrate findings from genes to behaviour across species, and improve the chances of translating preclinical findings to clinical practice. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  3. Role of L-type Ca(2+) channels, sarcoplasmic reticulum and Rho kinase in rat basilar artery contractile properties in a new model of subarachnoid hemorrhage.

    PubMed

    Egea-Guerrero, Juan José; Murillo-Cabezas, Francisco; Muñoz-Sánchez, María Ángeles; Vilches-Arenas, Angel; Porras-González, Cristina; Castellano, Antonio; Ureña, Juan; González-Montelongo, María del Carmen

    2015-09-01

    We have previously described that L-type Ca(2+) channels' (LTCCs) activation and metabotropic Ca(2+) release from the sarcoplasmic reticulum (SR) regulate RhoA/Rho kinase (ROCK) activity and sustained arterial contraction. We have investigated whether this signaling pathway can be altered in a new experimental model of subarachnoid hemorrhage (SAH). For this purpose, arterial reactivity was evaluated on days 1 to 5 after surgery. A significant increase of basal tone, measured 4 and 60min after normalization, was observed on day 5 after SAH and at 60min on days 2 and 3 after SAH. This phenomenon was suppressed with LTCCs and ROCK inhibitors. We have also studied arterial rings vasoreactivity in response to high K(+) solutions. Interestingly, there were no significant differences in the phasic component of the high K(+)-induced contraction between sham and SAH groups, whereas a significant increase in the sustained contraction was observed on day 5 after SAH. This latter component was sensitive to fasudil, and selectively reduced by low nifedipine concentration, and phospholipase C and SR-ATPase inhibitors. Therefore, our data suggest that the metabotropic function of LTCCs is potentiated in SAH. Our results could provide a new strategy to optimize the pharmacological treatment of this pathological process.

  4. FPL 64176 Modification of CaV1.2 L-Type Calcium Channels: Dissociation of Effects on Ionic Current and Gating Current

    PubMed Central

    McDonough, Stefan I.; Mori, Yasuo; Bean, Bruce P.

    2005-01-01

    FPL 64176 (FPL) is a nondihydropyridine compound that dramatically increases macroscopic inward current through L-type calcium channels and slows activation and deactivation. To understand the mechanism by which channel behavior is altered, we compared the effects of the drug on the kinetics and voltage dependence of ionic currents and gating currents. Currents from a homogeneous population of channels were obtained using cloned rabbit CaV1.2 (α1C, cardiac L-type) channels stably expressed in baby hamster kidney cells together with β1a and α2δ1 subunits. We found a striking dissociation between effects of FPL on ionic currents, which were modified strongly, and on gating currents, which were not detectably altered. Inward ionic currents were enhanced ∼5-fold for a voltage step from −90 mV to +10 mV. Kinetics of activation and deactivation were slowed dramatically at most voltages. Curiously, however, at very hyperpolarized voltages (<−250 mV), deactivation was actually faster in FPL than in control. Gating currents were measured using a variety of inorganic ions to block ionic current and also without blockers, by recording gating current at the reversal potential for ionic current (+50 mV). Despite the slowed kinetics of ionic currents, FPL had no discernible effect on the fundamental movements of gating charge that drive channel gating. Instead, FPL somehow affects the coupling of charge movement to opening and closing of the pore. An intriguing possibility is that the drug causes an inactivated state to become conducting without otherwise affecting gating transitions. PMID:15501945

  5. Tumor Necrosis Factor Alpha Inhibits L-Type Ca2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

    PubMed Central

    Reyes-García, Jorge; Flores-Soto, Edgar; Solís-Chagoyán, Héctor; Sommer, Bettina; Díaz-Hernández, Verónica; García-Hernández, Luz María

    2016-01-01

    Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca2+ channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway. PMID:27445440

  6. Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel*

    PubMed Central

    Szpyt, John; Lorenzon, Nancy; Perez, Claudio F.; Norris, Ethan; Allen, Paul D.; Beam, Kurt G.; Samsó, Montserrat

    2012-01-01

    The L-type Ca2+ channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α1s or CaV1.1, α2, β1a, δ1, and γ), we created transgenic mice expressing a recombinant β1a subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α2-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of CaV1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α1s subunit to the membrane and its suggested role in excitation-contraction coupling. PMID:23118233

  7. Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs

    PubMed Central

    Kim, Jae Gon; Sung, Dong Jun; Kim, Hyun-ji; Park, Sang Woong; Won, Kyung Jong; Kim, Bokyung; Shin, Ho Chul; Kim, Ki-Suk; Leem, Chae Hun; Zhang, Yin Hua; Cho, Hana; Bae, Young Min

    2016-01-01

    The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K+ current (IKr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific IKr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na+ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca2+ current (ICaL) caused by a decrease in Ca2+-dependent inactivation (CDI). Acute exposure to sparfloxacin, an IKr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack IKr due to E4031 pretreatment. Sparfloxacin reduced peak ICaL but increased late ICaL by slowing its inactivation. In contrast, ketoconazole, an IKr blocker without prolongation of QT interval and TdP produced reduction of both peak and late ICaL, suggesting the role of increased late ICaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca2+ with Ba2+ abolished the sparfloxacin effects on ICaL. In addition, sparfloxacin modulated ICaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native IKr, demonstrated similar increase in late ICaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late ICaL. Thus, drugs that cause delayed ICaL inactivation and IKr blockage may have more adverse effects than those that selectively block IKr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and IKr antagonist potencies. PMID:26930604

  8. Tumor Necrosis Factor Alpha Inhibits L-Type Ca(2+) Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway.

    PubMed

    Reyes-García, Jorge; Flores-Soto, Edgar; Solís-Chagoyán, Héctor; Sommer, Bettina; Díaz-Hernández, Verónica; García-Hernández, Luz María; Montaño, Luis M

    2016-01-01

    Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.

  9. Three-dimensional localization of the α and β subunits and of the II-III loop in the skeletal muscle L-type Ca2+ channel.

    PubMed

    Szpyt, John; Lorenzon, Nancy; Perez, Claudio F; Norris, Ethan; Allen, Paul D; Beam, Kurt G; Samsó, Montserrat

    2012-12-21

    The L-type Ca(2+) channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α(1s) or Ca(V)1.1, α(2), β(1a), δ1, and γ), we created transgenic mice expressing a recombinant β(1a) subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α(2)-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca(V)1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α(1s) subunit to the membrane and its suggested role in excitation-contraction coupling.

  10. The β1a subunit regulates the functional properties of adult frog and mouse L-type Ca2+ channels of skeletal muscle

    PubMed Central

    García, Rubén; Carrillo, Elba; Rebolledo, Santiago; García, María C; Sánchez, Jorge A

    2002-01-01

    The β1a subunit, one of the auxiliary subunits of CaV1.1 channels, was expressed in COS-1 cells, purified by electroelution and electrodialysis techniques and identified by Western blot using monoclonal antibodies. The purified β1a subunit strongly interacted in vitro with the alpha interaction domain (AID) of CaV1.1 channels. The actions of the purified β1a subunit on CaV1.1 channel currents were assessed in whole cell voltage clamp experiments performed in vesicles derived from frog and mouse adult skeletal muscle plasma membranes. L-type inward currents were recorded in solutions containing Ba2+ (IBa). Values of peak IBa were doubled by the β1a subunit in frog and mouse muscle vesicles and the amplitude of the slow component of tail currents was greatly increased. The actions of the β1a subunit on CaV1.1 channel currents reached a steady state within 20 min. The β1a subunit had no effect on the time courses of activation or inactivation of IBa or shifted the current-voltage relation. Non-linear capacitive currents were recorded in solutions that contained mostly impermeant ions. Charge movement depended on voltage with average Boltzmann parameters: Qmax + 28.0 ± 6.6 nC μF−1, V + −58.0 ± 2.0 mV and k + 15.3 ± 1.1 mV (n = 24). In the presence of the β1a subunit, these parameters remained unchanged: Qmax + 29.8 ± 3.5 nC μF−1, V + −54.5 ± 2.2 mV and k + 16.4 ± 1.3 mV (n = 21). Overall, the work describes a novel preparation to explore in situ the role of the β1a subunit on the function of adult CaV1.1 channels. PMID:12456821

  11. Electrophysiological properties of myocytes isolated from the mouse atrioventricular node: L-type ICa, IKr, If, and Na-Ca exchange

    PubMed Central

    Choisy, Stéphanie C; Cheng, Hongwei; Orchard, Clive H; James, Andrew F; Hancox, Jules C

    2015-01-01

    The atrioventricular node (AVN) is a key component of the cardiac pacemaker-conduction system. This study investigated the electrophysiology of cells isolated from the AVN region of adult mouse hearts, and compared murine ionic current magnitude with that of cells from the more extensively studied rabbit AVN. Whole-cell patch-clamp recordings of ionic currents, and perforated-patch recordings of action potentials (APs), were made at 35–37°C. Hyperpolarizing voltage commands from −40 mV elicited a Ba2+-sensitive inward rectifier current that was small at diastolic potentials. Some cells (Type 1; 33.4 ± 2.2 pF; n = 19) lacked the pacemaker current, If, whilst others (Type 2; 34.2 ± 1.5 pF; n = 21) exhibited a clear If, which was larger than in rabbit AVN cells. On depolarization from −40 mV L-type Ca2+ current, ICa,L, was elicited with a half maximal activation voltage (V0.5) of −7.6 ± 1.2 mV (n = 24). ICa,L density was smaller than in rabbit AVN cells. Rapid delayed rectifier (IKr) tail currents sensitive to E-4031 (5 μmol/L) were observed on repolarization to −40 mV, with an activation V0.5 of −10.7 ± 4.7 mV (n = 8). The IKr magnitude was similar in mouse and rabbit AVN. Under Na-Ca exchange selective conditions, mouse AVN cells exhibited 5 mmol/L Ni-sensitive exchange current that was inwardly directed negative to the holding potential (−40 mV). Spontaneous APs (5.2 ± 0.5 sec−1; n = 6) exhibited an upstroke velocity of 37.7 ± 16.2 V/s and ceased following inhibition of sarcoplasmic reticulum Ca2+ release by 1 μmol/L ryanodine, implicating intracellular Ca2+ cycling in murine AVN cell electrogenesis. PMID:26607172

  12. K(ATP) channel gain-of-function leads to increased myocardial L-type Ca(2+) current and contractility in Cantu syndrome.

    PubMed

    Levin, Mark D; Singh, Gautam K; Zhang, Hai Xia; Uchida, Keita; Kozel, Beth A; Stein, Phyllis K; Kovacs, Atilla; Westenbroek, Ruth E; Catterall, William A; Grange, Dorothy Katherine; Nichols, Colin G

    2016-06-14

    Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits. We show that patients with CS, as well as mice with constitutive (cGOF) or tamoxifen-induced (icGOF) cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts, with increased ejection fraction and increased contractility. Whole-cell voltage-clamp recordings from cGOF or icGOF ventricular myocytes (VM) show increased basal L-type Ca(2+) current (LTCC), comparable to that seen in WT VM treated with isoproterenol. Mice with vascular-specific expression (vGOF) show left ventricular dilation as well as less-markedly increased LTCC. Increased LTCC in KATP GOF models is paralleled by changes in phosphorylation of the pore-forming α1 subunit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928, suggesting enhanced protein kinase activity as a potential link between increased KATP current and CS cardiac pathophysiology.

  13. KATP channel gain-of-function leads to increased myocardial L-type Ca2+ current and contractility in Cantu syndrome

    PubMed Central

    Levin, Mark D.; Singh, Gautam K.; Zhang, Hai Xia; Uchida, Keita; Kozel, Beth A.; Stein, Phyllis K.; Kovacs, Atilla; Westenbroek, Ruth E.; Catterall, William A.; Grange, Dorothy Katherine; Nichols, Colin G.

    2016-01-01

    Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits. We show that patients with CS, as well as mice with constitutive (cGOF) or tamoxifen-induced (icGOF) cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts, with increased ejection fraction and increased contractility. Whole-cell voltage-clamp recordings from cGOF or icGOF ventricular myocytes (VM) show increased basal L-type Ca2+ current (LTCC), comparable to that seen in WT VM treated with isoproterenol. Mice with vascular-specific expression (vGOF) show left ventricular dilation as well as less-markedly increased LTCC. Increased LTCC in KATP GOF models is paralleled by changes in phosphorylation of the pore-forming α1 subunit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928, suggesting enhanced protein kinase activity as a potential link between increased KATP current and CS cardiac pathophysiology. PMID:27247394

  14. Isoform-specific regulation of mood behavior and pancreatic β cell and cardiovascular function by L-type Ca2+ channels

    PubMed Central

    Sinnegger-Brauns, Martina J.; Hetzenauer, Alfred; Huber, Irene G.; Renström, Erik; Wietzorrek, Georg; Berjukov, Stanislav; Cavalli, Maurizio; Walter, Doris; Koschak, Alexandra; Waldschütz, Ralph; Hering, Steffen; Bova, Sergio; Rorsman, Patrik; Pongs, Olaf; Singewald, Nicolas; Striessnig, Jörg

    2004-01-01

    Cav1.2 and Cav1.3 L-type Ca2+ channels (LTCCs) are believed to underlie Ca2+ currents in brain, pancreatic β cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause card iovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Cav1.2 α1 subunits (Cav1.2DHP–/–) without affecting function and expression. This allowed separation of the DHP effects of Cav1.2 from those of Cav1.3 and other LTCCs. DHP effects on pancreatic β cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Cav1.2DHP–/– mice, which rules out a direct role of Cav1.3 for these physiological processes. Using Cav1.2DHP–/– mice, we established DHPs as mood-modifying agents: LTCC activator–induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Cav1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects. PMID:15146240

  15. 2,5-Di-t-butyl-1,4-benzohydroquinone (BHQ) inhibits vascular L-type Ca2+ channel via superoxide anion generation

    PubMed Central

    Fusi, Fabio; Saponara, Simona; Gagov, Hristo; Sgaragli, Giampietro

    2001-01-01

    The aim of the present study was to investigate the effects of 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), an inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), on the whole-cell voltage-dependent L-type Ca2+ current (ICa(L)) of freshly isolated smooth muscle cells from the rat tail artery using the patch-clamp technique. BHQ, added to the perfusion solution, reduced ICa(L) in a concentration- (IC50=66.7 μM) and voltage-dependent manner. This inhibition was only partially reversible. BHQ shifted the voltage dependence of the steady-state inactivation curve to more negative potentials by 7 mV in the mid-potential of the curve, without affecting the activation curve as well as the time course of ICa(L) inactivation. Preincubation of the cells either with 10 μM cyclopiazonic acid, a SERCA inhibitor, or with 3 mM diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD), did not modify BHQ inhibition of ICa(L). On the contrary, this effect was no longer evident when SOD (250 u ml−1) was added to the perfusion medium. Either in the presence or in the absence of cells, BHQ gave rise to superoxide anion formation, which was markedly inhibited by the addition of SOD. These results indicate that, at micromolar concentrations, BHQ inhibits vascular ICa(L) by giving rise to the formation of superoxide anion which in turn impairs the channel function. PMID:11487507

  16. Co-localization of L-type voltage dependent calcium channel alpha 1D subunit (Ca(v)1.3) and calbindin (CB) in the mouse central nervous system.

    PubMed

    Xu, Jie Hua; Yang, Zhen Bang; Wang, Hui; Tang, Feng-Ru

    2014-02-21

    Previous study has shown that the co-localization of calbindin (CB) with L-type voltage dependent Ca(2+) channel (VDCC) alpha 1C subunit (Ca(v)1.2) in the rat insulinoma 1046-38 (RIN) beta cells may play an important regulatory role in Ca(2+) influx and exocytosis of insulin granules. In the present study, L-type voltage dependent Ca(2+) channel (VDCC) and calbindin (CB) were demonstrated in different regions of the mouse central nervous system (CNS). Double labeling immunofluorescence staining showed a co-localization of Ca(v)1.3 and CB. The co-localization of Ca(v)1.3 and CB in certain brain regions such as the hippocampus suggests their important roles in neuroplasticity. The relative high percentages of co-localization of Ca(v)1.3 with CB in the laminae II of the dorsal horn of the spinal cord indicate that the regulation mechanism of nociceptive transmission may be related with both VDCC and Ca(2+) binding protein.

  17. Rotenone-stimulated superoxide release from mitochondrial complex I acutely augments L-type Ca2+ current in A7r5 aortic smooth muscle cells.

    PubMed

    Ochi, Rikuo; Dhagia, Vidhi; Lakhkar, Anand; Patel, Dhara; Wolin, Michael S; Gupte, Sachin A

    2016-05-01

    Voltage-gated L-type Ca(2+) current (ICa,L) induces contraction of arterial smooth muscle cells (ASMCs), and ICa,L is increased by H2O2 in ASMCs. Superoxide released from the mitochondrial respiratory chain (MRC) is dismutated to H2O2 We studied whether superoxide per se acutely modulates ICa,L in ASMCs using cultured A7r5 cells derived from rat aorta. Rotenone is a toxin that inhibits complex I of the MRC and increases mitochondrial superoxide release. The superoxide content of mitochondria was estimated using mitochondrial-specific MitoSOX and HPLC methods, and was shown to be increased by a brief exposure to 10 μM rotenone. ICa,L was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 μM) resulted in a greater ICa,L increase in a dose-dependent manner to a maximum of 22.1% at 10 μM for 1 min, which gradually decreased to 9% after 5 min. The rotenone-induced ICa,L increase was associated with a shift in the current-voltage relationship (I-V) to a hyperpolarizing direction. DTT administration resulted in a 17.9% increase in ICa,L without a negative shift in I-V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited ICa,L by 13%, and additional rotenone induced an increase with a negative shift. Sustained treatment with Tempol (4-hydroxy tempo) led to a significant ICa,L increase but it inhibited the rotenone-induced increase. Staurosporine, a broad-spectrum protein kinase inhibitor, partially inhibited ICa,L and completely suppressed the rotenone-induced increase. Superoxide released from mitochondria affected protein kinases and resulted in stronger ICa,L preceding its dismutation to H2O2 The removal of nitric oxide is a likely mechanism for the increase in ICa,L. Copyright © 2016 the American Physiological Society.

  18. Enhancing VTA Cav1.3 L-type Ca2+ channel activity promotes cocaine and mood - related behaviors via overlapping AMPA receptor mechanisms in the nucleus accumbens

    PubMed Central

    Martínez-Rivera, Arlene; Hao, Jin; Tropea, Thomas F.; Giordano, Thomas P.; Kosovsky, Maria; Rice, Richard C.; Lee, Amy; Huganir, Richard L.; Striessnig, Joerg; Addy, Nii A.; Han, Shizhong; Rajadhyaksha, Anjali M.

    2016-01-01

    Genetic factors significantly influence susceptibility for substance abuse and mood disorders. Rodent studies have begun to elucidate a role of Cav1.3 L-type Ca2+ channels in neuropsychiatric-related behaviors, such as addictive and depressive-like behaviors. Human studies have also linked the CACNA1D gene, which codes for the Cav1.3 protein, with bipolar disorder (BD). However, the neurocircuitry and the molecular mechanisms underlying the role of Cav1.3 in neuropsychiatric phenotypes are not well established. In the present study, we directly manipulated Cav1.3 channels in Cav1.2 dihydropyridine (DHP) insensitive mutant mice and found that VTA Cav1.3 channels mediate cocaine-related and depressive-like behavior through a common nucleus accumbens (NAc) shell calcium permeable AMPA receptor (CP-AMPAR) mechanism that requires GluA1 phosphorylation at S831. Selective activation of VTA Cav1.3 with (±)-BayK-8644 (BayK) enhanced cocaine conditioned place preference (CPP) and cocaine psychomotor activity while inducing depressive-like behavior, an effect not observed in S831A phospho-mutant mice. Infusion of the CP-AMPAR-specific blocker Naspm into the NAc shell reversed the cocaine and depressive-like phenotypes. In addition, activation of VTA Cav1.3 channels resulted in social behavioral deficits. In contrast to the cocaine- and depression-related phenotypes, GluA1/A2 AMPARs in the NAc core mediated social deficits, independent of S831-GluA1 phosphorylation. Using a candidate gene analysis approach, we also identified single nucleotide polymorphisms in the CACNA1D gene associated with cocaine dependence (CD) in human subjects. Together, our findings reveal novel, overlapping mechanisms through which VTA Cav1.3 mediates cocaine-related, depressive-like and social phenotypes suggesting that Cav1.3 may serve as a target for the treatment of neuropsychiatric symptoms. PMID:28194001

  19. C-terminal modulatory domain controls coupling of voltage-sensing to pore opening in Cav1.3 L-type Ca(2+) channels.

    PubMed

    Lieb, Andreas; Ortner, Nadine; Striessnig, Jörg

    2014-04-01

    Activity of voltage-gated Cav1.3 L-type Ca(2+) channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON) of Cav1.3L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages

  20. Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes

    PubMed Central

    Verde, Ignacio; Vandecasteele, Grégoire; Lezoualc'h, Frank; Fischmeister, Rodolphe

    1999-01-01

    The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (ICa) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT–PCR.IBMX (100 μM), a broad-spectrum PDE inhibitor, increased basal ICa by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the β-adrenoceptor agonist isoprenaline (1 μM). However, MIMX (1 μM), a PDE1 inhibitor, EHNA (10 μM), a PDE2 inhibitor, cilostamide (0.1 μM), a PDE3 inhibitor, or Ro 20-1724 (0.1 μM), a PDE4 inhibitor, had no effect on basal ICa and little stimulatory effects on [cAMP]i (20–30%).Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on ICa or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40–50%), only cilostamide (0.1 μM)+Ro20-1724 (0.1 μM) produced a significant stimulation of ICa (50%). Addition of EHNA (10 μM) to this mix increased ICa to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 μM) or isoprenaline (1 μM).When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased ICa by (≈40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in ICa. Maximal effects on ICa were ≈8% for MIMX (3 μM), ≈20% for EHNA (1–3 μM), ≈30% for cilostamide (0.3–1 μM) and ≈50% for Ro20-1724 (0.1 μM).Our results demonstrate that PDE1-4 subtypes regulate ICa in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal ICa, all four PDE subtypes determine the response of ICa to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3

  1. Basal responses of the L-type Ca2+ and hyperpolarization-activated currents to autonomic agonists in the rabbit sino-atrial node.

    PubMed Central

    Zaza, A; Robinson, R B; DiFrancesco, D

    1996-01-01

    1. The dose dependence of the cholinergic agonist acetylcholine (ACh) and the beta-adrenergic agonist isoprenaline (Iso) were determined for the hyperpolarization-activated current (If) and the L-type Ca2+ current (ICa,L) in single cells isolated from the rabbit sino-atrial (SA) node. 2. ACh inhibited If by a negative shift of its activation curve with a maximal effect of -9.9 mV; half-maximal effect was produced by 0.019 microM ACh. High ACh concentrations were required to inhibit ICa,L only partially (31% inhibition at 300 microM). 3. In contrast, If and ICa,L responded to Iso over a similar dose range, with concentrations for half-maximal enhancement of 0.0136 and 0.0070 microM, respectively. 4. The effects on spontaneous activity of ACh (range 0.001-0.03 microM) and Iso (range 0.001-1 microM) were investigated. ACh decreased the slope of diastolic depolarization at concentrations similar to those inhibiting If (> 50% at 0.03 microM). Iso enhanced diastolic depolarization at concentrations similar to those affecting both If and ICa,L (half-maximal effect at 0.027 microM). 5. In a ramp-clamp protocol simulating diastolic depolarization, the threshold for activation of inward nifedipine-sensitive current was -41.22 +/- 0.68 mV. Although enhancing ICa,L, Iso did not affect this threshold. 6. Half-maximal ACh concentrations for inhibition of automaticity and If are similar and are lower than the threshold concentrations for modulation of ICa,L; this argues against a role of ICa,L in direct muscarinic modulation of pacemaking. In contrast, modulation of If, ICa,L and automaticity occur at similar Iso concentrations. The difference between maximum diastolic potential (-61.95 +/- 0.93 mV) and the threshold for Iso-stimulated ICa,L (-39.54 +/- 1.03 mV) suggests that this current plays a role only at later stages of diastolic depolarization. PMID:8866859

  2. C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to Pore Opening in Cav1.3 L-type Ca2+ Channels

    PubMed Central

    Lieb, Andreas; Ortner, Nadine; Striessnig, Jörg

    2014-01-01

    Activity of voltage-gated Cav1.3 L-type Ca2+ channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON) of Cav1.3L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages

  3. Regulation of N- and L-type Ca2+ channels in adult frog sympathetic ganglion B cells by nerve growth factor in vitro and in vivo.

    PubMed

    Lei, S; Dryden, W F; Smith, P A

    1997-12-01

    To examine mechanisms responsible for the long-term regulation of Ca2+-channels in an adult neuron, changes in whole cell Ba2+ current (IBa) were examined in adult bullfrog sympathetic ganglion B cells in vitro. Cells were cultured at low density in defined, serum free medium. After 15 days, total IBa was similar to the initial value, whereas IBa density was reduced by approximately 36%, presumably due to an increase in neuronal surface area. By contrast, IBa density remained constant after 6-15 days in the presence of murine beta-NGF (200 ng/ml), and total IBa was almost doubled. Inclusion of cytosine arabinoside (Ara-C; 10 microM) to inhibit proliferation of nonneuronal cells, did not affect the survival of neurons in the absence of nerve growth factor (NGF) nor did it attenuate IBa. Ara-C did not prevent the effect of NGF on IBa. There were three independent components to the action of NGF; during 6-9 days, it increased omega-conotoxin-GVIA-sensitive N-type IBa (IBa,N); increased nifedipine-sensitive L-type IBa (IBa,L) and decreased inactivation of the total Ba2+ conductance (gBa). The latter effect involved a selective decrease in the amplitude of one of the four kinetic components that describe the inactivation process. Total IBa was also 55.8% larger than control in the somata of B cells acutely dissociated from leopard frogs that had received prior subcutaneous injections of NGF. By contrast, injection of NGF antiserum decreased total IBa by 29.4%. There was less inactivation of gBa in B cells from NGF-injected animals than in cells from animals injected with NGF antiserum (P < 0.001). These data suggest that NGF-like molecule(s) play(s) a role in the maintenance of IBa in an adult amphibian sympathetic neuron; the presence of NGF may allow the neuron to maintain a constant relationship between cell size and current density. They also show that IBa inactivation in an adult neuron can be modulated in a physiologically relevant way by an extracellular ligand.

  4. Cold stress and corticotropin-releasing hormone induced changes in messenger ribonucleic acid for the alpha(1)-subunit of the L-type Ca(2+) channel in the rat anterior pituitary and enriched populations of corticotropes.

    PubMed

    Xie, J; Nagle, G T; Ritchie, A K; Collins, T J; Childs, G V

    1999-07-01

    In response to stress, adrenocorticotropin (ACTH) is secreted from anterior pituitary corticotropes. Corticotropin-releasing hormone (CRH) is a potent stimulator of ACTH secretion. The CRH stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. This study was designed to investigate whether the expression of L-type Ca(2+) channels in the rat anterior pituitary and in corticotropes is regulated by acute stress and CRH. RNase protection assays were used to quantify alpha(1C) mRNA of the L-type Ca(2+) channel. The alpha(1C) mRNA levels from stressed rats increased by 31% in anterior pituitaries of rats after 30 min of exposure to cold stress. Neither 60 min cold stress nor 30 min restraint stress had an effect on alpha(1C) mRNA levels. When alpha(1C) mRNA was detected by in situ hybridization in a population of corticotropes enriched to 90%, 0.5 nM CRH (3 h) stimulated a 36% increase in the average area of label/cell and a 10% increase in the average density of label. Our results suggest that (1) the expression of alpha(1C) subunit mRNA of L-type Ca(2+) channels is increased in the rat anterior pituitary with a stress-specific response that might reflect an increase both in thyrotropes and corticotropes (both are known to be stimulated by cold stress), and (2) the CRH-mediated increase in alpha(1C) mRNA expression in individual rat corticotropes, in vitro, supports the hypothesis that some of the increase in vivo is due to changes in corticotropes.

  5. Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca[superscript 2+]·calmodulins

    SciTech Connect

    Fallon, Jennifer L.; Baker, Mariah R.; Xiong, Liangwen; Loy, Ryan E.; Yang, Guojun; Dirksen, Robert T.; Hamilton, Susan L.; Quiocho, Florante A.

    2009-11-10

    Voltage-dependent calcium channels (Ca(V)) open in response to changes in membrane potential, but their activity is modulated by Ca(2+) binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca(V)1.2 alpha(1) subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca(2+).CaM in 2 distinct binding modes. The structure of the Ca(V)1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca(2+).CaMs interacting with the pre-IQ regions and a canonical Ca(V)1-IQ-Ca(2+).CaM complex. Native Ca(V)1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca(2+)-dependent inactivation of heterologously expressed Ca(V)1.2 channels.

  6. The L-Type Voltage-Gated Calcium Channel Ca[subscript v]1.3 Mediates Consolidation, but Not Extinction, of Contextually Conditioned Fear in Mice

    ERIC Educational Resources Information Center

    McKinney, Brandon C.; Murphy, Geoffrey G.

    2006-01-01

    Using pharmacological techniques, it has been demonstrated that both consolidation and extinction of Pavlovian fear conditioning are dependent to some extent upon L-type voltage-gated calcium channels (LVGCCs). Although these studies have successfully implicated LVGCCs in Pavlovian fear conditioning, they do not provide information about the…

  7. The L-Type Voltage-Gated Calcium Channel Ca[subscript v]1.3 Mediates Consolidation, but Not Extinction, of Contextually Conditioned Fear in Mice

    ERIC Educational Resources Information Center

    McKinney, Brandon C.; Murphy, Geoffrey G.

    2006-01-01

    Using pharmacological techniques, it has been demonstrated that both consolidation and extinction of Pavlovian fear conditioning are dependent to some extent upon L-type voltage-gated calcium channels (LVGCCs). Although these studies have successfully implicated LVGCCs in Pavlovian fear conditioning, they do not provide information about the…

  8. Ca2+–calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel α1C-subunit gene (Cacna1c) by DREAM translocation

    PubMed Central

    Ronkainen, Jarkko J; Hänninen, Sandra L; Korhonen, Topi; Koivumäki, Jussi T; Skoumal, Reka; Rautio, Sini; Ronkainen, Veli-Pekka; Tavi, Pasi

    2011-01-01

    Abstract Recent studies have demonstrated that changes in the activity of calcium–calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation–contraction (E–C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM–GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+–CaMKII–DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII. PMID:21486818

  9. Ca2+-calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel alpha(1C)-subunit gene (Cacna1c) by DREAM translocation.

    PubMed

    Ronkainen, Jarkko J; Hänninen, Sandra L; Korhonen, Topi; Koivumäki, Jussi T; Skoumal, Reka; Rautio, Sini; Ronkainen, Veli-Pekka; Tavi, Pasi

    2011-06-01

    Recent studies have demonstrated that changes in the activity of calcium-calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation-contraction (E-C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM-GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

  10. L-type calcium channels contribute to 5-HT3-receptor-evoked CaMKIIα and ERK activation and induction of emesis in the least shrew (Cryptotis parva).

    PubMed

    Hutchinson, Tarun E; Zhong, Weixia; Chebolu, Seetha; Wilson, Sean M; Darmani, Nissar A

    2015-05-15

    Activation of serotonergic 5-HT3 receptors by its selective agonist 2-methyl serotonin (2-Me-5-HT) induces vomiting, which is sensitive to selective antagonists of both 5-HT3 receptors (palonosetron) and L-type calcium channels (LTCC) (amlodipine or nifedipine). Previously we demonstrated that 5-HT3 receptor activation also causes increases in a palonosetron-sensitive manner in: i) intracellular Ca(2+) concentration, ii) attachment of calmodulin (CaM) to 5-HT3 receptor, and iii) phosphorylation of Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) and extracellular-signal-regulated kinase 1/2 (ERK1/2). Here, we investigate the role of the short-acting LTCC blocker nifedipine on 2-Me-5-HT-evoked intracellular Ca(2+) increase and on downstream intracellular emetic signaling, which have been shown to be coupled with 2-Me-5-HT׳s emetic effects in the least shrew. Using the cell-permeant Ca(2+) indicator fluo-4 AM, here we present evidence for the contribution of Ca(2+) influx through LTCCs (sensitive to nifedipine) in 2-Me-5-HT (1µM) -evoked rise in cytosolic Ca(2+) levels in least shrew brainstem slices. Nifedipine pretreatment (10mg/kg, s.c.) also suppressed 2-Me-5-HT-evoked interaction of 5-HT3 receptors with CaM as well as phosphorylation of CaMKIIα and ERK1/2 in the least shrew brainstem, and 5-HT3 receptors -CaM colocalization in jejunum of the small intestine. In vitro exposure of isolated enterochromaffin cells of the small intestine to 2-Me-5-HT (1µM) caused CaMKIIα phosphorylation, which was also abrogated by nifedipine pretreatment (0.1µM). In addition, pretreatment with the CaMKII inhibitor KN62 (10mg/kg, i.p.) suppressed emesis and also the activation of CaMKIIα, and ERK in brainstem caused by 2-Me-5-HT (5mg/kg, i.p.). This study provides further mechanistic explanation for our published findings that nifedipine can dose-dependently protect shrews from 2-Me-5-HT-induced vomiting.

  11. Central Nervous System-Toxic Lidocaine Concentrations Unmask L-Type Ca²⁺ Current-Mediated Action Potentials in Rat Thalamocortical Neurons: An In Vitro Mechanism of Action Study.

    PubMed

    Putrenko, Igor; Ghavanini, Amer A; Meyer Schöniger, Katrin S; Schwarz, Stephan K W

    2016-05-01

    High systemic lidocaine concentrations exert well-known toxic effects on the central nervous system (CNS), including seizures, coma, and death. The underlying mechanisms are still largely obscure, and the actions of lidocaine on supraspinal neurons have received comparatively little study. We recently found that lidocaine at clinically neurotoxic concentrations increases excitability mediated by Na-independent, high-threshold (HT) action potential spikes in rat thalamocortical neurons. Our goal in this study was to characterize these spikes and test the hypothesis that they are generated by HT Ca currents, previously implicated in neurotoxicity. We also sought to identify and isolate the specific underlying subtype of Ca current. We investigated the actions of lidocaine in the CNS-toxic concentration range (100 μM-1 mM) on ventrobasal thalamocortical neurons in rat brain slices in vitro, using whole-cell patch-clamp recordings aided by differential interference contrast infrared videomicroscopy. Drugs were bath applied; action potentials were generated using current clamp protocols, and underlying currents were identified and isolated with ion channel blockers and electrolyte substitution. Lidocaine (100 μM-1 mM) abolished Na-dependent tonic firing in all neurons tested (n = 46). However, in 39 of 46 (85%) neurons, lidocaine unmasked evoked HT action potentials with lower amplitudes and rates of de-/repolarization compared with control. These HT action potentials remained during the application of tetrodotoxin (600 nM), were blocked by Cd (50 μM), and disappeared after superfusion with an extracellular solution deprived of Ca. These features implied that the unmasked potentials were generated by high-voltage-activated Ca channels and not by Na channels. Application of the L-type Ca channel blocker, nifedipine (5 μM), completely blocked the HT potentials, whereas the N-type Ca channel blocker, ω-conotoxin GVIA (1 μM), had little effect. At clinically CNS

  12. A novel antihypertension agent, sargachromenol D from marine brown algae, Sargassum siliquastrum, exerts dual action as an L-type Ca(2+) channel blocker and endothelin A/B2 receptor antagonist.

    PubMed

    Park, Byong-Gon; Shin, Woon-Seob; Oh, Sangtae; Park, Gab-Man; Kim, Nam Ik; Lee, Seokjoon

    2017-09-01

    We isolated the novel vasoactive marine natural products, (5E,10E)-14-hydroxy-2,6,10-trimethylpentadeca-5,10-dien-4-one (4) and sargachromenol D (5), from Sargassum siliquastrum collected from the coast of the East Sea in South Korea by using activity-guided HPLC purification. The compounds effectively dilated depolarization (50mMK(+))-induced basilar artery contraction with EC50 values of 3.52±0.42 and 1.62±0.63μM, respectively, but only sargachromenol D (5) showed a vasodilatory effect on endothelin-1 (ET-1)-induced basilar artery contraction (EC50=9.8±0.6μM). These results indicated that sargachromenol D (5) could act as a dual antagonist of l-type Ca(2+) channel and endothelin A/B2 receptors. Moreover, sargachromenol D (5) lowered blood pressure in spontaneous hypertensive rats (SHRs) 2h after oral treatment at a dose of 80mg/kg dose and the effect was maintained for 24h. Based on our ex vivo and in vivo experiments, we propose that sargachromenol D (5) is a strong candidate for the treatment of hypertension that is not controlled by conventional drugs, in particular, severe-, type II diabetes-, salt-sensitive, and metabolic disease-induced hypertension. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Psychostimulants, antidepressants and neurokinin-1 receptor antagonists ('motor disinhibitors') have overlapping, but distinct, effects on monoamine transmission: the involvement of L-type Ca2+ channels and implications for the treatment of ADHD.

    PubMed

    Stanford, S Clare

    2014-12-01

    Both psychostimulants and antidepressants target monoamine transporters and, as a consequence, augment monoamine transmission. These two groups of drugs also increase motor activity in preclinical behavioural screens for antidepressants. Substance P-preferring receptor (NK1R) antagonists similarly increase both motor activity in these tests and monoamine transmission in the brain. In this article, the neurochemical and behavioural responses to these three groups of drugs are compared. It becomes evident that NK1R antagonists represent a distinct class of compounds ('motor disinhibitors') that differ substantially from both psychostimulants and antidepressants, especially during states of heightened arousal or stress. Also, all three groups of drugs influence the activation of voltage-gated Ca(v)1.2 and Ca(v)1.3 L-type channels (LTCCs) in the brain, albeit in different ways. This article discusses evidence that points to disruption of these functional interactions between NK1R and LTCCs as a contributing factor in the cognitive and behavioural abnormalities that are prominent features of Attention Deficit Hyperactivity Disorder (ADHD). Arising from this is the interesting possibility that the hyperactivity and impulsivity (as in ADHD) and psychomotor retardation (as in depression) reflect opposite poles of a behavioural continuum. A better understanding of this pharmacological network could help explain why psychostimulants augment motor behaviour during stress (e.g., in preclinical screens for antidepressants) and yet reduce locomotor activity and impulsivity in ADHD. This article is part of the Special Issue entitled 'CNS Stimulants'. Copyright © 2014 The Author. Published by Elsevier Ltd.. All rights reserved.

  14. Saxitoxin blocks L-type ICa.

    PubMed

    Su, Zhi; Sheets, Michael; Ishida, Hideyuki; Li, Fenghua; Barry, William H

    2004-01-01

    Saxitoxin (STX) and tetrodotoxin (TTX) are frequently used to selectively block sodium channels. In this study, we provide evidence that commercial STX also inhibits L-type Ca2+ currents (I(Ca,L)) in adult mouse ventricular myocytes (VMs) and tsA-201 cells that were transiently cotransfected with three calcium channel subunits. We measured inhibition of sodium currents (INa) in mouse VMs, of I(Ca,L) in mouse VM and tsA-201 cells, and intracellular calcium concentration ([Ca2+]i) transients in single mouse VMs. STX or TTX was abruptly applied before the test voltage pulse using a rapid solution switcher device. STX (10 microM; Calbiochem) and TTX (60 microM; Sigma-Aldrich) completely blocked INa in mouse VMs. However, STX at 10 microM also reduced I(Ca,L) in mouse VM by 39% (P < 0.0001; n = 14), whereas TTX at 60 microM had no effect on I(Ca,L). STX (10 microM; Calbiochem) reduced the amplitude of the [Ca2+]i transients in mouse VMs by 36% (P < 0.0001; n = 10). In contrast, TTX (60 microM; Sigma-Aldrich) only reduced the amplitude of the [Ca2+]i transients by 9% (P = 0.003; n = 5). STX (10 microM) obtained from Sigma-Aldrich showed a similar inhibitory effect on I(Ca,L) (33%) (P < 0.0001; n = 5) in mouse VMs. STX (Calbiochem) inhibited the calcium currents of tsA-201 cells in a dose-dependent manner. This inhibition was voltage-independent. The current-voltage relationship of calcium currents in tsA-201 cells was not altered by STX. These results indicate that STX partially blocks L-type Ca2+ channels and thus provide further evidence that its effects are not specific for Na+ channels.

  15. Actions of Two Main Metabolites of Propiverine (M-1 and M-2) on Voltage-Dependent L-Type Ca2+ Currents and Ca2+ Transients in Murine Urinary Bladder Myocytes

    PubMed Central

    Zhu, Hai-Lei; Brain, Keith L.; Aishima, Manami; Shibata, Atsushi; Young, John S.; Sueishi, Katsuo; Teramoto, Noriyoshi

    2008-01-01

    The anticholinergic propiverine, used for the treatment of overactive bladder (OAB) syndrome, has functionally active metabolites (M-1 and M-2), but the site of actions of these metabolites is uncertain. Propiverine is rapidly absorbed after oral administration and is extensively biotransformed in the liver, giving rise to several active metabolites (M-1, the propiverine N-oxide, and M-2, the N-oxide lacking the aliphatic side chain). This study determines the effect of M-1 and M-2 on voltage-dependent nifedipine-sensitive inward Ca2+ currents (ICa) using patch-clamp techniques and fluorescent Ca2+ imaging (following electrical field stimulation (EFS) and acetylcholine (ACh)) in the murine urinary bladder. In conventional whole-cell recording, propiverine and M-1 but not M-2 inhibited the peak amplitude of ICa in a concentration-dependent manner at a holding potential of −60 mV (propiverine, Ki = 10 μM; M-1, Ki = 118 μM). M-1 shifted the steady-state inactivation curve of ICa to the left at −90 mV by 7 mV. Carbachol (CCh) reversibly inhibited ICa. This inhibition probably occurred through muscarinic type 3 receptors, coupling with G-proteins, since nanomolar concentrations of 4-DAMP greatly reduced this inhibition, while pirenzepine or AF-DX 116 at concentrations up to 1 μM were almost ineffective. In the presence of M-2, the CCh-induced inhibition of ICa was blocked. In fluorescent Ca2+ imaging, M-2 inhibited EFS-induced and ACh-induced Ca2+ transients. These results suggest that M-1 acts, at least in part, as a Ca2+ channel antagonist (as it inhibited ICa), while M-2 has more direct antimuscarinic actions. PMID:17928569

  16. A 1-D dusty plasma photonic crystal

    SciTech Connect

    Mitu, M. L.; Ticoş, C. M.; Toader, D.; Banu, N.; Scurtu, A.

    2013-09-21

    It is demonstrated numerically that a 1-D plasma crystal made of micron size cylindrical dust particles can, in principle, work as a photonic crystal for terahertz waves. The dust rods are parallel to each other and arranged in a linear string forming a periodic structure of dielectric-plasma regions. The dispersion equation is found by solving the waves equation with the boundary conditions at the dust-plasma interface and taking into account the dielectric permittivity of the dust material and plasma. The wavelength of the electromagnetic waves is in the range of a few hundred microns, close to the interparticle separation distance. The band gaps of the 1-D plasma crystal are numerically found for different types of dust materials, separation distances between the dust rods and rod diameters. The distance between levitated dust rods forming a string in rf plasma is shown experimentally to vary over a relatively wide range, from 650 μm to about 1350 μm, depending on the rf power fed into the discharge.

  17. Pertussis toxin nullifies the depolarization of the membrane potential and the stimulation of the rapid phase of Ca entry through L-type calcium channels that are produced by follicle stimulating hormone in 10- to 12-day-old rat Sertoli cells.

    PubMed

    Jacobus, Ana Paula; Loss, Eloísa Silveira; Wassermann, Guillermo Federico

    2010-01-01

    The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca(2+) into cells and to the Ca(2+)-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of (45)Ca(2+) uptake and [(14)C]-methyl aminoisobutyric acid ([(14)C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca(2+) channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of (45)Ca(2+) entry and [(14)C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca(2+) channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/βγ/PI3Kγ pathway, independent of the effects of IGF-I.

  18. Pertussis Toxin Nullifies the Depolarization of the Membrane Potential and the Stimulation of the Rapid Phase of 45Ca2+ Entry Through L-type Calcium Channels that are Produced by Follicle Stimulating Hormone in 10- to 12-Day-Old Rat Sertoli Cells

    PubMed Central

    Jacobus, Ana Paula; Loss, Eloísa Silveira; Wassermann, Guillermo Federico

    2010-01-01

    The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca2+ into cells and to the Ca2+-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of 45Ca2+ uptake and [14C]-methyl aminoisobutyric acid ([14C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca2+ channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of 45Ca2+ entry and [14C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca2+ channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/βγ/PI3Kγ pathway, independent of the effects of IGF-I. PMID:21423378

  19. High-Frequency Stimulation-Induced Synaptic Potentiation in Dorsal and Ventral CA1 Hippocampal Synapses: The Involvement of NMDA Receptors, mGluR5, and (L-Type) Voltage-Gated Calcium Channels

    ERIC Educational Resources Information Center

    Papatheodoropoulos, Costas; Kouvaros, Stylianos

    2016-01-01

    The ability of the ventral hippocampus (VH) for long-lasting long-term potentiation (LTP) and the mechanisms underlying its lower ability for shortlasting LTP compared with the dorsal hippocampus (DH) are unknown. Using recordings of field excitatory postsynaptic potentials (EPSPs) from the CA1 field of adult rat hippocampal slices, we found that…

  20. High-Frequency Stimulation-Induced Synaptic Potentiation in Dorsal and Ventral CA1 Hippocampal Synapses: The Involvement of NMDA Receptors, mGluR5, and (L-Type) Voltage-Gated Calcium Channels

    ERIC Educational Resources Information Center

    Papatheodoropoulos, Costas; Kouvaros, Stylianos

    2016-01-01

    The ability of the ventral hippocampus (VH) for long-lasting long-term potentiation (LTP) and the mechanisms underlying its lower ability for shortlasting LTP compared with the dorsal hippocampus (DH) are unknown. Using recordings of field excitatory postsynaptic potentials (EPSPs) from the CA1 field of adult rat hippocampal slices, we found that…

  1. cAMP-dependent protein kinase and Ca2+ influx through L-type voltage-gated calcium channels mediate Raf-independent activation of extracellular regulated kinase in response to glucagon-like peptide-1 in pancreatic beta-cells.

    PubMed

    Gomez, Edith; Pritchard, Catrin; Herbert, Terence P

    2002-12-13

    Glucagon like peptide-1 (GLP1) is a G(s)-coupled receptor agonist that exerts multiple effects on pancreatic beta-cells, including the stimulation of insulin gene expression and secretion. In this report, we show that treatment of the mouse pancreatic beta-cell line MIN6 with GLP1 leads to the glucose-dependent activation of Erk. These effects are mimicked by forskolin, a direct activator of adenylate cyclase, and blocked by H89, an inhibitor of cAMP-dependent protein kinase. Additionally, we provide evidence that GLP1-stimulated activation of Erk requires an influx of calcium through L-type voltage-gated calcium channels and the activation of calcium/calmodulin-dependent protein kinase II. GLP1-stimulated activation of Erk is blocked by inhibitors of MEK, but GLP1 does not induce the activation of A-Raf, B-Raf, C-Raf, or Ras. Additionally, dominant negative forms of Ras(N17) and Rap1(N17) fail to block GLP1-stimulated activation of Erk. In conclusion, our results indicate that, in the presence of stimulatory concentrations of glucose, GLP1 stimulates the activation of Erk through a mechanism dependent on MEK but independent of both Raf and Ras. This requires 1) the activation of cAMP-dependent protein kinase, 2) an influx of extracellular Ca(2+) through L-type voltage-gated calcium channels, and 3) the activation of CaM kinase II.

  2. Dihydropyridine receptor of L-type Ca sup 2+ channels: Identification of binding domains for ( sup 3 H)(+)-PN200-110 and ( sup 3 H)azidopine within the. alpha. 1 subunit

    SciTech Connect

    Striessnig, J.; Murphy, B.J.; Catterall, W.A. )

    1991-12-01

    To identify the binding domain for dihydropyridine Ca{sup 2{plus}} antagonists, skeletal muscle Ca{sup 2{plus}} channels were photolabeled with ({sup 3}H)({plus})-PN200-110 and ({sup 3}H) azidopine. Regions of {alpha}1 photolabeled by these ligands were then identified by antibody mapping of proteolytic fragments. Approximately 50% of the specific labeling by both ligands was incorporated in domain III. ({sup 3}H)Azidopine labeled peptide Gln-989-Arg-1022, which contains a portion of the connecting loop between transmembrane segments IIIS5 and IIIS6 (IIIS5/S6), and peptide Ala-1023-Lys-1077, which contains IIIS6 itself and some adjacent amino acid residues. In contrast, ({sup 3}H)({plus})-PN200-110 labeling occurred almost exclusively in the fragment containing IIIS6. A second site labeled by both ligands was identified in transmembrane segment S6 of domain IV and adjacent residues. It is proposed, based on physiological studies, that these three peptide segments interact to form a receptor site accessible from the extracellular surface of the Ca{sup 2{plus}} channel.

  3. Otilonium bromide inhibits calcium entry through L-type calcium channels in human intestinal smooth muscle.

    PubMed

    Strege, P R; Evangelista, S; Lyford, G L; Sarr, M G; Farrugia, G

    2004-04-01

    Otilonium bromide (OB) is used as an intestinal antispasmodic. The mechanism of action of OB is not completely understood. As Ca(2+) entry into intestinal smooth muscle is required to trigger contractile activity, our hypothesis was that OB blocked Ca(2+) entry through L-type Ca(2+) channels. Our aim was to determine the effects of OB on Ca(2+), Na(+) and K(+) ion channels in human jejunal circular smooth muscle cells and on L-type Ca(2+) channels expressed heterologously in HEK293 cells. Whole cell currents were recorded using standard patch clamp techniques. Otilonium bromide (0.09-9 micromol L(-1)) was used as this reproduced clinical intracellular concentrations. In human circular smooth muscle cells, OB inhibited L-type Ca(2+) current by 25% at 0.9 micromol L(-1) and 90% at 9 micromol L(-1). Otilonium bromide had no effect on Na(+) or K(+) currents. In HEK293 cells, 1 micromol L(-1) OB significantly inhibited the expressed L-type Ca(2+) channels. Truncation of the alpha(1C) subunit C and N termini did not block the inhibitory effects of OB. Otilonium bromide inhibited Ca(2+) entry through L-type Ca(2+) at concentrations similar to intestinal tissue levels. This effect may underlie the observed muscle relaxant effects of the drug.

  4. Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells.

    PubMed

    Alvarez-Collazo, Julio; Alonso-Carbajo, Lucía; López-Medina, Ana I; Alpizar, Yeranddy A; Tajada, Sendoa; Nilius, Bernd; Voets, Thomas; López-López, José Ramón; Talavera, Karel; Pérez-García, María Teresa; Alvarez, Julio L

    2014-11-01

    Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca(2+) channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca(2+) currents in mouse ventricular cardiomyocytes (VCM, with Ca(2+) as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba(2+) as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca(2+) channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca(2+) channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

  5. Calcium entry through L-type calcium channels is essential for neurite regeneration in cultured sympathetic neurons.

    PubMed

    Kulbatski, Iris; Cook, Douglas J; Tator, Charles H

    2004-03-01

    Previous work showed that a post-neuritotomy rise in [Ca2+]i is required for regeneration. We tested the following hypotheses in cultured sympathetic neurons: (1) blocking L-type channels at the time of injury inhibits regeneration; (2) enhancing Ca2+ entry through L-type Ca2+ channels enhances regeneration; (3) L-type Ca2+ channel distribution is predominantly on the soma and proximal neurites of uninjured and injured neurons. To visualize L-type Ca2+ channels and block Ca2+ influx, the fluorescent dihydropyridine antagonist, DM-BODIPY, was used. Our results show that regeneration is markedly inhibited by the antagonist when administered 20 min. prior to injury, in the presence or absence of nerve growth factor (NGF) (p < 0.0001). Severe degeneration of proximal and distal neurites was seen 48 h after injury. Regeneration was minimally inhibited by the antagonist when administered 5 min after injury (p < 0.05), but not inhibited when administered 2 or 24 h after injury (p > 0.05). We found that L-type channels are distributed ubiquitously on the soma and neurites of uninjured and injured cells, and on regenerating neurites. The addition of the L-type channel agonist, BayK8644, (1 microM) 20 min prior to injury enhanced neurite length at 24 h post-injury (p = 0.002). Blocking L-type channels did not affect the viability of uninjured or injured cells. For the first time, it has been shown that Ca2+ entry through L-type Ca2+ channels is essential for post-neuritotomy sympathetic neurite regeneration, and that this effect shows a strict temporal dependency. We also demonstrated that regeneration can be enhanced by increasing Ca2+ influx through L-type channels.

  6. AKAP5 keeps L-type channels and NFAT on their toes.

    PubMed

    Navedo, Manuel F; Hell, Johannes W

    2014-06-12

    In this issue of Cell Reports, Murphy et al. and Dittmer et al. present exciting new insight into the regulation of Ca2+ influx via the L-type Ca2+ channel Cav1.2 and how increased Ca2+ influx translates into localized activation of the nuclear transcription factor NFAT upon depolarization in neurons. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Calcium signaling in mast cells: focusing on L-type calcium channels.

    PubMed

    Suzuki, Yoshihiro; Inoue, Toshio; Ra, Chisei

    2012-01-01

    Mast cells play central roles in adaptive and innate immunity. IgE-dependent stimulation of the high-affinity IgE receptor (FcεRI) results in rapid secretion of various proinflammatory chemical mediators and cytokines. All of the outputs depend to certain degrees on an increase in the intracellular Ca(2+) concentration, and influx of Ca(2+) from the extracellular space is often required for their full activation. There is strong evidence that FcεRI stimulation induces two different modes of Ca(2+) influx, store-operated Ca(2+) entry (SOCE) and non-SOCE, which are activated in response to endoplasmic reticulum Ca(2+) store depletion and independently of Ca(2+) store depletion, respectively, in mast cells. Although Ca(2+) release-activated Ca(2+) channels are the major route of SOCE, recent evidence indicates that they are not the only Ca(2+) channels activated by Ca(2+) store depletion. The recent data suggest that L-type Ca(2+) channels, which were thought to be a characteristic feature of excitable cells, exist in mast cells to mediate non-SOCE, which is critical for protecting mast cells against activation-induced mitochondrial cell death. In this chapter, we provide an overview of recent advances in our understanding of Ca(2+) signaling in mast cells with a special attention to the emerging role for the L-type Ca(2+) channels as a regulator of mast cell survival.

  8. 5E- and 5Z-farnesylacetones from Sargassum siliquastrum as novel selective L-type calcium channel blockers.

    PubMed

    Shin, Woon-Seob; Oh, Sangtae; An, Sung-Wan; Park, Gab-Man; Kwon, Daeho; Ham, Jungyeob; Lee, Seokjoon; Park, Byong-Gon

    2013-04-01

    A specific blocker of L-type Ca(2+) channels may be useful in decreasing arterial tone by reducing the open-state probability of L-type Ca(2+) channels. The aim of the present study was to evaluate the farnesylacetones, which are major active constituents of Sargassum siliquastrum, regarding their vasodilatation efficacies, selectivities toward L-type Ca(2+) channels, and in vivo antihypertensive activities. The application of 5E-(farnesylacetone 311) or 5Z-farnesylacetone (farnesylacetone 312) induced concentration-dependent vasodilatation effects on the basilar artery that was pre-contracted with depolarization and showed an ignorable potential role of endothelial-derived nitric oxide. We also tested farnesylacetone 311 or 312 to determine their pharmacological profiles for the blockade of native L-type Ca(2+) channels in basilar arterial smooth muscle cells (BASMCs) and ventricular myocytes (VMCs), cloned L- (α1C/β2a/α2δ), N- (α1B/β1b/α2δ), and T-type Ca(2+) channels (α1G, α1H, and α1I). Farnesylacetone 311 or 312 showed greater selectivity toward the L-type Ca(2+) channels among the tested voltage-gated Ca(2+) channels. The ranked order of the potency for farnesylacetone 311 was cloned α1C≒L-type (BASMC)≒L-type (VMCs)>α1B>α1H>α1I>α1G and that for farnesylacetone 312 was cloned α1C≒L-type (BASMCs)≒L-type (VMCs)>α1H>α1G>α1B>α1I. The oral administration of the farnesylacetone 311 (80mg/kg) conferred potent, long-lasting antihypertensive activity in spontaneous hypertensive rats, but it did not alter the heart rate. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Cardiac L-type calcium current is increased in a model of hyperaldosteronism in the rat.

    PubMed

    Martin-Fernandez, Beatriz; Miana, María; De Las Heras, Natalia; Ruiz-Hurtado, Gema; Fernandez-Velasco, María; Bas, Manuel; Ballesteros, Sandra; Lahera, Vicente; Cachofeiro, Victoria; Delgado, Carmen

    2009-06-01

    Accumulating evidence supports the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease. It has been postulated that aldosterone could contribute to ventricular arrhythmogeneity by modulation of cardiac ionic channels. The aim of this study was to analyse ex vivo the electrophysiological characteristics of the L-type cardiac calcium current (I(CaL)) in a model of hyperaldosteronism in the rat. Aldosterone was administered for 3 weeks, and cardiac collagen deposition and haemodynamic parameters were analysed. In addition, RT-PCR and patch-clamp techniques were applied to study cardiac L-type Ca(2+) channels in isolated cardiomyocytes. Administration of aldosterone induced maladaptive cardiac remodelling that was related to increased collagen deposition, diastolic dysfunction and cardiac hypertrophy. In addition, ventricular myocytes isolated from the aldosterone-treated group showed increased I(CaL) density and conductance and prolongation of the action potential duration. No changes in kinetics or in voltage dependence of activation and inactivation of I(CaL) were observed, but relative expression of Ca(V)1.2 mRNA levels was higher in cardiomyocytes isolated from the aldosterone-treated group. The present study demonstrates that aldosterone treatment induces myocardial fibrosis, cardiac hypertrophy, increase of I(CaL) density, upregulation of L-type Ca(2+) channels and prolongation of action potential duration. It could be proposed that aldosterone, through these mechanisms, might exert pro-arrhythmic effects in the pathological heart.

  10. Hydrogen Sulfide Inhibits L-Type Calcium Currents Depending upon the Protein Sulfhydryl State in Rat Cardiomyocytes

    PubMed Central

    Tsai, Haojan; Tang, Chaoshu; Jin, Hongfang; Du, Junbao

    2012-01-01

    Hydrogen sulfide (H2S) is a novel gasotransmitter that inhibits L-type calcium currents (I Ca, L). However, the underlying molecular mechanisms are unclear. In particular, the targeting site in the L-type calcium channel where H2S functions remains unknown. The study was designed to investigate if the sulfhydryl group could be the possible targeting site in the L-type calcium channel in rat cardiomyocytes. Cardiac function was measured in isolated perfused rat hearts. The L-type calcium currents were recorded by using a whole cell voltage clamp technique on the isolated cardiomyocytes. The L-type calcium channel containing free sulfhydryl groups in H9C2 cells were measured by using Western blot. The results showed that sodium hydrosulfide (NaHS, an H2S donor) produced a negative inotropic effect on cardiac function, which could be partly inhibited by the oxidant sulfhydryl modifier diamide (DM). H2S donor inhibited the peak amplitude of I Ca, L in a concentration-dependent manner. However, dithiothreitol (DTT), a reducing sulfhydryl modifier markedly reversed the H2S donor-induced inhibition of I Ca, L in cardiomyocytes. In contrast, in the presence of DM, H2S donor could not alter cardiac function and L type calcium currents. After the isolated rat heart or the cardiomyocytes were treated with DTT, NaHS could markedly alter cardiac function and L-type calcium currents in cardiomyocytes. Furthermore, NaHS could decrease the functional free sulfhydryl group in the L-type Ca2+ channel, which could be reversed by thiol reductant, either DTT or reduced glutathione. Therefore, our results suggest that H2S might inhibit L-type calcium currents depending on the sulfhydryl group in rat cardiomyocytes. PMID:22590646

  11. Modifying L-type calcium current kinetics: consequences for cardiac excitation and arrhythmia dynamics.

    PubMed

    Mahajan, Aman; Sato, Daisuke; Shiferaw, Yohannes; Baher, Ali; Xie, Lai-Hua; Peralta, Robert; Olcese, Riccardo; Garfinkel, Alan; Qu, Zhilin; Weiss, James N

    2008-01-15

    The L-type Ca current (I(Ca,L)), essential for normal cardiac function, also regulates dynamic action potential (AP) properties that promote ventricular fibrillation. Blocking I(Ca,L) can prevent ventricular fibrillation, but only at levels suppressing contractility. We speculated that, instead of blocking I(Ca,L), modifying its shape by altering kinetic features could produce equivalent anti-fibrillatory effects without depressing contractility. To test this concept experimentally, we overexpressed a mutant Ca-insensitive calmodulin (CaM(1234)) in rabbit ventricular myocytes to inhibit Ca-dependent I(Ca,L) inactivation, combined with the ATP-sensitive K current agonist pinacidil or I(Ca,L) blocker verapamil to maintain AP duration (APD) near control levels. Cell shortening was enhanced in pinacidil-treated myocytes, but depressed in verapamil-treated myocytes. Both combinations flattened APD restitution slope and prevented APD alternans, similar to I(Ca,L) blockade. To predict the arrhythmogenic consequences, we simulated the cellular effects using a new AP model, which reproduced flattening of APD restitution slope and prevention of APD/Ca(i) transient alternans but maintained a normal Ca(i) transient. In simulated two-dimensional cardiac tissue, these changes prevented the arrhythmogenic spatially discordant APD/Ca(i) transient alternans and spiral wave breakup. These findings provide a proof-of-concept test that I(Ca,L) can be targeted to increase dynamic wave stability without depressing contractility, which may have promise as an antifibrillatory strategy.

  12. A potent 1,4-dihydropyridine L-type calcium channel blocker, benidipine, promotes osteoblast differentiation.

    PubMed

    Nishiya, Y; Kosaka, N; Uchii, M; Sugimoto, S

    2002-01-01

    During their differentiation, osteoblasts sequentially express type I collagen, alkaline phosphatase (ALP), and osteocalcin, and then undergo mineral deposition. Among dihydropyridine-type calcium channel blockers, only benidipine stimulated ALP activity of osteoblastic cells derived from neonatal mouse calvaria. To identify the molecular target of benidipine and elucidate the mechanism of action of the drug in osteoblasts, the mouse osteoblastic cell line MC3T3-E1 was used. Benidipine prompted ALP activity and ALP transcription induced by ascorbic acid, and mineral deposition by ascorbic acid and b-glycerophosphate. Benidipine, however, did not change collagen accumulation. MC3T3-E1 cells expressed the L-type Ca channel a1C subunit throughout the differentiation process, and Ca influx by potassium ions and Bay K 8644, an agonist, was strongly attenuated by benidipine. Each one of three structurally different classes of Ca channel blockers, nifedipine, verapamil, and diltiazem stimulated ALP activity, although at much higher concentrations of ca. 100 nM than benidipine, 1 pM. These results suggest that benidipine directly exerts its effect on osteoblasts and promotes osteoblast differentiation after the step of collagen accumulation by blocking the L-type Ca channel. Since benidipine blocked Ca influx more potently than the three other Ca channel blockers, the unique and potent osteoblast differentiating ability of benidipine may be due to its high affinity for Ca channel together with its high membrane retaining ability, as has been previously reported.

  13. Effect of resveratrol on L-type calcium current in rat ventricular myocytes.

    PubMed

    Zhang, Li-ping; Yin, Jing-xiang; Liu, Zheng; Zhang, Yi; Wang, Qing-shan; Zhao, Juan

    2006-02-01

    To study the effect of resveratrol on L-type calcium current (I(Ca-L)) in isolated rat ventricular myocytes and the mechanisms underlying these effects. I(Ca-L) was examined in isolated single rat ventricular myocytes by using the whole cell patch-clamp recording technique. Resveratrol (10-40 micromol/L) reduced the peak amplitude of I(Ca-L) and shifted the current-voltage (I-V) curve upwards in a concentration-dependent manner. Resveratrol (10, 20, 40 micromol/L) decreased the peak amplitude of I(Ca-L) from -14.2+/-1.5 pA/pF to -10.5+/-1.5 pA/pF (P<0.05), -7.5+/-2.4 pA/pF (P<0.01), and -5.2+/-1.2 pA/pF (P<0.01), respectively. Resveratrol (40 micromol/L) shifted the steady-state activation curve of I(Ca-L) to the right and changed the half-activation potential (V0.5) from -19.4+/-0.4 mV to -15.4+/-1.9 mV (P<0.05). Resveratrol at a concentration of 40 micromol/L did not affect the steady-state inactivation curve of I(Ca-L), but did markedly shift the time-dependent recovery curve of I(Ca-L) to the right, and slow down the recovery of I(Ca-L) from inactivation. Sodium orthovanadate (Na(3)VO(4); 1 mmol/L), a potent inhibitor of tyrosine phosphatase, significantly inhibited the effects of resveratrol (P<0.01). Resveratrol inhibited I(Ca-L) mainly by inhibiting the activation of L-type calcium channels and slowing down the recovery of L-type calcium channels from inactivation. This inhibitory effect of resveratrol was mediated by the inhibition of protein tyrosine kinase in rat ventricular myocytes.

  14. Lateral mobility of L-type calcium channels in synaptic terminals of retinal bipolar cells.

    PubMed

    Thoreson, Wallace B; Mercer, Aaron J; Cork, Karlene M; Szalewski, Robert J

    2013-01-01

    Efficient and precise release of glutamate from retinal bipolar cells is ensured by the positioning of L-type Ca(2+) channels close to release sites at the base of the synaptic ribbon. We investigated whether Ca(2+) channels at bipolar cell ribbon synapses are fixed in position or capable of moving in the membrane. We tracked the movements of individual L-type Ca(2+) channels in bipolar cell terminals after labeling channels with quantum dots (QDs) attached to α(2)δ(4) accessory Ca(2+) channel subunits via intermediary antibodies. We found that individual Ca(2+) channels moved within a confined domain of 0.13-0.15 μm(2) in bipolar cell terminals, similar to ultrastructural estimates of the surface area of the active zone beneath the ribbon. Disruption of actin expanded the confinement domain indicating that cytoskeletal interactions help to confine channels at the synapse, but the relatively large diffusion coefficients of 0.3-0.45 μm(2)/s suggest that channels are not directly anchored to actin. Unlike photoreceptor synapses, removing membrane cholesterol did not change domain size, indicating that lipid rafts are not required to confine Ca(2+) channels at bipolar cell ribbon synapses. The ability of Ca(2+) channels to move within the presynaptic active zone suggests that regulating channel mobility may affect release from bipolar cell terminals.

  15. L-type calcium channels in exocytosis and endocytosis of chromaffin cells.

    PubMed

    Nanclares, Carmen; Baraibar, Andrés M; Gandía, Luis

    2017-09-02

    The coexistence of different subtypes of voltage-dependent calcium channels (VDCC) within the same chromaffin cell (CC) and the marked interspecies variability in the proportion of VDCC subtypes that are present in the plasmalemma of the CCs raises the question on their roles in controlling different physiological functions. Particularly relevant seems to be the role of VDCCs in the regulation of the exocytotic neurotransmitter release process, and its tightly coupled membrane retrieval (endocytosis) process since both are Ca(2+)-dependent processes. This review is focused on the role of Ca(2+) influx through L-type VDCC in the regulation of these two processes. It is currently accepted that the different VDCC subtypes (i.e., T, L, N, P/Q, R) contribute to exocytosis proportionally to their density of expression and gating properties. However, the pattern of stimulation defines a preferential role of the different subtypes of VDCC on exocytosis and endocytosis. Thus, L-type channels seem to control catecholamine release induced by prolonged stimuli while fast exocytosis in response to short square depolarizing pulses or action potentials is mediated by Ca(2+) entering CCs through P/Q channels. The pattern of stimulation also influences the endocytotic process, and thus, electrophysiological data suggest the sustained Ca(2+) entry through slow-inactivating L-type channels could be responsible for the activation of fast endocytosis.

  16. Contribution of downregulation of L-type calcium currents to delayed neuronal death in rat hippocampus after global cerebral ischemia and reperfusion.

    PubMed

    Li, Xiao-Ming; Yang, Jian-Ming; Hu, De-Hui; Hou, Feng-Qing; Zhao, Miao; Zhu, Xin-Hong; Wang, Ying; Li, Jian-Guo; Hu, Ping; Chen, Liang; Qin, Lu-Ning; Gao, Tian-Ming

    2007-05-09

    Transient forebrain ischemia induces delayed, selective neuronal death in the CA1 region of the hippocampus. The underlying molecular mechanisms are as yet unclear, but it is known that activation of L-type Ca2+ channels specifically increases the expression of a group of genes required for neuronal survival. Accordingly, we examined temporal changes in L-type calcium-channel activity in CA1 and CA3 pyramidal neurons of rat hippocampus after transient forebrain ischemia by patch-clamp techniques. In vulnerable CA1 neurons, L-type Ca2+-channel activity was persistently downregulated after ischemic insult, whereas in invulnerable CA3 neurons, no change occurred. Downregulation of L-type calcium channels was partially caused by oxidation modulation in postischemic channels. Furthermore, L-type but neither N-type nor P/Q-type Ca2+-channel antagonists alone significantly inhibited the survival of cultured hippocampal neurons. In contrast, specific L-type calcium-channel agonist remarkably reduced neuronal cell death and restored the inhibited channels induced by nitric oxide donor. More importantly, L-type calcium-channel agonist applied after reoxygenation or reperfusion significantly decreased neuronal injury in in vitro oxygen-glucose deprivation ischemic model and in animals subjected to forebrain ischemia-reperfusion. Together, the present results suggest that ischemia-induced inhibition of L-type calcium currents may give rise to delayed death of neurons in the CA1 region, possibly via oxidation mechanisms. Our findings may lead to a new perspective on neuronal death after ischemic insult and suggest that a novel therapeutic approach, activation of L-type calcium channels, could be tested at late stages of reperfusion for stroke treatment.

  17. Phospholemman Modulates the Gating of Cardiac L-Type Calcium Channels

    PubMed Central

    Wang, Xianming; Gao, Guofeng; Guo, Kai; Yarotskyy, Viktor; Huang, Congxin; Elmslie, Keith S.; Peterson, Blaise Z.

    2010-01-01

    Ca2+ entry through L-type calcium channels (CaV1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of CaV1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with CaV1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed CaV1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca2+-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca2+ influx via CaV1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of CaV1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca2+ overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca2+ influx during the cardiac action potential waveform plateau phase. PMID:20371314

  18. Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

    PubMed Central

    Bardy, G; Virsolvy, A; Quignard, J F; Ravier, M A; Bertrand, G; Dalle, S; Cros, G; Magous, R; Richard, S; Oiry, C

    2013-01-01

    Background and Purpose Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca2+]i) in beta cells, in the absence of any co-stimulating factor. Experimental Approach Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca2+]i were measured using the ratiometric fluorescent Ca2+ indicator Fura-2. Ca2+ channel currents were recorded with the whole-cell patch-clamp technique. Key Results Quercetin concentration-dependently increased insulin secretion and elevated [Ca2+]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L−1), but were nearly abolished by the L-type Ca2+ channel antagonist nifedipine (1 μmol·L−1). Similar to the L-type Ca2+ channel agonist Bay K 8644, quercetin enhanced the L-type Ca2+ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca2+]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L−1), with the two drugs having cumulative effects on [Ca2+]i. Conclusions and Implications Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca2+ influx through an interaction with L-type Ca2+ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion. PMID:23530660

  19. Inactivation of Gating Currents of L-Type Calcium Channels

    PubMed Central

    Shirokov, Roman; Ferreira, Gonzalo; Yi, Jianxun; Ríos, Eduardo

    1998-01-01

    In studies of gating currents of rabbit cardiac Ca channels expressed as α1C/β2a or α1C/β2a/α2δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V1/2 ≃ −90 mV) is generated in inactivated channels. In α1C/β2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α2δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α2δ subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α2δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode. PMID:9607938

  20. Comparative analysis of the kinetic characteristics of L-type calcium channels in cardiac cells of hibernators.

    PubMed Central

    Alekseev, A E; Markevich, N I; Korystova, A F; Terzic, A; Kokoz, Y M

    1996-01-01

    An undefined property of L-type Ca2+ channels is believed to underlie the unique phenotype of hibernating hearts. Therefore, L-type Ca2+ channels in single cardiomyocytes isolated from hibernating versus awake ground-squirrels (Citellus undulatus) were compared using the perforated mode of the patch-clamp technique, and interpreted by way of a kinetic model of Ca2+ channel behavior based upon the concept of independence of the activation and inactivation processes. We find that, in hibernating ground-squirrels, the cardiac L-type Ca2+ current is lower in magnitude when compared to awake animals. Both in the awake or hibernating states, kinetics of L-type Ca2+ channels could be described by a d2f1(2)f2 model with an activation and two inactivation processes. The activation (or d) process relates to the movement of the gating charge. The slow (or f1) inactivation is associated with movement of gating charge and is current-dependent. The rapid (or f2) inactivation is a complex process which cannot be represented as a single-step conformational transition induced by the gating charge movement, and is regulated by beta-adrenoceptor stimulation. When compared to awake animals, the kinetic properties of Ca2+ channels from hibernating ground-squirrels differed in the following parameters: (1) pronounced shift (15-20 mV) toward depolarization in the normalized conductance of both inactivation components, and moderate shift in the activation component; (2) 1.5-2-fold greater time constants; and (3) two-fold greater activation gating charge. Thus, L-type Ca2+ channels apparently switch their phenotype during the hibernating transition. Stimulation of beta-adrenoceptors by isoproterenol, reversed the hibernating kinetic- (but not amplitude-) phenotype toward the awake type. Therefore, an aberrance in the beta-adrenergic system can not fully explain the observed changes in the L-type Ca2+ current. This suggests that during hibernation additional mechanisms may reduce the single

  1. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    PubMed

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses.

  2. Myoscape controls cardiac calcium cycling and contractility via regulation of L-type calcium channel surface expression.

    PubMed

    Eden, Matthias; Meder, Benjamin; Völkers, Mirko; Poomvanicha, Montatip; Domes, Katrin; Branchereau, M; Marck, P; Will, Rainer; Bernt, Alexander; Rangrez, Ashraf; Busch, Matthias; Hrabě de Angelis, Martin; Heymes, Christophe; Rottbauer, Wolfgang; Most, Patrick; Hofmann, Franz; Frey, Norbert

    2016-04-28

    Calcium signalling plays a critical role in the pathogenesis of heart failure. Here we describe a cardiac protein named Myoscape/FAM40B/STRIP2, which directly interacts with the L-type calcium channel. Knockdown of Myoscape in cardiomyocytes decreases calcium transients associated with smaller Ca(2+) amplitudes and a lower diastolic Ca(2+) content. Likewise, L-type calcium channel currents are significantly diminished on Myoscape ablation, and downregulation of Myoscape significantly reduces contractility of cardiomyocytes. Conversely, overexpression of Myoscape increases global Ca(2+) transients and enhances L-type Ca(2+) channel currents, and is sufficient to restore decreased currents in failing cardiomyocytes. In vivo, both Myoscape-depleted morphant zebrafish and Myoscape knockout (KO) mice display impairment of cardiac function progressing to advanced heart failure. Mechanistically, Myoscape-deficient mice show reduced L-type Ca(2+)currents, cell capacity and calcium current densities as a result of diminished LTCC surface expression. Finally, Myoscape expression is reduced in hearts from patients suffering of terminal heart failure, implying a role in human disease.

  3. Lateral Mobility of Presynaptic L-Type Calcium Channels at Photoreceptor Ribbon Synapses

    PubMed Central

    Mercer, Aaron J.; Chen, Minghui; Thoreson, Wallace B.

    2011-01-01

    At most synapses, presynaptic Ca2+ channels are positioned near vesicle release sites, and increasing this distance reduces synaptic strength. We examined the lateral membrane mobility of presynaptic L-type Ca2+ channels at photoreceptor ribbon synapses of the tiger salamander (Ambystoma tigrinum) retina. Movements of individual Ca2+ channels were tracked by coupling quantum dots to an antibody against the extracellular α2δ4 Ca2+ channel subunit. α2δ4 antibodies labeled photoreceptor terminals and co-localized with antibodies to synaptic vesicle protein SV2 and Ca2+ channel CaV1.4 α1 subunits. The results show that Ca2+ channels are dynamic and move within a confined region beneath the synaptic ribbon. The size of this confinement area is regulated by actin and membrane cholesterol. Fusion of nearby synaptic vesicles caused jumps in Ca2+ channel position, propelling them towards the outer edge of the confinement domain. Channels rebounded rapidly towards the center. Thus, although CaV channels are mobile, molecular scaffolds confine them beneath the ribbon to maintain neurotransmission even at high release rates. PMID:21430141

  4. AKAP-Anchored PKA Maintains Neuronal L-type Calcium Channel Activity and NFAT Transcriptional Signaling

    PubMed Central

    Murphy, Jonathan G.; Sanderson, Jennifer L.; Gorski, Jessica A.; Scott, John D.; Catterall, William A.; Sather, William A.; Dell’Acqua, Mark L.

    2014-01-01

    Summary In neurons, Ca2+ influx through L-type voltage-gated Ca2+ channels (LTCC) couples electrical activity to changes in transcription. LTCC activity is elevated by the cAMP-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), with both enzymes localized to the channel by A-kinase anchoring protein (AKAP) 79/150. AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T-cells (NFAT). We report here that genetic disruption of PKA anchoring to AKAP79/150 also interferes with LTCC activation of CaN-NFAT signaling in neurons. Disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Our findings support a model wherein basal activity of AKAP79/150-anchored PKA opposes CaN to preserve LTCC phosphorylation, thereby sustaining LTCC activation of CaN-NFAT signaling to the neuronal nucleus. PMID:24835999

  5. Effect of propionyl-L-carnitine on L-type calcium channels in human heart sarcolemma

    SciTech Connect

    Bevilacqua, M.; Vago, T.; Norbiato, G. )

    1991-02-01

    Propionyl-L-carnitine (PC) protects perfused rat hearts against damage by ischemia-reperfusion. Activation of L-type calcium channel play a role on ischemia-reperfusion damage. Therefore, we studied the effect of PC on some properties of L-type calcium channels in an in vitro preparation from human myocardium sarcolemma (from patients with idiopathic dilated cardiomyopathy). Binding of the L-type calcium channel blockers isradipine ({sup 3}H)-PN 200-110 (PN) to plasma membrane preparations revealed a single population of binding sites (total number: Bmax = 213 +/- 34 fM/mg protein and affinity: Kd = 152 +/- 19 nM; n = 6). The characteristics of these binding sites were evaluated in the presence and in the absence of Ca{sup 2}{sup +} and of calcium blockers (D-888, a verapamillike drug, and diltiazem). Incubation in a Ca{sup 2}{sup +}-containing buffer increased the affinity of PN binding sites. Binding sites for PN were modulated by organic calcium channel blockers; in competition isotherms at 37{degree}C, D-888 (desmethoxyverapamil) decreased the PN binding, whereas diltiazem increased it. These results strongly suggest that the site labelled by PN is the voltage-operated calcium channel of the human myocardium. The addition of PC (1 mM) to plasma membranes labelled with PN at 37{degree}C decreased the affinity of the binding; this effect was counteracted by the addition of Ca{sup 2}{sup +} to the medium. This result was consistent with a competition between Ca{sup 2}{sup +} and PC. The effect of PC incubation at 4{degree}C was the opposite; at this temperature PC increased the affinity of the binding sites and the effect was obscured by Ca{sup 2}{sup +}.

  6. AKAP-anchored PKA maintains neuronal L-type calcium channel activity and NFAT transcriptional signaling.

    PubMed

    Murphy, Jonathan G; Sanderson, Jennifer L; Gorski, Jessica A; Scott, John D; Catterall, William A; Sather, William A; Dell'Acqua, Mark L

    2014-06-12

    L-type voltage-gated Ca2+ channels (LTCC) couple neuronal excitation to gene transcription. LTCC activity is elevated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), and both enzymes are localized to the channel by A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T cells (NFAT). We report here that the basal activity of AKAP79/150-anchored PKA maintains neuronal LTCC coupling to CaN-NFAT signaling by preserving LTCC phosphorylation in opposition to anchored CaN. Genetic disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of postsynaptic dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Thus, LTCC-NFAT transcriptional signaling in neurons requires precise organization and balancing of PKA and CaN activities in the channel nanoenvironment, which is only made possible by AKAP79/150 scaffolding.

  7. Regulation of L-type calcium channel by phospholemman in cardiac myocytes.

    PubMed

    Zhang, Xue-Qian; Wang, JuFang; Song, Jianliang; Rabinowitz, Joseph; Chen, Xiongwen; Houser, Steven R; Peterson, Blaise Z; Tucker, Amy L; Feldman, Arthur M; Cheung, Joseph Y

    2015-07-01

    We evaluated whether phospholemman (PLM) regulates L-type Ca(2+) current (ICa) in mouse ventricular myocytes. Expression of α1-subunit of L-type Ca(2+) channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak ICa (at -10 mV) from KO myocytes was ~41% larger, the inactivation time constant (τ(inact)) of ICa was ~39% longer, but deactivation time constant (τ(deact)) was similar. In the presence of isoproterenol (1 μM), peak ICa was ~48% larger and τ(inact) was ~144% higher in KO myocytes. With Ba(2+) as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τ(deact). To dissect the molecular determinants by which PLM regulated ICa, we expressed PLM mutants by adenovirus-mediated gene transfer in cultured KO myocytes. After 24h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak ICa and longer τ(inact) than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylatable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak ICa. Expressing PLM mutants did not alter expression of α1-subunit of L-type Ca(2+) channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak ICa amplitude. We conclude that PLM limits Ca(2+) influx in cardiac myocytes by reducing maximal ICa and accelerating voltage-dependent inactivation.

  8. Regulation of L-type calcium channel by phospholemman in cardiac myocytes

    PubMed Central

    Zhang, Xue-Qian; Wang, JuFang; Song, Jianliang; Rabinowitz, Joseph; Chen, Xiongwen; Houser, Steven R.; Peterson, Blaise Z.; Tucker, Amy L.; Feldman, Arthur M.; Cheung, Joseph Y.

    2015-01-01

    We evaluated whether phospholemman (PLM) regulates L-type Ca2+ current (ICa) in mouse ventricular myocytes. Expression of α1-subunit of L-type Ca2+ channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak ICa (at −10 mV) from KO myocytes was ~41% larger, the inactivation time constant (τinact) of ICa was ~39% longer, but deactivation time constant (τdeact) was similar. In the presence of isoproterenol (1 µM), peak ICa was ~48% larger and τinact was ~144% higher in KO myocytes. With Ba2+ as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τdeact. To dissect the molecular determinants by which PLM regulated ICa, we expressed PLM mutants by adenovirus- mediated gene transfer in cultured KO myocytes. After 24 h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak ICa and longer τinact than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak ICa. Expressing PLM mutants did not alter expression of α1-subunit of L-type Ca2+ channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak ICa amplitude. We conclude that PLM limits Ca2+ influx in cardiac myocytes by reducing maximal ICa and accelerating voltage-dependent inactivation. PMID:25918050

  9. Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance.

    PubMed Central

    Guia, A; Stern, M D; Lakatta, E G; Josephson, I R

    2001-01-01

    Little is known about the native properties of unitary cardiac L-type calcium currents (i(Ca)) measured with physiological calcium (Ca) ion concentration, and their role in excitation-contraction (E-C) coupling. Our goal was to chart the concentration-dependence of unitary conductance (gamma) to physiological Ca concentration and compare it to barium ion (Ba) conductance in the absence of agonists. In isolated, K-depolarized rat myocytes, i(Ca) amplitudes were measured using cell-attached patches with 2 to 70 mM Ca or 2 to 105 mM Ba in the pipette. At 0 mV, 2 mM of Ca produced 0.12 pA, and 2 mM of Ba produced 0.19 pA unitary currents. Unitary conductance was described by a Langmuir isotherm relationship with a maximum gammaCa of 5.3 +/- 0.2 pS (n = 15), and gammaBa of 15 +/- 1 pS (n = 27). The concentration producing half-maximal gamma, Kd(gamma), was not different between Ca (1.7 +/- 0.3 mM) and Ba (1.9 +/- 0.4 mM). We found that quasi-physiological concentrations of Ca produced currents that were as easily resolvable as those obtained with the traditionally used higher concentrations. This study leads to future work on the molecular basis of E-C coupling with a physiological concentration of Ca ions permeating the Ca channel. PMID:11371449

  10. Melanocortin 4 receptor constitutive activity inhibits L-type voltage-gated calcium channels in neurons.

    PubMed

    Agosti, F; Cordisco Gonzalez, S; Martinez Damonte, V; Tolosa, M J; Di Siervi, N; Schioth, H B; Davio, C; Perello, M; Raingo, J

    2017-03-27

    The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain nuclei playing a crucial role in the regulation of energy balance controlling the homeostasis of the organism. It displays both agonist-evoked and constitutive activity, and moreover, it can couple to different G proteins. Most of the research on MC4R has been focused on agonist-induced activity, while the molecular and cellular basis of MC4R constitutive activity remains scarcely studied. We have previously shown that neuronal N-type voltage-gated calcium channels (CaV2.2) are inhibited by MC4R agonist-dependent activation, while the CaV subtypes that carry L- and P/Q-type current are not. Here, we tested the hypothesis that MC4R constitutive activity can affect CaV, with focus on the channel subtypes that can control transcriptional activity coupled to depolarization (L-type, CaV1.2/1.3) and neurotransmitter release (N- and P/Q-type, CaV2.2 and CaV2.1). We found that MC4R constitutive activity inhibits specifically CaV1.2/1.3 and CaV2.1 subtypes of CaV. We also explored the signaling pathways mediating this inhibition, and thus propose that agonist-dependent and basal MC4R activation modes signal differentially through Gs and Gi/o pathways to impact on different CaV subtypes. In addition, we found that chronic incubation with MC4R endogenous inverse agonist, agouti and agouti-related peptide (AgRP), occludes CaV inhibition in a cell line and in amygdaloid complex cultured neurons as well. Thus, we define new mechanisms of control of the main mediators of depolarization-induced calcium entry into neurons by a GPCR that displays constitutive activity.

  11. Expression and 1,4-dihydropyridine-binding properties of brain L-type calcium channel isoforms.

    PubMed

    Sinnegger-Brauns, Martina J; Huber, Irene G; Koschak, Alexandra; Wild, Claudia; Obermair, Gerald J; Einzinger, Ursula; Hoda, Jean-Charles; Sartori, Simone B; Striessnig, Jörg

    2009-02-01

    The L-type calcium channel (LTCC) isoforms Ca(v)1.2 and Ca(v)1.3 display similar 1,4-dihydropyridine (DHP) binding properties and are both expressed in mammalian brain. Recent work implicates Ca(v)1.3 channels as interesting drug targets, but no isoform-selective modulators exist. It is also unknown to what extent Ca(v)1.1 and Ca(v)1.4 contribute to L-type-specific DHP binding activity in brain. To address this question and to determine whether DHPs can discriminate between Ca(v)1.2 and Ca(v)1.3 binding pockets, we combined radioreceptor assays and quantitative polymerase chain reaction (qPCR). We bred double mutants (Ca(v)-DM) from mice expressing mutant Ca(v)1.2 channels [Ca(v)1.2DHP(-/-)] lacking high affinity for DHPs and from Ca(v)1.3 knockouts [Ca(v)1.3(-/-)]. (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) and Ca(v)-DM brains was reduced to 15.1 and 4.4% of wild type, respectively, indicating that Ca(v)1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Ca(v)1.1 and Ca(v)1.4 alpha(1) subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low-affinity binding (127-fold K(d) increase) to the mutated Ca(v)1.2 channels. Inhibition of (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) (predominantly Ca(v)1.3) and wild-type (predominantly Ca(v)1.2) brain membranes by unlabeled DHPs revealed a 3- to 4-fold selectivity of nitrendipine and nifedipine for the Ca(v)1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, because of their very low expression, Ca(v)1.1 and Ca(v)1.4 are unlikely to serve as drug targets to treat CNS diseases.

  12. L-Type Calcium Channels Are Required for One Form of Hippocampal Mossy Fiber LTP

    PubMed Central

    Kapur, Ajay; Yeckel, Mark F.; Gray, Richard; Johnston, Daniel

    2010-01-01

    The requirement of postsynaptic calcium influx via L-type channels for the induction of long-term potentiation (LTP) of mossy fiber input to CA3 pyramidal neurons was tested for two different patterns of stimulation. Two types of LTP-inducing stimuli were used based on the suggestion that one of them, brief high-frequency stimulation (B-HFS), induces LTP postsynaptically, whereas the other pattern, long high-frequency stimulation (L-HFS), induces mossy fiber LTP presynaptically. To test whether or not calcium influx into CA3 pyramidal neurons is necessary for LTP induced by either pattern of stimulation, nimodipine, a L-type calcium channel antagonist, was added during stimulation. In these experiments nimodipine blocked the induction of mossy fiber LTP when B-HFS was given [34 ± 5% (mean ± SE) increase in control versus 7 ± 4% in nimodipine, P < 0.003]; in contrast, nimodipine did not block the induction of LTP with L-HFS (107 ± 10% in control vs. 80 ± 9% in nimodipine, P > 0.05). Administration of nimodipine after the induction of LTP had no effect on the expression of LTP. In addition, B- and L-HFS delivered directly to commissural/ associational fibers in stratum radiatum failed to induce a N-methyl-d-aspartate-independent form of LTP, obviating the possibility that the presumed mossy fiber LTP resulted from potentiation of other synapses. Nimodipine had no effect on calcium transients recorded from mossy fiber presynaptic terminals evoked with the B-HFS paradigm but reduced postsynaptic calcium transients. Our results support the hypothesis that induction of mossy fiber LTP by B-HFS is mediated postsynaptically and requires entry of calcium through L-type channels into CA3 neurons. PMID:9535977

  13. Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.

    PubMed

    de Weille, J R; Schweitz, H; Maes, P; Tartar, A; Lazdunski, M

    1991-03-15

    The venom of the black mamba contains a 60-amino acid peptide called calciseptine. The peptide has been fully sequenced. It is a smooth muscle relaxant and an inhibitor of cardiac contractions. Its physiological action resembles that of drugs, such as the 1,4-dihydropyridines, which are important in the treatment of cardiovascular diseases. Calciseptine, like the 1,4-dihydropyridines, selectively blocks L-type Ca2+ channels and is totally inactive on other voltage-dependent Ca2+ channels such as N-type and T-type channels. To our knowledge, it is the only natural polypeptide that has been shown to be a specific inhibitor of L-type Ca2+ channels.

  14. Novel 1, 4-dihydropyridines for L-type calcium channel as antagonists for cadmium toxicity

    PubMed Central

    Saddala, Madhu Sudhana; Kandimalla, Ramesh; Adi, Pradeepkiran Jangampalli; Bhashyam, Sainath Sri; Asupatri, Usha Rani

    2017-01-01

    The present study, we design and synthesize the novel dihydropyridine derivatives, i.e., 3 (a-e) and 5 (a-e) and evaluated, anticonvulsant activity. Initially due to the lacuna of LCC, we modeled the protein through modeller 9.15v and evaluated through servers. Docking studies were performed with the synthesized compounds and resulted two best compounds, i.e., 5a, 5e showed the best binding energies. The activity of intracellular Ca2+ measurements was performed on two cell lines: A7r5 (rat aortic smooth muscle cells) and SH-SY5Y (human neuroblastoma cells). The 5a and 5e compounds was showing the more specific activity on L-type calcium channels, i.e. A7r5 (IC50 = 0.18 ± 0.02 and 0.25 ± 0.63 μg/ml, respectively) (containing only L-type channels) than SH-SY5Y (i.e. both L-type and T-type channels) (IC50 = 8 ± 0.23 and 10 ± 0.18 μg/ml, respectively) with intracellular calcium mobility similar to amlodipine. Finally, both in silico and in vitro results exploring two derivatives 5a and 5e succeeded to treat cadmium toxicity. PMID:28345598

  15. Effect of shenmai injection on L-type calcium current of diaphragmatic muscle in rats.

    PubMed

    Zhao, Limin; Xiong, Shengdao; Niu, Ruji; Xu, Yongjian; Zhang, Zhengxiang

    2004-01-01

    In this study, whole cell patch clamp recording technique was employed to investigate the effect of Shenmai Injection (SMI) on L-type calcium current of diaphragmatic muscle in rats. The result showed that when the diaphragmatic muscle cell was held at -80 mV and depolarized to +60 mV, 10 microl/ml, 50 microl/ml and 100 microl/ml SMI enhanced the inner peak L-type calcium current from -(6.8 +/- 0.7) pA/pF (n=7) to -(7.3 +/- 0.8) pA/pF (P>0.05, n=7), -(8.6 +/- 1.0) pA/pF (P<0.05, n=7) and -(9.4 +/- 1.2) pA/pF (P<0.05, n=7), respectively, The rates of L-type calcium current were increased by (7.34 +/- 2.37)%, (25.72 +/- 5.94)%, and (38.16 +/- 7.33)%, respectively. However, it had no significant effect on maximal activation potential and reversal potential. Our results suggested that SMI could activate the calcium channel of the diaphragmatic fibers of the rats, increase the influx of Ca2+, and enhance the contractility of diaphragmatic muscles.

  16. A role for L-type calcium channels in the maturation of parvalbumin-containing hippocampal interneurons.

    PubMed

    Jiang, M; Swann, J W

    2005-01-01

    While inhibitory interneurons are well recognized to play critical roles in the brain, relatively little is know about the molecular events that regulate their growth and differentiation. Calcium ions are thought to be important in neuronal development and L-type voltage gated Ca(+2) channels have been implicated in activity-dependent mechanisms of early-life. However, few studies have examined the role of these channels in the maturation of interneurons. The studies reported here were conducted in hippocampal slice cultures and indicate that the L-type Ca(+2) channel agonists and antagonists accelerate and suppress respectively the growth of parvalbumin-containing interneurons. The effects of channel blockade were reversible suggesting they are not the result of interneuronal cell death. Results from immunoblotting showed that these drugs have similar effects on the expression of the GABA synthetic enzymes, glutamic acid decarboxylase65, glutamic acid decarboxylase67 and the vesicular GABA transporter. This suggests that L-type Ca(+2) channels regulate not only parvalbumin expression but also interneuron development. These effects are likely mediated by actions on the interneurons themselves since the alpha subunits of L-type channels, voltage-gated calcium channel subunit 1.2 and voltage-gated calcium channel subunit 1.3 were found to be highly expressed in neonatal mouse hippocampus and co-localized with parvalbumin in interneurons. Results also showed that while these interneurons can contain either subunit, voltage-gated calcium channel subunit 1.3 was more widely expressed. Taken together results suggest that an important subset of developing interneurons expresses L-type Ca(+2) channels alpha subunits, voltage-gated calcium channel subunit 1.2 and especially voltage-gated calcium channel subunit 1.3 and that these channels likely regulate the development of these interneurons in an activity-dependent manner.

  17. L-type calcium channel blockers enhance 5-HTP-induced antinociception in mice.

    PubMed

    Liang, Jian-hui; Li, Jun-xu; Wang, Xu-hua; Chen, Bi; Lu, Ying; Zhang, Pan; Han, Rong; Ye, Xiang-feng

    2004-05-01

    To investigate the involvement of L-type Ca(2+) channels in antinociceptive action induced by the 5-HT precursor, 5-hydroxytryptophan (5-HTP). Female Kunming mice were treated with either 5-HTP (20-80 mg/kg, ip) alone, or the combination of 5-HTP and fluoxetine (2-8 mg/kg, ip), pargyline (15-60 mg/kg, ip), nimodipine (2.5-10 mg/kg, ip), nifedipine (2.5-10 mg/kg, ip), verapamil (2.5-10 mg/kg, ip), CaCl(2) (5-20 mmol/L, icv), or EGTA (0.5-3 mmol/L, icv) prior to the hot-plate test (55 degree, hind-paw licking latency). In addition, locomotor activity in mice treated with 5-HTP alone was measured using an ambulometer with five activity boxes. Ip injection of 5-HTP alone had no influence on the spontaneous locomotor activity, whereas dose-dependently increased the latency to licking hind-paw in the hot-plate test in mice. The inhibitory effects of 5-HTP on nociceptive response were significantly enhanced by fluoxetine in the mouse hot-plate test. At a sub-effective dose, pargyline could cause a leftward shift in the dose-response curve of 5-HTP-induced antinociception. Co-administration with 5-HTP and nimodipine, nifedipine, or verapamil obviously potentiated the antinociceptive effects elicited by 5-HTP. Interestingly, 5-HTP-induced antinociception was antagonized by CaCl(2) and enhanced by EGTA injected icv in the mouse hot-plate test. These findings suggest that systemic administration of 5-HTP may yield the antinociceptive effects, which are related to Ca(2+) influx from extracellular fluid through L-type Ca(2+) channels.

  18. A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes.

    PubMed

    Galvis-Pareja, David; Zapata-Torres, Gerald; Hidalgo, Jorge; Ayala, Pedro; Pedrozo, Zully; Ibarra, Cristián; Diaz-Araya, Guillermo; Hall, Andrew R; Vicencio, Jose Miguel; Nuñez-Vergara, Luis; Lavandero, Sergio

    2014-08-15

    Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca(2+) channels and their renowned antioxidant properties. We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca(2+) channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca(2+) channel-blocking activity and antioxidant properties. The Ca(2+) channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca(2+) channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca(2+) channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Broad-spectrum antiemetic efficacy of the L-type calcium channel blocker amlodipine in the least shrew (Cryptotis parva).

    PubMed

    Zhong, Weixia; Chebolu, Seetha; Darmani, Nissar A

    2014-05-01

    The dihydropyridine l-type calcium (Ca(2+)) channel blockers nifedipine and amlodipine reduce extracellular Ca(2+) entry into cells. They are widely used for the treatment of hypertensive disorders. We have recently demonstrated that extracellular Ca(2+) entry via l-type Ca(2+) channels is involved in emesis and that nifedipine has broad-spectrum antiemetic activity. The aim of this study was to evaluate the antiemetic efficacy of the longer-acting l-type Ca(2+) channel blocker, amlodipine. Fully effective emetic doses of diverse emetogens such as the l-type Ca(2+) channel agonist (FPL 64176) as well as selective and/or nonselective agonists of serotonergic 5-HT3 (e.g. 5-HT or 2-Me-5-HT)-, dopamine D2 (e.g. apomorphine or quinpirole)-, cholinergic M1 (e.g. pilocarpine or McN-A343)- and tachykininergic NK1 (e.g. GR73632)-receptors, were administered intraperitoneally (i.p.) in the least shrew to induce vomiting. The broad-spectrum antiemetic potential of amlodipine was evaluated against these emetogens. Subcutaneous (s.c.) administration of amlodipine (0.5-10mg/kg) attenuated in a dose-dependent and potent manner both the frequency and percentage of shrews vomiting in response to intraperitoneal (i.p.) administration of FPL 64176 (10mg/kg), 5-HT (5mg/kg), 2-Me-5-HT (5mg/kg), apomorphine (2mg/kg), quinpirole (2mg/kg), pilocarpine (2mg/kg), McN-A343 (2mg/kg), or GR73632 (5mg/kg). A combination of non-effective doses of amlodipine (0.5mg/kg, s.c.) and the 5-HT3 receptor antagonist palonosetron (0.05 mg/kg, s.c.) was more effective against FPL 64176-induced vomiting than their corresponding doses tested alone. Amlodipine by itself suppressed the frequency of acute cisplatin (10mg/kg, i.p)-induced vomiting in a dose-dependent manner. Moreover, a combination of a non-effective dose of amlodipine (1mg/kg) potentiated the antiemetic efficacy of a semi-effective dose of palonosetron (0.5mg/kg, s.c.) against acute vomiting caused by cisplatin. We confirm that influx of

  20. Mapping of dihydropyridine binding residues in a less sensitive invertebrate L-type calcium channel (LCa v 1).

    PubMed

    Senatore, Adriano; Boone, Adrienne; Lam, Stanley; Dawson, Taylor F; Zhorov, Boris; Spafford, J David

    2011-01-01

    Invertebrate L-type calcium channel, LCa(v) 1, isolated from the pond snail Lymnaea stagnalis is nearly indistinguishable from mammalian Ca(v) 1.2 (α1C) calcium channel in biophysical characteristics observed in vitro. These L-type channels are likely constrained within a narrow range of biophysical parameters to perform similar functions in the snail and mammalian cardiovascular systems. What distinguishes snail and mammalian L-type channels is a difference in dihydropyridine sensitivity: 100 nM isradipine exhibits a significant block of mammalian Ca(v) 1.2 currents without effect on snail LCa(v)1 currents. The native snail channel serves as a valuable surrogate for validating key residue differences identified from previous experimental and molecular modeling work. As predicted, three residue changes in LCa(v)1 (N_3o18, F_3i10, and I_4i12) replaced with DHP-sensing residues in respective positions of Ca(v) 1.2, (Q_3o18, Y_3i10, and M_4i12) raises the potency of isradipine block of LCa(v)1 channels to that of mammalian Ca(v) 1.2. Interestingly, the single N_3o18_Q mutation in LCa(v) 1 channels lowers DHP sensitivity even further and the triple mutation bearing enhanced isradipine sensitivity, still retains a reduced potency of agonist, (S)-Bay K8644.

  1. Long term regulation of cardiac L-type calcium channel by small G proteins.

    PubMed

    Magyar, J; Jenes, A; Kistamás, K; Ruzsnavszky, F; Nánási, P P; Satin, J; Szentandrássy, N; Bányász, T

    2011-01-01

    Calcium ions are crucial elements of excitation-contraction coupling in cardiac myocytes. The intracellular Ca(2+ ) concentration changes continously during the cardiac cycle, but the Ca(2+ ) entering to the cell serves as an intracellular second messenger, as well. The Ca(2+ ) as a second messenger influences the activity of many intracellular signalling pathways and regulates gene expression. In cardiac myocytes the major pathway for Ca(2+ ) entry into cells is L-type calcium channel (LTCC). The precise control of LTCC function is essential for maintaining the calcium homeostasis of cardiac myocytes. Dysregulation of LTCC may result in different diseases like cardiac hypertrophy, arrhytmias, heart failure. The physiological and pathological structural changes in the heart are induced in part by small G proteins. These proteins are involved in wide spectrum of cell biological functions including protein transport, regulation of cell proliferation, migration, apoptosis, and cytoskeletal rearrangement. Understanding the crosstalk between small G proteins and LTCC may help to understand the pathomechanism of different cardiac diseases and to develop a new generation of genetically-encoded Ca(2+ ) channel inhibitors.

  2. Rem-GTPase regulates cardiac myocyte L-type calcium current

    PubMed Central

    Magyar, Janos; Kiper, Carmen E.; Sievert, Gail; Cai, Weikang; Shi, Geng-Xian; Crump, Shawn M.; Li, Liren; Niederer, Steven; Smith, Nic; Andres, Douglas A.; Satin, Jonathan

    2012-01-01

    Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca2+-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK–LTCC interactions is untested. Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca2+ current (ICa,L) via LTCC in murine cardiomyocytes. Methods and Results: Rem knockout mice (Rem−/−) were engineered, and ICa,L and Ca2+-handling properties were assessed. Rem−/− ventricular cardiomyocytes displayed increased ICa,L density. ICa,L activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type ICa,L. Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem−/−. Cell shortening was not significantly different. Increased ICa,L density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger ICa,L density, Rem−/− cardiomyocyte Ca2+ twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem−/− LTCC can account for the paradoxical decrease of Ca2+ transients. Conclusions: This is the first demonstration that loss of an RGK protein influences ICa,L in vivo in cardiac myocytes. PMID:22854599

  3. L-type calcium channels refine the neural population code of sound level.

    PubMed

    Grimsley, Calum Alex; Green, David Brian; Sivaramakrishnan, Shobhana

    2016-12-01

    The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (CaL: CaV1.1-1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of CaL to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. CaL is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, CaL activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, CaL boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, CaL either suppresses or enhances firing at sound levels that evoke maximum firing. CaL multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level. Copyright © 2016 the American Physiological Society.

  4. L-type calcium channel: Clarifying the "oxygen sensing hypothesis".

    PubMed

    Cserne Szappanos, Henrietta; Viola, Helena; Hool, Livia C

    2017-03-18

    The heart is able to respond acutely to changes in oxygen tension. Since ion channels can respond rapidly to stimuli, the "ion channel oxygen sensing hypothesis" has been proposed to explain acute adaptation of cells to changes in oxygen demand. However the exact mechanism for oxygen sensing continues to be debated. Mitochondria consume the lion's share of oxygen in the heart, fuelling the production of ATP that drives excitation and contraction. Mitochondria also produce reactive oxygen species that are capable of altering the redox state of proteins. The cardiac L-type calcium channel is responsible for maintaining excitation and contraction. Recently, the reactive cysteine on the cardiac L-type calcium channel was identified. These data clarified that the channel does not respond directly to changes in oxygen tension, but rather responds to cellular redox state. This leads to acute alterations in cell signalling responsible for the development of arrhythmias and pathology.

  5. Modulation by extracellular ATP of L-type calcium channels in guinea-pig single sinoatrial nodal cell.

    PubMed Central

    Qi, A. D.; Kwan, Y. W.

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate ([ATP]zero) on the L-type Ca2+ channel currents in guinea-pig single sinoatrial nodal (SAN) cells, isolated by enzymatic dissociation, were investigated by use of whole-cell patch-clamp techniques. 2. The application of [ATP]zero (2 microM-1 mM) produced an inhibitory effect on the L-type Ca2+ channel current peak amplitude (10 mM Ba2+ as charge carrier) in a concentration-dependent and reversible manner with an IC50 of 100 microM and a Hill coefficient of 1.83. 3. The presence of the adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM) and 8-phenyltheophylline (10 microM) did not affect the [ATP]zero-induced inhibition of the Ca2+ channel currents. Adenosine (100 microM) had little effect on the basal Ca2+ channel currents. Adenosine 500 microM, caused 23% inhibition of the Ca2+ channel current, which was abolished by 0.1 microM DPCPX. 4. The presence of the P2-purinoceptor antagonists, suramin (1, 10 and 100 microM), reactive blue 2 (1 and 10 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 50 and 100 microM) failed to affect the inhibitory action of [ATP]zero on Ca2+ channel currents. 5. The relative rank order of potency of different nucleotides and nucleosides, at a concentration of 100 microM, on the inhibition of the Ca2+ channel currents is as follows: adenosine 5'-triphosphate (ATP) = alpha,beta-methylene-ATP (alpha,beta MeATP) > > 2-methylthioATP (2-MeSATP) > or = adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > > uridine 5'-triphosphate (UTP) = adenosine 5'-diphosphate (ADP) > adenosine 5'-monophosphate (AMP) > or = adenosine. 6. These results suggest that [ATP]zero may play an important role in the heart beat by inhibiting the L-type Ca2+ channel currents in single SAN cells. This inhibitory effect is not due to the formation of adenosine resulting from the enzymatic degradation of [ATP]zero. Based on the relative order of inhibitory

  6. A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

    SciTech Connect

    Galvis-Pareja, David; Zapata-Torres, Gerald; Hidalgo, Jorge; Ayala, Pedro; and others

    2014-08-15

    Rationale: Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca{sup 2+} channels and their renowned antioxidant properties. Methods: We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca{sup 2+} channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca{sup 2+} channel-blocking activity and antioxidant properties. The Ca{sup 2+} channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. Results: Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca{sup 2+} channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca{sup 2+} channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Conclusions: Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring. - Highlights: • Dihydropyridine (DHP) molecules are widely used in cardiovascular disease. • DHPs block Ca{sup 2+} entry through LTCC—some DHPs have antioxidant activity as well. • We synthesized 6 new DHPs and tested their Ca{sup 2+} blocking and antioxidant activities. • 3-Aryl meta-hydroxyl substitution strongly increases their Ca{sup 2+} blocking activity. • 3-Aryl meta-hydroxyl substitution did not affect the antioxidant properties.

  7. Fluoride affects calcium homeostasis and osteogenic transcription factor expressions through L-type calcium channels in osteoblast cell line.

    PubMed

    Duan, Xiao-Qin; Zhao, Zhi-Tao; Zhang, Xiu-Yun; Wang, Ying; Wang, Huan; Liu, Da-Wei; Li, Guang-Sheng; Jing, Ling

    2014-12-01

    Osteoblast L-type voltage-dependent calcium channels (VDCC) play important roles in maintaining intracellular homeostasis and influencing multiple cellular processes. In particular, they contribute to the activities and functions of osteoblasts (OBs). In order to study how L-type VDCC modulate calcium ion (Ca(2+)) homeostasis and the expression of osteogenic transcription factors in OBs exposed to fluoride, MC3T3-E1 cells were exposed to a gradient of concentrations of fluoride (0, 2.0, 5.0, 10.0 mg/L) in combination with 10 μM nifedipine, a specific inhibitor of VDCC, for 48 h. We examined messenger RNA (mRNA) and protein levels of Cav1.2, the main subunit of VDCC, and c-fos, c-jun, runt-related transcription factor 2 (Runx2), osterix (OSX), and intracellular free Ca(2+) ([Ca(2+)]i) concentrations in MC3T3-E1 cells. Our results showed that [Ca(2+)]i levels increased in a dose-dependent manner with increase in concentration of fluoride. Meantime, results indicated that lower concentrations of fluoride (less than 5 mg/L, especially 2 mg/L) can lead to high expression of Cav1.2 and enhance osteogenic function, while high concentration of fluoride (10 mg/L) can induce decreased Cav1.2 and osteogenic transcriptional factors in MC3T3E1 cells exposed to fluoride. However, the levels of [Ca(2+)]i, Cav1.2, c-fos, c-jun, Runx2, and OSX induced by fluoride were significantly altered and even reversed in the presence of nifedipine. These results demonstrate that L-type calcium channels play a crucial role in Ca(2+) homeostasis and they affect the expression of osteogenic transcription factors in fluoride-treated osteoblasts.

  8. The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

    PubMed Central

    Bachnoff, Niv; Cohen-Kutner, Moshe; Atlas, Daphne

    2011-01-01

    A PKA consensus phosphorylation site S1928 at the α11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α11.2 or α11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α11.2, the pore forming subunit of CaV1.2. PMID:22216029

  9. Irradiation behavior of Ti-stabilized 316L type steel

    NASA Astrophysics Data System (ADS)

    Rodchenkov, B. S.; Kalinin, G. M.; Strebkov, Yu. S.; Shamardin, V. K.; Prokhorov, V. I.; Bulanova, T. M.

    2009-04-01

    Type 316L austenitic steels are widely used for the in-vessel internal structures of fission reactors (core, core support, etc.) and for experimental irradiation facilities. The modifications of 316L Type steel (316L, 316L(N), US 316, J 316, JPCA, etc.) have been considered as structural material for International Thermonuclear Experimental Reactor (ITER). The results of investigation the irradiation behaviour of Ti-stabilized 316 L type steel (0.04 C-15 Cr-11 Ni-2.5 Mo-0.5 Ti) are presented in this work. The specimens cut out from 316L-Ti steel forging were irradiated in the SM-2 reactor up to a dose ˜4 and 10 dpa at 265 ± 15 °C. The tensile properties, fracture toughness and changes in resistance to intergranular stress corrosion cracking (IGSCC) have been investigated after irradiation. The results for Ti-stabilized 316L steel were compared with those for 316L(N)-IG steel irradiated at the same condition.

  10. Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK

    PubMed Central

    Edwards, Brian S.; Dang, An K.; Murtazina, Dilyara A.; Dozier, Melissa G.; Whitesell, Jennifer D.; Khan, Shaihla A.; Cherrington, Brian D.; Amberg, Gregory C.; Clay, Colin M.

    2016-01-01

    We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3–1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3–1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca2+ influx via the L-type Ca2+ channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca2+ influx through L-type Ca2+ channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca2+ influx via L-type channels to facilitate ERK phosphorylation. PMID:26696122

  11. A rational route to SCM materials based on a 1-D cobalt selenocyanato coordination polymer.

    PubMed

    Boeckmann, Jan; Näther, Christian

    2011-07-07

    Thermal annealing of a discrete complex with terminal SeCN anions and monodentate coligands enforces the formation of a 1D cobalt selenocyanato coordination polymer that shows slow relaxation of the magnetization. Therefore, this approach offers a rational route to 1D materials that might show single chain magnetic behaviour.

  12. A comparison between calcium channel blocking drugs with different potencies for T- and L-type channels in preventing atrial electrical remodeling.

    PubMed

    Ohashi, Narutaka; Mitamura, Hideo; Tanimoto, Kojiro; Fukuda, Yukiko; Kinebuchi, Osamu; Kurita, Yasuo; Shiroshita-Takeshita, Akiko; Miyoshi, Shunichiro; Hara, Motoki; Takatsuki, Seiji; Ogawa, Satoshi

    2004-09-01

    Calcium overload plays a key role in the development of atrial electrical remodeling. The effect of an L-type Ca channel blocker in preventing this remodeling has been reported to be short lasting, partly due to down-regulation of this channel and persisting Ca entry through the T-type Ca channel. To prove if efonidipine, a dual L- and T-type Ca channel blocker exerts a greater effect than an L-type Ca channel blocker verapamil, 21 dogs underwent rapid atrial pacing at 400 bpm for 14 days, pretreatment with efonidipine in 7 (E), verapamil in 7 (V), and none in 7 (C). We measured the atrial effective refractory period (ERP) serially during 14 days of rapid pacing. In response to rapid pacing, ERP decreased progressively in C. In contrast, in E and V, ERP remained greater than ERP in C (P < 0.01) on days 2 through 7. However, on the 14th day, ERP in V decreased to the level seen in C, whereas ERP in E remained significantly longer than ERPs in C or V (P < 0.01). The blockade L-type Ca channel alone is not sufficient, but the addition of a T-type Ca channel blockade shows a more sustained effect to prevent atrial electrical remodeling.

  13. Spectroscopy and photometry of L-type asteroids

    NASA Astrophysics Data System (ADS)

    Tanga, P.; Devogele, M.; Cellino, A.; Campins, H.; Pinilla-Alonso, N.; Abe, L.; Bendjoya, Ph.; Rivet, J. P.

    2015-10-01

    Among L-type asteroids, a peculiar category of objects exists: it includes the so-called "Barbarians", known to have very specific features in the phasepolarization curve. Such objects are thought to contain a high percentage of Calcium-Aluminumrich- Inclusions, responsible of a spinel absorption feature in the near infrared (around 2.1-2.2 μm). However, Barbarians are also peculiar in some other physical properties: in particular they seem to have unusually high rotation periods, and large amplitude light curves. We started a campaign of NIR spectroscopy and photometry to shed a light on such properties and to compare the Barbarians to the other objects belonging to the same taxonomic type.

  14. The L-type channel antagonist isradipine is neuroprotective in a mouse model of Parkinson’s disease

    PubMed Central

    Ilijic, E; Guzman, JN; Surmeier, DJ

    2011-01-01

    The motor symptoms of Parkinson’s disease (PD) are due to the progressive loss of dopamine (DA) neurons in substantia nigra pars compacta (SNc). Nothing is known to slow the progression of the disease, making the identification of potential neuroprotective agents of great clinical importance. Previous studies using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD have shown that antagonism of L-type Ca2+ channels protects SNc DA neurons. However, this was not true in a 6-hydroxydopamine (6-OHDA) model. One potential explanation for this discrepancy is that protection in the 6-OHDA model requires greater antagonism of Cav1.3 L-type Ca2+ channels thought to underlie vulnerability and this was not achievable with the low affinity dihydropyridine (DHP) antagonist used. To test this hypothesis, the DHP with the highest affinity for Cav1.3 L-type channels – isradipine – was systemically administered and then the DA toxin 6-OHDA injected intrastriatally. Twenty-five days later, neuroprotection and plasma concentration of isradipine were determined. This analysis revealed that isradipine produced a dose-dependent sparing of DA fibers and cell bodies at concentrations achievable in humans, suggesting that isradipine is a potentially viable neuroprotective agent for PD. PMID:21515375

  15. Binding and selectivity in L-type calcium channels: a mean spherical approximation.

    PubMed Central

    Nonner, W; Catacuzzeno, L; Eisenberg, B

    2000-01-01

    L-type calcium channels are Ca(2+) binding proteins of great biological importance. They generate an essential intracellular signal of living cells by allowing Ca(2+) ions to move across the lipid membrane into the cell, thereby selecting an ion that is in low extracellular abundance. Their mechanism of selection involves four carboxylate groups, containing eight oxygen ions, that belong to the side chains of the "EEEE" locus of the channel protein, a setting similar to that found in many Ca(2+)-chelating molecules. This study examines the hypothesis that selectivity in this locus is determined by mutual electrostatic screening and volume exclusion between ions and carboxylate oxygens of finite diameters. In this model, the eight half-charged oxygens of the tethered carboxylate groups of the protein are confined to a subvolume of the pore (the "filter"), but interact spontaneously with their mobile counterions as ions interact in concentrated bulk solutions. The mean spherical approximation (MSA) is used to predict ion-specific excess chemical potentials in the filter and baths. The theory is calibrated using a single experimental observation, concerning the apparent dissociation constant of Ca(2+) in the presence of a physiological concentration of NaCl. When ions are assigned their independently known crystal diameters and the carboxylate oxygens are constrained, e.g., to a volume of 0.375 nm(3) in an environment with an effective dielectric coefficient of 63.5, the hypothesized selectivity filter produces the shape of the calcium binding curves observed in experiment, and it predicts Ba(2+)/Ca(2+) and Na(+)/Li(+) competition, and Cl(-) exclusion as observed. The selectivities for Na(+), Ca(2+), Ba(2+), other alkali metal ions, and Cl(-) thus can be predicted by volume exclusion and electrostatic screening alone. Spontaneous coordination of ions and carboxylates can produce a wide range of Ca(2+) selectivities, depending on the volume density of carboxylate

  16. Methamphetamine acutely inhibits voltage-gated calcium channels but chronically up-regulates L-type channels.

    PubMed

    Andres, Marilou A; Cooke, Ian M; Bellinger, Frederick P; Berry, Marla J; Zaporteza, Maribel M; Rueli, Rachel H; Barayuga, Stephanie M; Chang, Linda

    2015-07-01

    In neurons, calcium (Ca(2+) ) channels regulate a wide variety of functions ranging from synaptic transmission to gene expression. They also induce neuroplastic changes that alter gene expression following psychostimulant administration. Ca(2+) channel blockers have been considered as potential therapeutic agents for the treatment of methamphetamine (METH) dependence because of their ability to reduce drug craving among METH users. Here, we studied the effects of METH exposure on voltage-gated Ca(2+) channels using SH-SY5Y cells as a model of dopaminergic neurons. We found that METH has different short- and long-term effects. A short-term effect involves immediate (< 5 min) direct inhibition of Ca(2+) ion movements through Ca(2+) channels. Longer exposure to METH (20 min or 48 h) selectively up-regulates the expression of only the CACNA1C gene, thus increasing the number of L-type Ca(2+) channels. This up-regulation of CACNA1C is associated with the expression of the cAMP-responsive element-binding protein (CREB), a known regulator of CACNA1C gene expression, and the MYC gene, which encodes a transcription factor that putatively binds to a site proximal to the CACNA1C gene transcription initiation site. The short-term inhibition of Ca(2+) ion movement and later, the up-regulation of Ca(2+) channel gene expression together suggest the operation of cAMP-responsive element-binding protein- and C-MYC-mediated mechanisms to compensate for Ca(2+) channel inhibition by METH. Increased Ca(2+) current density and subsequent increased intracellular Ca(2+) may contribute to the neurodegeneration accompanying chronic METH abuse. Methamphetamine (METH) exposure has both short- and long-term effects. Acutely, methamphetamine directly inhibits voltage-gated calcium channels. Chronically, neurons compensate by up-regulating the L-type Ca(2+) channel gene, CACNA1C. This compensatory mechanism is mediated by transcription factors C-MYC and CREB, in which CREB is linked to the

  17. Ion-dependent Inactivation of Barium Current through L-type Calcium Channels

    PubMed Central

    Ferreira, Gonzalo; Yi, Jianxun; Ríos, Eduardo; Shirokov, Roman

    1997-01-01

    It is widely believed that Ba2+ currents carried through L-type Ca2+ channels inactivate by a voltage- dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca2+ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba2+ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, IBa decayed as the sum of two exponentials. The slow phase (τ ≈ 6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (τ ≈ 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na+ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba2+ current. The reduction of Ba2+ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba2+ entry during the pulse did not change the reversal potential for Ba2+. A simple model of Ca2+-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba2+ current decay assuming the affinity of the inactivation site on the α1 subunit to be 100 times lower for Ba2+ than Ca2+. PMID:9101404

  18. Coherent Synchrotron Radiation and Space Charge for a 1-D Bunch on an Arbitrary Planar Orbit

    SciTech Connect

    Warnock, R.L.; /SLAC

    2008-01-08

    Realistic modeling of coherent synchrotron radiation (CSR) and the space charge force in single-pass systems and rings usually requires at least a two-dimensional (2-D) description of the charge/current density of the bunch. Since that leads to costly computations, one often resorts to a 1-D model of the bunch for first explorations. This paper provides several improvements to previous 1-D theories, eliminating unnecessary approximations and physical restrictions.

  19. Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues

    PubMed Central

    Imamura, Morikazu; Matsuura, Yuichi; Masujin, Kentaro; Shimizu, Yoshihisa; Shu, Yujing; Kurachi, Megumi; Kasai, Kazuo; Murayama, Yuichi; Fukuda, Shigeo; Onoe, Sadao; Hagiwara, Ken’ichi; Yamakawa, Yoshio; Sata, Tetsutaro; Mohri, Shirou; Okada, Hiroyuki; Yokoyama, Takashi

    2010-01-01

    We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues. PMID:20587193

  20. L-type calcium channels and calcium/calmodulin-dependent kinase II differentially mediate behaviors associated with nicotine withdrawal in mice.

    PubMed

    Jackson, K J; Damaj, M I

    2009-07-01

    Smoking is a widespread health problem. Because the nicotine withdrawal syndrome is a major contributor to continued smoking and relapse, it is important to understand the molecular and behavioral mechanisms of nicotine withdrawal to generate more effective smoking cessation therapies. Studies suggest a role for calcium-dependent mechanisms, such as L-type calcium channels and calcium/calmodulin-dependent protein kinase II (CaMKII), in the effects of nicotine dependence; however, the role of these mechanisms in nicotine-mediated behaviors is unclear. Thus, the goal of this study was to elucidate the role of L-type calcium channels and CaMKII in nicotine withdrawal behaviors. Using both pharmacological and genetic methods, our results show that L-type calcium channels are involved in physical, but not affective, nicotine withdrawal behaviors. Although our data do provide evidence of a role for CaMKII in nicotine withdrawal behaviors, our pharmacological and genetic assessments yielded different results concerning the specific role of the kinase. Pharmacological data suggest that CaMKII is involved in somatic signs and affective nicotine withdrawal, and activity level is decreased after nicotine withdrawal, whereas the genetic assessments yielded results suggesting that CaMKII is involved only in the anxiety-related response, yet the kinase activity may be increased after nicotine withdrawal; thus, future studies are necessary to clarify the precise behavioral specifics of the relevance of CaMKII in nicotine withdrawal behaviors. Overall, our data show that L-type calcium channels and CaMKII are relevant in nicotine withdrawal and differentially mediate nicotine withdrawal behaviors.

  1. Short-term exposure to L-type calcium channel blocker, verapamil, alters the expression pattern of calcium-binding proteins in the brain of goldfish, Carassius auratus.

    PubMed

    Palande, Nikhil V; Bhoyar, Rahul C; Biswas, Saikat P; Jadhao, Arun G

    2015-01-01

    The influx of calcium ions (Ca(2+)) is responsible for various physiological events including neurotransmitter release and synaptic modulation. The L-type voltage dependent calcium channels (L-type VDCCs) transport Ca(2+) across the membrane. Calcium-binding proteins (CaBPs) bind free cytosolic Ca(2+) and prevent excitotoxicity caused by sudden increase in cytoplasmic Ca(2+). The present study was aimed to understand the regulation of expression of neuronal CaBPs, namely, calretinin (CR) and parvalbumin (PV) following blockade of L-type VDCCs in the CNS of Carassius auratus. Verapamil (VRP), a potent L-type VDCC blocker, selectively blocks Ca(2+) entry at the plasma membrane level. VRP present in the aquatic environment at a very low residual concentration has shown ecotoxicological effects on aquatic animals. Following acute exposure for 96h, median lethal concentration (LC50) for VRP was found to be 1.22mg/L for goldfish. At various doses of VRP, the behavioral alterations were observed in the form of respiratory difficulty and loss of body balance confirming the cardiovascular toxicity caused by VRP at higher doses. In addition to affecting the cardiovascular system, VRP also showed effects on the nervous system in the form of altered expression of PV. When compared with controls, the pattern of CR expression did not show any variations, while PV expression showed significant alterations in few neuronal populations such as the pretectal nucleus, inferior lobes, and the rostral corpus cerebellum. Our result suggests possible regulatory effect of calcium channel blockers on the expression of PV. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. [Effect of shenmai injection on L-type calcium channel of diaphragmatic muscle cells in rats].

    PubMed

    Zhao, Li-min; Xiong, Sheng-dao; Niu, Ru-ji

    2003-08-01

    To explore the effect of Shenmai Injection (SMI) on L-type calcium channel of diaphragmatic muscle cells in rats. Single diaphragmatic muscle cell of rats was obtained by the acute enzyme isolation method and the standard whole-cell patch clamp technique was used to record the inward peak L-type calcium current (IPLC) and current-voltage relationship curve of diaphragmatic muscle cells of 7 rats, and to compare the effects of SMI in various concentrations on them. When keeping the electric potential at -80 mV, stimulation frequency 0.5 Hz, clamp time 300 ms, stepped voltage 10 mV, and depolarized to +60 mV, 10 microliters/ml of SMI could only cause the mean IPLC of rat's diaphragmatic muscle cells increased from -6.9 +/- 0.6 pA/pF to -7.5 +/- 0.7 pA/pF, the amplification being (9.2 +/- 2.8)%, comparison between those of pre-treatment and post-treatment showed insignificant difference. But when the concentration of SMI increased to 50 microliters/ml and 100 microliters/ml, the mean IPLC increased to -8.4 +/- 0.6 pA/pF and -9.2 +/- 0.6 pA/pF, respectively, and the amplification was (22.4 +/- 1.7)% and (34.6 +/- 4.6)% respectively, showing significant difference to that of pre-treatment (P < 0.05). However, SMI showed no significant effect on maximal activation potential and reversal potential. SMI can activate the calcium channel of diaphragmatic muscle cells in rats, increase the influx of Ca2+, so as to strengthen the contraction of diaphragmatic muscle, which may be one of the ionic channel mechanisms of SMI in treating diaphragmatic muscle fatigue in clinical practice.

  3. Exhaustive exercise decreases L-type calcium current by activating endoplasmic reticulum stress.

    PubMed

    Ma, Yanzhuo; Kong, Lingfeng; Qi, Shuying; Wang, Dongmei

    2017-04-01

    This study investigated effects of exhaustive exercise on L-type calcium current (ICa,L) and the putative intracellular cascade responsible for the effects. Rats were randomly divided into three treatment groups: sedentary (without exercise), exercised to exhaustion and salubrinal injection before each exhaustive exercise period. Exercise group rats were forced to swim until exhaustion each time for 9 days with 5% body weight attached to the head. Salubrinal (1 mg/kg) or an equivalent volume of placebo solution (dimethyl sulfoxide) was injected via the intraperitoneal route daily for the first 3 days, followed by subcutaneous injections of salubrinal (0.5 mg/kg) or placebo solution daily for 9 days (starting 30 min before exercise). After a 1-day recovery period, whole-cell patch clamping was used to investigate the L-type Ca2+ current (ICa,L), with sedentary control rats. Additionally, endoplasmic reticulum (ER) chaperone protein levels were analyzed. Exhaustive exercise triggered ER stress, demonstrated by elevated expression of ER stress markers: phospho-eIF2α, CCAT/enhancer-binding homologous protein (CHOP) and caspase-12. Compared to controls, ICa,L was inhibited by exhaustive exercise, which was blocked by salubrinal, a selective eIF2α dephosphorylation inhibitor used to inhibit ER stress. These results suggest that ER stress participates in regulation of ICa,L. However, exhaustive exercise did not change the voltage dependence of steady-state activation and inactivation of ICaL, and salubrinal infusion caused no difference in voltage dependence of steady-state activation and inactivation of ICa,L. Exhaustive exercise activates ER stress, thus inhibiting ICaL, which may change the action potential duration and contribute to proarrhythmia.

  4. Otilonium bromide inhibits muscle contractions via L-type calcium channels in the rat colon.

    PubMed

    Martin, M T; Hove-Madsen, L; Jimenez, M

    2004-04-01

    The aim of this study is to evaluate in vitro the effect of otilonium bromide (OB) on the mechanical and electrical activities of the rat colonic smooth muscle using muscle bath, microelectrodes and patch-clamp techniques. Otilonium bromide dose dependently inhibited the spontaneous activity (logIC(50) +/- SE: -5.31 +/- 0.05). This effect was not modified by TTX (10(-6) mol L(-1)). Cyclic depolarizations were abolished by OB (10(-4) mol L(-1)). Electrical field stimulation induced inhibitory junction potentials (IJPs) followed by a depolarization with superimposed spikes causing a contraction. In the presence of OB (10(-4) mol L(-1)) IJPs were recorded, but spikes and contractions were abolished. Otilonium bromide (3 x 10(-6) mol L(-1)) inhibited inward current obtained in isolated cells (amphotericin perforated patch technique). The otilonium-sensitive current amplitude was maximal (75pA) around 0 mV. The effect of different doses of OB was tested by depolarizing cells from -70 mV to 0 mV. OB dose dependently inhibited the inward current with an EC(50) of 885 nmol L(-1). Abolishment of the otilonium-sensitive current by 3 x 10(-6) mol L(-1) nifedipine confirmed that it was an L-type Ca(2+) current. Our results show that OB inhibits the spontaneous and triggered muscular contractions. This effect is produced by the inhibition of muscular action potentials carried by L-type calcium current, confirming the spasmolytic properties of OB.

  5. Spexin Enhances Bowel Movement through Activating L-type Voltage-dependent Calcium Channel via Galanin Receptor 2 in Mice

    PubMed Central

    Lin, Cheng-yuan; Zhang, Man; Huang, Tao; Yang, Li-ling; Fu, Hai-bo; Zhao, Ling; Zhong, Linda LD; Mu, Huai-xue; Shi, Xiao-ke; Leung, Christina FP; Fan, Bao-min; Jiang, Miao; Lu, Ai-ping; Zhu, Li-xin; Bian, Zhao-xiang

    2015-01-01

    A novel neuropeptide spexin was found to be broadly expressed in various endocrine and nervous tissues while little is known about its functions. This study investigated the role of spexin in bowel movement and the underlying mechanisms. In functional constipation (FC) patients, serum spexin levels were significantly decreased. Consistently, in starved mice, the mRNA of spexin was significantly decreased in intestine and colon. Spexin injection increased the velocity of carbon powder propulsion in small intestine and decreased the glass beads expulsion time in distal colon in mice. Further, spexin dose-dependently stimulated the intestinal/colonic smooth muscle contraction. Galanin receptor 2 (GALR2) antagonist M871, but not Galanin receptor 3 (GALR3) antagonist SNAP37899, effectively suppressed the stimulatory effects of spexin on intestinal/colonic smooth muscle contraction, which could be eliminated by extracellular [Ca2+] removal and L-type voltage-dependentCa2+ channel (VDCC) inhibitor nifedipine. Besides, spexin dramatically increased the [Ca2+]i in isolated colonic smooth muscle cells. These data indicate that spexin can act on GALR2 receptor to regulate bowel motility by activating L-type VDCC. Our findings provide evidence for important physiological roles of spexin in GI functions. Selective action on spexin pathway might have therapeutic effects on GI diseases with motility disorders. PMID:26160593

  6. The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

    PubMed Central

    Taiakina, Valentina; Boone, Adrienne N.; Fux, Julia; Senatore, Adriano; Weber-Adrian, Danielle

    2013-01-01

    NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels. PMID:23626724

  7. Raised activity of L-type calcium channels renders neurons prone to form paroxysmal depolarization shifts.

    PubMed

    Rubi, Lena; Schandl, Ulla; Lagler, Michael; Geier, Petra; Spies, Daniel; Gupta, Kuheli Das; Boehm, Stefan; Kubista, Helmut

    2013-09-01

    Neuronal L-type voltage-gated calcium channels (LTCCs) are involved in several physiological functions, but increased activity of LTCCs has been linked to pathology. Due to the coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, such as KCa or non-specific cation channels, LTCCs act as important regulators of neuronal excitability. Augmentation of after-hyperpolarizations may be one mechanism that shows how elevated LTCC activity can lead to neurological malfunctions. However, little is known about other impacts on electrical discharge activity. We used pharmacological up-regulation of LTCCs to address this issue on primary rat hippocampal neurons. Potentiation of LTCCs with Bay K8644 enhanced excitatory postsynaptic potentials to various degrees and eventually resulted in paroxysmal depolarization shifts (PDS). Under conditions of disturbed Ca(2+) homeostasis, PDS were evoked frequently upon LTCC potentiation. Exposing the neurons to oxidative stress using hydrogen peroxide also induced LTCC-dependent PDS. Hence, raising LTCC activity had unidirectional effects on brief electrical signals and increased the likeliness of epileptiform events. However, long-lasting seizure-like activity induced by various pharmacological means was affected by Bay K8644 in a bimodal manner, with increases in one group of neurons and decreases in another group. In each group, isradipine exerted the opposite effect. This suggests that therapeutic reduction in LTCC activity may have little beneficial or even adverse effects on long-lasting abnormal discharge activities. However, our data identify enhanced activity of LTCCs as one precipitating cause of PDS. Because evidence is continuously accumulating that PDS represent important elements in neuropathogenesis, LTCCs may provide valuable targets for neuroprophylactic therapy.

  8. Quantum Nucleation of Phase Slips in a 1D Model of a Superfluid

    SciTech Connect

    Freire, J.A.; Arovas, D.P.; Levine, H.

    1997-12-01

    We use a 1D model of a superfluid based on the Gross-Pitaevskii Lagrangian to illustrate a general numerical method designed to find quantum tunneling rates in extended bosonic systems. Specifically, we study flow past an obstacle and directly solve the imaginary time dynamics to find the {open_quotes}bounce{close_quotes} solution connected with the decay of the metastable laminar state via phase slip nucleation. The action for the tunneling configuration goes to zero at the threshold (in superfluid velocity) for classical production of these slips. Applications to other processes are briefly discussed. {copyright} {ital 1997} {ital The American Physical Society}

  9. Delocalization of Weakly Interacting Bosons in a 1D Quasiperiodic Potential

    NASA Astrophysics Data System (ADS)

    Michal, V. P.; Altshuler, B. L.; Shlyapnikov, G. V.

    2014-07-01

    We consider weakly interacting bosons in a 1D quasiperiodic potential (Aubry-Azbel-Harper model) in the regime where all single-particle states are localized. We show that the interparticle interaction may lead to the many-body delocalization and we obtain the finite-temperature phase diagram. Counterintuitively, in a wide range of parameters the delocalization requires stronger coupling as the temperature increases. This means that the system of bosons can undergo a transition from a fluid to insulator (glass) state under heating.

  10. Phosphorylation of the Consensus Sites of Protein Kinase A on α1D L-type Calcium Channel*

    PubMed Central

    Ramadan, Omar; Qu, Yongxia; Wadgaonkar, Raj; Baroudi, Ghayath; Karnabi, Eddy; Chahine, Mohamed; Boutjdir, Mohamed

    2009-01-01

    The novel α1D L-type Ca2+ channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased α1D Ca2+ channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the α1 subunit of the α1D Ca2+ channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the α1 subunit of α1D Ca2+ channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the α1 subunit of α1D Ca2+ channel. These novel findings provide new insights into the autonomic regulation of the α1D Ca2+ channel in the heart. PMID:19074150

  11. PKC-mediated modulation of L-type calcium channels may contribute to fat-induced insulin resistance.

    PubMed

    McCarty, Mark F

    2006-01-01

    Increased intracellular free calcium [Ca2+]i has been noted in adipocytes, platelets, and leukocytes of subjects with insulin resistance syndrome or allied disorders. In rodent studies, measures which increase [Ca2+]i in adipocytes and skeletal muscle are associated with impaired insulin signaling, attributable at least in part to diminished ability of insulin to activate phosphoserine phosphatase-1 (PP-1). In fat-fed insulin resistant rats, pre-treatment with a drug that selectively chelates intracellular calcium eliminates about half of the decrement in insulin-stimulated glucose uptake induced by fat feeding; since this chelator does not influence the insulin sensitivity of chow-fed rats, it is reasonable to suspect that fat feeding boosts [Ca2+]i in skeletal muscle, and that this effect is partially responsible for the associated reduction in insulin sensitivity. Clinical insulin resistance is associated with increased levels of triglycerides and other fatty acid metabolites in muscle fibers; this can give rise to diacylglycerol-mediated activation of PKC, which in turn compromises insulin signaling by triggering kinase cascades that phosphorylate IRS-1 on key serine residues. Yet there is also evidence that, in skeletal muscle, PKC activity up-regulates the function of L-type calcium channels, increasing their maximal conductance while left-shifting their voltage dependence. Thus, the PKC activation associated with fat overexposure might be expected to boost basal [Ca2+]i in skeletal muscle, potentially impeding insulin-mediated activation of PP-1. This hypothesis is consistent with several clinical studies demonstrating that long-acting inhibitors of L-type calcium channels can improve insulin sensitivity in overweight hypertensives; it should be readily testable in rodent models of fat-induced insulin resistance. Since parathyroid hormone can act on adipocytes and muscle to boost [Ca2+]i, mild secondary hyperparathyroidism associated with low calcium intakes

  12. L-type calcium channels and MAP kinase contribute to thyrotropin-releasing hormone-induced depolarization in thalamic paraventricular nucleus neurons

    PubMed Central

    Kolaj, Miloslav; Zhang, Li

    2016-01-01

    In rat paraventricular thalamic nucleus (PVT) neurons, activation of thyrotropin-releasing hormone (TRH) receptors enhances neuronal excitability via concurrent decrease in a G protein-coupled inwardly rectifying K (GIRK)-like conductance and opening of a cannabinoid receptor-sensitive transient receptor potential canonical (TRPC)-like conductance. Here, we investigated the calcium (Ca2+) contribution to the components of this TRH-induced response. TRH-induced membrane depolarization was reduced in the presence of intracellular BAPTA, also in media containing nominally zero [Ca2+]o, suggesting a critical role for both intracellular Ca2+ release and Ca2+ influx. TRH-induced inward current was unchanged by T-type Ca2+ channel blockade, but was decreased by blockade of high-voltage-activated Ca2+ channels (HVACCs). Both the pharmacologically isolated GIRK-like and the TRPC-like components of the TRH-induced response were decreased by nifedipine and increased by BayK8644, implying Ca2+ influx via L-type Ca2+ channels. Only the TRPC-like conductance was reduced by either thapsigargin or dantrolene, suggesting a role for ryanodine receptors and Ca2+-induced Ca2+ release in this component of the TRH-induced response. In pituitary and other cell lines, TRH stimulates MAPK. In PVT neurons, only the GIRK-like component of the TRH-induced current was selectively decreased in the presence of PD98059, a MAPK inhibitor. Collectively, the data imply that TRH-induced depolarization and inward current in PVT neurons involve both a dependency on extracellular Ca2+ influx via opening of L-type Ca2+ channels, a sensitivity of a TRPC-like component to intracellular Ca2+ release via ryanodine channels, and a modulation by MAPK of a GIRK-like conductance component. PMID:27009047

  13. Mechanisms of NMDA Receptor- and Voltage-Gated L-Type Calcium Channel-Dependent Hippocampal LTP Critically Rely on Proteolysis That Is Mediated by Distinct Metalloproteinases.

    PubMed

    Wiera, Grzegorz; Nowak, Daria; van Hove, Inge; Dziegiel, Piotr; Moons, Lieve; Mozrzymas, Jerzy W

    2017-02-01

    Long-term potentiation (LTP) is widely perceived as a memory substrate and in the hippocampal CA3-CA1 pathway, distinct forms of LTP depend on NMDA receptors (nmdaLTP) or L-type voltage-gated calcium channels (vdccLTP). LTP is also known to be effectively regulated by extracellular proteolysis that is mediated by various enzymes. Herein, we investigated whether in mice hippocampal slices these distinct forms of LTP are specifically regulated by different metalloproteinases (MMPs). We found that MMP-3 inhibition or knock-out impaired late-phase LTP in the CA3-CA1 pathway. Interestingly, late-phase LTP was also decreased by MMP-9 blockade. When both MMP-3 and MMP-9 were inhibited, both early- and late-phase LTP was impaired. Using immunoblotting, in situ zymography, and immunofluorescence, we found that LTP induction was associated with an increase in MMP-3 expression and activity in CA1 stratum radiatum. MMP-3 inhibition and knock-out prevented the induction of vdccLTP, with no effect on nmdaLTP. L-type channel-dependent LTP is known to be impaired by hyaluronic acid digestion. We found that slice treatment with hyaluronidase occluded the effect of MMP-3 blockade on LTP, further confirming a critical role for MMP-3 in this form of LTP. In contrast to the CA3-CA1 pathway, LTP in the mossy fiber-CA3 projection did not depend on MMP-3, indicating the pathway specificity of the actions of MMPs. Overall, our study indicates that the activation of perisynaptic MMP-3 supports L-type channel-dependent LTP in the CA1 region, whereas nmdaLTP depends solely on MMP-9. Various types of long-term potentiation (LTP) are correlated with distinct phases of memory formation and retrieval, but the underlying molecular signaling pathways remain poorly understood. Extracellular proteases have emerged as key players in neuroplasticity phenomena. The present study found that L-type calcium channel-dependent LTP in the CA3-CA1 hippocampal projection is critically regulated by the activity

  14. Vitamin E isomer δ-tocopherol enhances the efficiency of neural stem cell differentiation via L-type calcium channel.

    PubMed

    Deng, Sihao; Hou, Guoqiang; Xue, Zhiqin; Zhang, Longmei; Zhou, Yuye; Liu, Chao; Liu, Yanqing; Li, Zhiyuan

    2015-01-12

    The effects of the vitamin E isomer δ-tocopherol on neural stem cell (NSC) differentiation have not been investigated until now. Here we investigated the effects of δ-tocopherol on NSC neural differentiation, maturation and its possible mechanisms. Neonatal rat NSCs were grown in suspended neurosphere cultures, and were identified by their expression of nestin protein and their capacity for self-renewal. Treatment with a low concentration of δ-tocopherol induced a significant increase in the percentage of β-III-tubulin-positive cells. δ-Tocopherol also stimulated morphological maturation of neurons in culture. We further observed that δ-tocopherol stimulation increased the expression of voltage-dependent Ca(2+) channels. Moreover, a L-type specific Ca(2+) channel blocker verapamil reduced the percentage of differentiated neurons after δ-tocopherol treatment, and blocked the effects of δ-tocopherol on NSC differentiation into neurons. Together, our study demonstrates that δ-tocopherol may act through elevation of L-type calcium channel activity to increase neuronal differentiation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  15. Cholinergic modulation of L-type calcium current in isolated sensory hair cells of the statocyst of octopus, Eledone cirrhosa.

    PubMed

    Chrachri, Abdesslam; Williamson, Roddy

    2004-04-22

    Whole-cell voltage-clamp recordings from dissociated hair cells of the statocyst of octopus, Eledone cirrhosa, demonstrated that application of ACh, carbachol or muscarine (10 microM) reversibly decreased the amplitude of L-type calcium current (I(Ca,L)), while nicotine (10-100 microM) did not have any effect. Furthermore, atropine blocked the effect of ACh and agonists suggesting that ACh reduces I(Ca,L) through activation of muscarinic receptors. Internal dialysis of these cells with guanosine 5'-O-3-thiotriphosphate (GTPgammaS), a non-hydrolysable GTP analogue, mimicked the ACh-induced inhibition of I(Ca,L) and occluded any further ACh-induced inhibition. Internal dialysis of these hair cells with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) reduced the ACh-induced inhibition I(Ca,L). The inhibitory effects of ACh were abolished by pre-incubation of these cells with pretussis toxin (PTX) suggesting that ACh-induced inhibition of I(Ca,L) involves a PTX-sensitive G protein pathway.

  16. Science Signaling Podcast for 24 January 2017: Tissue-specific regulation of L-type calcium channels

    PubMed Central

    Hell, Johannes W.; Navedo, Manuel F.; VanHook, Annalisa M.

    2017-01-01

    This Podcast features an interview with Johannes Hell and Manuel Navedo, senior authors of two Research Articles that appear in the 24 January 2017 issue of Science Signaling, about tissue-specific regulation of the L-type calcium channel CaV1.2. This channel is present in many tissues, including the heart, vasculature, and brain, and allows calcium to flow into cells when it is activated. Signaling through the β-adrenergic receptor (βAR) stimulates CaV1.2 activity in heart cells and neurons to accelerate heart rate and increase neuronal excitability, respectively. Using mouse models, Qian et al. found that βAR-mediated enhancement of CaV1.2 activity in the brain required phosphorylation of Ser1928, whereas βAR-mediated enhancement of CaV1.2 activity in the heart did not require phosphorylation of this residue. In a related study, Nystoriak et al. demonstrated that phosphorylation of Ser1928 in arterial myocytes was required for vasoconstriction during acute hyperglycemia and in diabetic mice. These findings demonstrate tissue-specific differences in CaV1.2 regulation and suggest that it may be possible to design therapies to target this channel in specific tissues. PMID:28119457

  17. Science Signaling Podcast for 24 January 2017: Tissue-specific regulation of L-type calcium channels.

    PubMed

    Hell, Johannes W; Navedo, Manuel F; VanHook, Annalisa M

    2017-01-24

    This Podcast features an interview with Johannes Hell and Manuel Navedo, senior authors of two Research Articles that appear in the 24 January 2017 issue of Science Signaling, about tissue-specific regulation of the L-type calcium channel CaV1.2. This channel is present in many tissues, including the heart, vasculature, and brain, and allows calcium to flow into cells when it is activated. Signaling through the β-adrenergic receptor (βAR) stimulates CaV1.2 activity in heart cells and neurons to accelerate heart rate and increase neuronal excitability, respectively. Using mouse models, Qian et al found that βAR-mediated enhancement of CaV1.2 activity in the brain required phosphorylation of Ser(1928), whereas βAR-mediated enhancement of CaV1.2 activity in the heart did not require phosphorylation of this residue. In a related study, Nystoriak et al demonstrated that phosphorylation of Ser(1928) in arterial myocytes was required for vasoconstriction during acute hyperglycemia and in diabetic mice. These findings demonstrate tissue-specific differences in CaV1.2 regulation and suggest that it may be possible to design therapies to target this channel in specific tissues.Listen to Podcast. Copyright © 2017, American Association for the Advancement of Science.

  18. Quantum simulation of 2D topological physics in a 1D array of optical cavities.

    PubMed

    Luo, Xi-Wang; Zhou, Xingxiang; Li, Chuan-Feng; Xu, Jin-Shi; Guo, Guang-Can; Zhou, Zheng-Wei

    2015-07-06

    Orbital angular momentum of light is a fundamental optical degree of freedom characterized by unlimited number of available angular momentum states. Although this unique property has proved invaluable in diverse recent studies ranging from optical communication to quantum information, it has not been considered useful or even relevant for simulating nontrivial physics problems such as topological phenomena. Contrary to this misconception, we demonstrate the incredible value of orbital angular momentum of light for quantum simulation by showing theoretically how it allows to study a variety of important 2D topological physics in a 1D array of optical cavities. This application for orbital angular momentum of light not only reduces required physical resources but also increases feasible scale of simulation, and thus makes it possible to investigate important topics such as edge-state transport and topological phase transition in a small simulator ready for immediate experimental exploration.

  19. Verification and comparison of four numerical schemes for a 1D viscoelastic blood flow model.

    PubMed

    Wang, Xiaofei; Fullana, Jose-Maria; Lagrée, Pierre-Yves

    2015-01-01

    A reliable and fast numerical scheme is crucial for the 1D simulation of blood flow in compliant vessels. In this paper, a 1D blood flow model is incorporated with a Kelvin-Voigt viscoelastic arterial wall. This leads to a nonlinear hyperbolic-parabolic system, which is then solved with four numerical schemes, namely: MacCormack, Taylor-Galerkin, monotonic upwind scheme for conservation law and local discontinuous Galerkin. The numerical schemes are tested on a single vessel, a simple bifurcation and a network with 55 arteries. The numerical solutions are checked favorably against analytical, semi-analytical solutions or clinical observations. Among the numerical schemes, comparisons are made in four important aspects: accuracy, ability to capture shock-like phenomena, computational speed and implementation complexity. The suitable conditions for the application of each scheme are discussed.

  20. TAU: A 1D radiative transfer code for transmission spectroscopy of extrasolar planet atmospheres

    NASA Astrophysics Data System (ADS)

    Hollis, M. D. J.; Tessenyi, M.; Tinetti, G.

    2013-10-01

    The TAU code is a 1D line-by-line radiative transfer code, which is generally applicable for modelling transmission spectra of close-in extrasolar planets. The inputs are the assumed pressure-temperature profile of the planetary atmosphere, the continuum absorption coefficients and the absorption cross-sections for the trace molecular absorbers present in the model, as well as the fundamental system parameters taken from the published literature. The program then calculates the optical path through the planetary atmosphere of the radiation from the host star, and quantifies the absorption due to the modelled composition in a transmission spectrum of transit depth as a function of wavelength. The code is written in C++, parallelised using OpenMP, and is available for public download and use from http://www.ucl.ac.uk/exoplanets/. Running time: From 0:5 to 500 s, depending on run parameters

  1. TAU: A 1D radiative transfer code for transmission spectroscopy of extrasolar planet atmospheres

    NASA Astrophysics Data System (ADS)

    Hollis, M. D. J.; Tessenyi, M.; Tinetti, G.

    2014-02-01

    The TAU code is a 1D line-by-line radiative transfer code, which is generally applicable for modeling transmission spectra of close-in extrasolar planets. The inputs are the assumed temperature-pressure profile of the planetary atmosphere, the continuum absorption coefficients and the absorption cross-sections for the trace molecular absorbers present in the model, as well as the fundamental system parameters taken from the published literature. The program then calculates the optical path through the planetary atmosphere of the radiation from the host star, and quantifies the absorption due to the modeled composition in a transmission spectrum of transit depth as a function of wavelength. The code is written in C++, parallelized using OpenMP, and is available for public download and use from http://www.ucl.ac.uk/exoplanets/.

  2. Quantum simulation of 2D topological physics in a 1D array of optical cavities

    PubMed Central

    Luo, Xi-Wang; Zhou, Xingxiang; Li, Chuan-Feng; Xu, Jin-Shi; Guo, Guang-Can; Zhou, Zheng-Wei

    2015-01-01

    Orbital angular momentum of light is a fundamental optical degree of freedom characterized by unlimited number of available angular momentum states. Although this unique property has proved invaluable in diverse recent studies ranging from optical communication to quantum information, it has not been considered useful or even relevant for simulating nontrivial physics problems such as topological phenomena. Contrary to this misconception, we demonstrate the incredible value of orbital angular momentum of light for quantum simulation by showing theoretically how it allows to study a variety of important 2D topological physics in a 1D array of optical cavities. This application for orbital angular momentum of light not only reduces required physical resources but also increases feasible scale of simulation, and thus makes it possible to investigate important topics such as edge-state transport and topological phase transition in a small simulator ready for immediate experimental exploration. PMID:26145177

  3. Progress towards alkaline-earth fermions in a 1D uniform potential

    NASA Astrophysics Data System (ADS)

    Reschovsky, Benjamin J.; Barker, Daniel S.; Pisenti, Neal C.; Campbell, Gretchen K.

    2016-05-01

    We present our progress towards realizing a 1D uniform ''box trap'' potential for degenerate fermionic alkaline-earth atoms in order to study highly symmetric SU(N) spin models. Our experiment first generates a degenerate gas of 87 Sr atoms via evaporation in a crossed dipole trap. Next, we plan to load the atoms into an array of 1D box traps formed by a red-detuned 2D optical lattice and blue-detuned end-caps. The end-caps are generated by direct imaging of a digital micromirror device (DMD), which gives us dynamic control of the potential. We report initial characterization of the blue traps and heating rate measurements.

  4. On a 1D nonlocal transport equation with nonlocal velocity and subcritical or supercritical diffusion

    NASA Astrophysics Data System (ADS)

    Lazar, Omar

    2016-11-01

    We study a 1D transport equation with nonlocal velocity with subcritical or supercritical dissipation. For all data in the weighted Sobolev space Hk (wλ,κ) ∩L∞, where k = max ⁡ (0 , 3 / 2 - α) and wλ,κ is a given family of Muckenhoupt weights, we prove a global existence result in the subcritical case α ∈ (1 , 2). We also prove a local existence theorem for large data in H2 (wλ,κ) ∩L∞ in the supercritical case α ∈ (0 , 1). The proofs are based on the use of the weighted Littlewood-Paley theory, interpolation along with some new commutator estimates.

  5. Erk1/2 inhibit synaptic vesicle exocytosis through L type calcium channels

    PubMed Central

    Subramanian, Jaichandar; Morozov, Alexei

    2011-01-01

    L type calcium channels play only a minor role in basal neurotransmitter release in brain neurons, but contribute significantly after induction of plasticity. Very little is known about mechanisms that enable L type calcium channel participation in neurotransmitter release. Here, using mouse primary cortical neurons, we found that inhibition of Erk1/2 enhanced synaptic vesicle exocytosis by increasing calcium influx through L type calcium channels. Furthermore, inhibition of Erk1/2 increased the surface fraction of these channels. These findings indicate a novel inhibitory effect of Erk1/2 on synaptic transmission through L type calcium channels. PMID:21430174

  6. L-Type Calcium Channel Inhibition Contributes to the Proarrhythmic Effects of Aconitine in Human Cardiomyocytes

    PubMed Central

    Wu, Jianjun; Wang, Xiangchong; Chung, Ying Ying; Koh, Cai Hong; Liu, Zhenfeng; Guo, Huicai; Yuan, Qiang; Wang, Chuan; Su, Suwen; Wei, Heming

    2017-01-01

    Aconitine (ACO) is well-known for causing lethal ventricular tachyarrhythmias. While cardiac Na+ channel opening during repolarization has long been documented in animal cardiac myocytes, the cellular effects and mechanism of ACO in human remain unexplored. This study aimed to assess the proarrhythmic effects of ACO in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ACO concentration-dependently (0.3 ~ 3.0 μM) shortened the action potentials (AP) durations (APD) in ventricular-like hiPSC-CMs by > 40% and induced delayed after-depolarization. Laser-scanning confocal calcium imaging analysis showed that ACO decreased the duration and amplitude of [Ca2+]i transients and increased in the beating frequencies by over 60%. Moreover, ACO was found to markedly reduce the L-type calcium channel (LTCC) currents (ICa,L) in hiPSC-CMs associated with a positive-shift of activation and a negative shift of inactivation. ACO failed to alter the peak and late Na+ currents (INa) in hiPSC-CMs while it drastically increased the late INa in Guinea-pig ventricular myocytes associated with enhanced activation/delayed inactivation of INa at -55 mV~ -85 mV. Further, the effects of ACO on ICa,L, INa and the rapid delayed rectifier potassium current (Ikr) were validated in heterologous expression systems by automated voltage-clamping assays and a moderate suppression of Ikr was observed in addition to concentration-dependent ICa,L inhibition. Lastly, increased beating frequency, decreased Ca2+ wave and shortened field potential duration were recorded from hiPSC-CMs by microelectrode arrays assay. In summary, our data demonstrated that LTCC inhibition could play a main role in the proarrhythmic action of ACO in human cardiomyocytes. PMID:28056022

  7. Novel function of α1D L-type calcium channel in the atria.

    PubMed

    Srivastava, Ujala; Aromolaran, Ademuyiwa S; Fabris, Frank; Lazaro, Deana; Kassotis, John; Qu, Yongxia; Boutjdir, Mohamed

    2017-01-22

    Ca entry through atrial L-type Calcium channels (α1C and α1D) play an important role in muscular contraction, regulation of gene expression, and release of hormones including atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP). α1D Ca channel is exclusively expressed in atria, and has been shown to play a key role in the pathogenesis of atrial fibrillation. Recent data have shown that the small conductance calcium-activated potassium channel, SK4 is also atrial specific and also contributes prominently to the secretion of ANP and BNP. However, its functional role in the heart is still poorly understood. Here we used α1D gene heterozygous (α1D(+/-)) mice and HL-1 cells to determine the functional contribution of SK4 channels to α1D-dependent regulation of ANP and BNP secretion in response to endothelin (ET), and/or mechanical stretch. Immunoprecipitation with α1D specific antibody and western blotting with SK4 specific antibody on the immuno-precipitated protein complex showed a band at 50 KDa confirming the presence of SK4 in the complex and provided evidence of interaction between SK4 and α1D channels. Using RT-PCR, we observed a 2.9 fold decrease in expression of Cacna1d (gene encoding α1D) mRNA in atria from α1D(+/-)mice. The decrease in α1D mRNA corresponded with a 4.2 fold decrease in Kcnn4 (gene encoding SK4) mRNA from α1D(+/-) mice. These changes were paralleled with a 77% decrease in BNP serum levels from α1D(+/-) mice. When α1D was knocked down in HL-1cardiomyocytes using CRISPR/Cas9 technology, a 97% decrease in secreted BNP was observed even in cells subjected to stretch and endothelin. In conclusion, our data are first to show that α1D Ca and SK4 channels are coupled in the atria, and that deletion of α1D leads to decreased SK4 mRNA and BNP secretion providing evidence for a novel role of α1D in atrial endocrine function. Elucidating the regulatory factors that underlie the secretory function of atria will identify

  8. Orexin-A potentiates L-type calcium/barium currents in rat retinal ganglion cells.

    PubMed

    Liu, F; Weng, S-J; Yang, X-L; Zhong, Y-M

    2015-10-01

    Two neuropeptides, orexin-A and orexin-B (also called hypocretin-1 and -2), have been implicated in sleep/wake regulation, feeding behaviors via the activation of two subtypes of G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). While the expression of orexins and orexin receptors is immunohistochemically revealed in retinal neurons, the function of these peptides in the retina is largely unknown. Using whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that orexin-A increased L-type-like barium currents (IBa,L) in ganglion cells (GCs), and the effect was blocked by the selective OX1R antagonist SB334867, but not by the OX2R antagonist TCS OX2 29. The orexin-A effect was abolished by intracellular dialysis of GDP-β-S/GPAnt-2A, a Gq protein inhibitor, suggesting the mediation of Gq. Additionally, during internal dialysis of the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor U73122, orexin-A did not change the IBa,L of GCs, whereas the orexin-A effect persisted in the presence of the phosphatidylcholine (PC)-PLC inhibitor D609. The orexin-A-induced potentiation was not seen with internal infusion of Ca(2+)-free solution or when inositol 1,4,5-trisphosphate (IP3)-sensitive Ca(2+) release from intracellular stores was blocked by heparin/xestospongins-C. Moreover, the orexin-A effect was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, but was eliminated when PKC was inhibited by bisindolylmaleimide IV (Bis-IV)/Gö6976. Neither adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) nor guanosine 3',5'-cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway was likely involved, as orexin-A persisted to potentiate the IBa,L of GCs no matter these two pathways were activated or inhibited. These results suggest that, by activating OX1R, orexin-A potentiates the IBa,L of rat GCs through a distinct Gq/PI-PLC/IP3/Ca(2+)/PKC signaling pathway.

  9. Controlled structures of a 1D chiral metallosalen polymer by photo- and solvent-induced partial depolymerization.

    PubMed

    Xi, Xiaobing; Dong, Taiwei; Li, Gao; Cui, Yong

    2011-04-07

    A 1D chiral metallosalen polymer with free pyridine groups is self-assembled and its molecular weight, conformation, architecture and optical property are controlled by depolymerization through sunlight irradiation or alcohol inclusions.

  10. Sensitivity of transpiration to subsurface properties: Exploration with a 1-D model

    DOE PAGES

    Vrettas, Michail D.; Fung, Inez Y.

    2017-05-04

    The amount of moisture transpired by vegetation is critically tied to the moisture supply accessible to the root zone. In a Mediterranean climate, integrated evapotranspiration (ET) is typically greater in the dry summer when there is an uninterrupted period of high insolation. We present a 1-D model to explore the subsurface factors that may sustain ET through the dry season. The model includes a stochastic parameterization of hydraulic conductivity, root water uptake efficiency, and hydraulic redistribution by plant roots. Model experiments vary the precipitation, the magnitude and seasonality of ET demand, as well as rooting profiles and rooting depths ofmore » the vegetation. The results show that the amount of subsurface moisture remaining at the end of the wet winter is determined by the competition among abundant precipitation input, fast infiltration, and winter ET demand. The weathered bedrock retains math formula of the winter rain and provides a substantial moisture reservoir that may sustain ET of deep-rooted (>8 m) trees through the dry season. A small negative feedback exists in the root zone, where the depletion of moisture by ET decreases hydraulic conductivity and enhances the retention of moisture. Hence, hydraulic redistribution by plant roots is impactful in a dry season, or with a less conductive subsurface. Suggestions for implementing the model in the CESM are discussed.« less

  11. Sensitivity of transpiration to subsurface properties: Exploration with a 1-D model

    NASA Astrophysics Data System (ADS)

    Vrettas, Michail D.; Fung, Inez Y.

    2017-06-01

    The amount of moisture transpired by vegetation is critically tied to the moisture supply accessible to the root zone. In a Mediterranean climate, integrated evapotranspiration (ET) is typically greater in the dry summer when there is an uninterrupted period of high insolation. We present a 1-D model to explore the subsurface factors that may sustain ET through the dry season. The model includes a stochastic parameterization of hydraulic conductivity, root water uptake efficiency, and hydraulic redistribution by plant roots. Model experiments vary the precipitation, the magnitude and seasonality of ET demand, as well as rooting profiles and rooting depths of the vegetation. The results show that the amount of subsurface moisture remaining at the end of the wet winter is determined by the competition among abundant precipitation input, fast infiltration, and winter ET demand. The weathered bedrock retains ˜30% of the winter rain and provides a substantial moisture reservoir that may sustain ET of deep-rooted (>8 m) trees through the dry season. A small negative feedback exists in the root zone, where the depletion of moisture by ET decreases hydraulic conductivity and enhances the retention of moisture. Hence, hydraulic redistribution by plant roots is impactful in a dry season, or with a less conductive subsurface. Suggestions for implementing the model in the CESM are discussed.

  12. Perforated patch recording of L-type calcium current with beta-escin in guinea pig ventricular myocytes.

    PubMed

    Fu, Li-Ying; Wang, Fang; Chen, Xue-Song; Zhou, Hong-Yi; Yao, Wei-Xing; Xia, Guo-Jin; Jiang, Ming-Xing

    2003-11-01

    To establish a perforated patch recording (PPR) mode with beta-escin and compare L-type calcium current (I(Ca,L)) recorded under PPR and normal whole-cell recording (WCR) condition in isolated guinea-pig ventricular myocytes. Single myocytes were dissociated by enzymatic dissociation method. beta-escin was added to the pipette solution to perforate the cell membrane and obtain PPR mode. I(Ca,L) was recorded using PPR and WCR techniques. beta-Escin 20, 25, and 30 micromol/L could permeabilize the cell membrane and obtain PPR mode. With beta-escin 25 micromol/L, the success rate was highest (16/17, 94 %) and the time required for permibilization was 2-15 (8+/-4) min. Run-down of I(Ca,L) was considerably slower in PPR than in WCR condition. The amplitude of I(Ca,L) was decreased by 36 % at 20 min after the formation of WCR, while it was slowly decreased by 8 % at 30 min after the formation of PPR. The current-voltage relation (I-V) curves, activation and inactivation curves of I(Ca,L) were not significantly different between WCR and PPR. The inactivation rate of ICa,L was slower in PPR than in WCR, the faster inactivation time constant (tau(f)) was longer in PPR than in WCR at membrane potentials of -20 mV -- +10 mV (n=6, P<0.05), and the slower time constant (tau(s)) was also longer in PPR than in WCR at membrane potentials of -10 mV to +10 mV (n=6, P<0.05). There was no significant difference between the activation rate in WCR and PPR. Using beta-escin 25 micromol/L can easily obtain stable PPR in isolated guinea-pig ventricular myocytes, and this method is useful in dealing with channels, which show run-down under normal WCR such as L-type Ca channel.

  13. Metformin enhances anti-tumor effect of L-type amino acid transporter 1 (LAT1) inhibitor.

    PubMed

    Ueno, Seiji; Kimura, Toru; Yamaga, Takashi; Kawada, Akihiko; Ochiai, Toshiaki; Endou, Hitoshi; Sakurai, Hiroyuki

    2016-06-01

    In many cancer cells, L-type amino acid transporter 1 (LAT1) transports neutral amino acids with bulky side chain, which activate mammalian target of rapamycin (mTOR) to cause cell proliferation. An anti-diabetic drug, metformin, has been shown to activate AMP-activated protein kinase (AMPK), which leads to inhibition of mTOR. LAT1 inhibition in combination with metformin could result in more prominent suppression of mTOR activity. Anti-proliferative effect of a newly developed LAT1 specific inhibitor JPH203 in combination with metformin is evaluated in 2 head and neck cancer cell lines, Ca9-22 and HEp-2 cells and in nude mice inoculated with Ca9-22 cells. By MTT assay, 0.5 mM metformin inhibited proliferation of Ca9-22 cells to 70% of control. In the presence of 100 μM JPH203, proliferation of Ca9-22 cells was inhibited to 60% of control. By combining these 2 drugs, proliferation of Ca9-22 was significantly inhibited to 40% of control. However, this regimen was not very effective against HEp-2 cells. This combination also suppressed in vivo growth of Ca9-22 cells in a xenotransplant model. A combination of anti-LAT1 drug with metformin may be an effective anti-proliferative therapy for certain subsets of cancers. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  14. A 1D plug flow reactor as validation tool for reactive transport simulations

    NASA Astrophysics Data System (ADS)

    Battaïa, G.; Garcia, D.

    2012-04-01

    Predictions in CO2 geological sequestration involve a broad range of earth sciences linked in complex models. Amongst the processes commonly described, fluid-rock interactions are both a central issue and a source of discomfort for modelers since it has to deal with 1) kinetics data obtained through experimental procedures that dramatically differ from natural systems and 2) reactive surface model that are very diverse and often empirical. This study presents a new type of plug flow reactor developed to provide an experimental validation of reactive transport simulations. This is a 1D pressurized packed-bed plug-flow reactor containing a granular mixture as a porous medium. This mixture is composed of a reactive solids and unreactive quartz used to set an adequate ratio between fluid and reactive mineral to control the front velocity. A seven sampling valve unit allows concentration profiles of the reacting fluid to be captured at any time. One the one side, a low reaction rate (diopside, HNO3, pH 2) produces linear profile resulting from a constant dissolution rate along the reactor length. But on the other side, when performing the reaction of CO2 saturated solutions (5 bar) at 40°C with dolomite it gives rise to dissolution fronts migrating downstream. A proper projection of experimental data reveals a dynamic steady state of front shape is reached. Texture of the mineral recovered at the end of the experiment is quantified by Hg-porosimetry and these results are linked to SEM observations. Altogether, this provides a robust way for the parameterization of a reactive surface area model.

  15. Testing the accuracy of a 1-D volcanic plume model in estimating mass eruption rate

    NASA Astrophysics Data System (ADS)

    Mastin, Larry G.

    2014-03-01

    During volcanic eruptions, empirical relationships are used to estimate mass eruption rate from plume height. Although simple, such relationships can be inaccurate and can underestimate rates in windy conditions. One-dimensional plume models can incorporate atmospheric conditions and give potentially more accurate estimates. Here I present a 1-D model for plumes in crosswind and simulate 25 historical eruptions where plume height Hobs was well observed and mass eruption rate Mobs could be calculated from mapped deposit mass and observed duration. The simulations considered wind, temperature, and phase changes of water. Atmospheric conditions were obtained from the National Center for Atmospheric Research Reanalysis 2.5° model. Simulations calculate the minimum, maximum, and average values (Mmin, Mmax, and Mavg) that fit the plume height. Eruption rates were also estimated from the empirical formula Mempir = 140Hobs4.14 (Mempir is in kilogram per second, Hobs is in kilometer). For these eruptions, the standard error of the residual in log space is about 0.53 for Mavg and 0.50 for Mempir. Thus, for this data set, the model is slightly less accurate at predicting Mobs than the empirical curve. The inability of this model to improve eruption rate estimates may lie in the limited accuracy of even well-observed plume heights, inaccurate model formulation, or the fact that most eruptions examined were not highly influenced by wind. For the low, wind-blown plume of 14-18 April 2010 at Eyjafjallajökull, where an accurate plume height time series is available, modeled rates do agree better with Mobs than Mempir.

  16. Testing the accuracy of a 1-D volcanic plume model in estimating mass eruption rate

    USGS Publications Warehouse

    Mastin, Larry G.

    2014-01-01

    During volcanic eruptions, empirical relationships are used to estimate mass eruption rate from plume height. Although simple, such relationships can be inaccurate and can underestimate rates in windy conditions. One-dimensional plume models can incorporate atmospheric conditions and give potentially more accurate estimates. Here I present a 1-D model for plumes in crosswind and simulate 25 historical eruptions where plume height Hobs was well observed and mass eruption rate Mobs could be calculated from mapped deposit mass and observed duration. The simulations considered wind, temperature, and phase changes of water. Atmospheric conditions were obtained from the National Center for Atmospheric Research Reanalysis 2.5° model. Simulations calculate the minimum, maximum, and average values (Mmin, Mmax, and Mavg) that fit the plume height. Eruption rates were also estimated from the empirical formula Mempir = 140Hobs4.14 (Mempir is in kilogram per second, Hobs is in kilometer). For these eruptions, the standard error of the residual in log space is about 0.53 for Mavg and 0.50 for Mempir. Thus, for this data set, the model is slightly less accurate at predicting Mobs than the empirical curve. The inability of this model to improve eruption rate estimates may lie in the limited accuracy of even well-observed plume heights, inaccurate model formulation, or the fact that most eruptions examined were not highly influenced by wind. For the low, wind-blown plume of 14–18 April 2010 at Eyjafjallajökull, where an accurate plume height time series is available, modeled rates do agree better with Mobs than Mempir.

  17. Modelling turbulent vertical mixing sensitivity using a 1-D version of NEMO

    NASA Astrophysics Data System (ADS)

    Reffray, G.; Bourdalle-Badie, R.; Calone, C.

    2015-01-01

    Through two numerical experiments, a 1-D vertical model called NEMO1D was used to investigate physical and numerical turbulent-mixing behaviour. The results show that all the turbulent closures tested (k+l from Blanke and Delecluse, 1993, and two equation models: generic length scale closures from Umlauf and Burchard, 2003) are able to correctly reproduce the classical test of Kato and Phillips (1969) under favourable numerical conditions while some solutions may diverge depending on the degradation of the spatial and time discretization. The performances of turbulence models were then compared with data measured over a 1-year period (mid-2010 to mid-2011) at the PAPA station, located in the North Pacific Ocean. The modelled temperature and salinity were in good agreement with the observations, with a maximum temperature error between -2 and 2 °C during the stratified period (June to October). However, the results also depend on the numerical conditions. The vertical RMSE varied, for different turbulent closures, from 0.1 to 0.3 °C during the stratified period and from 0.03 to 0.15 °C during the homogeneous period. This 1-D configuration at the PAPA station (called PAPA1D) is now available in NEMO as a reference configuration including the input files and atmospheric forcing set described in this paper. Thus, all the results described can be recovered by downloading and launching PAPA1D. The configuration is described on the NEMO site (http://www.nemo-ocean.eu/Using-NEMO/Configurations/C1D_PAPA). This package is a good starting point for further investigation of vertical processes.

  18. Modelling turbulent vertical mixing sensitivity using a 1-D version of NEMO

    NASA Astrophysics Data System (ADS)

    Reffray, G.; Bourdalle-Badie, R.; Calone, C.

    2014-08-01

    Through two numerical experiments, a 1-D vertical model called NEMO1D was used to investigate physical and numerical turbulent-mixing behaviour. The results show that all the turbulent closures tested (k + l from Blanke and Delecluse, 1993 and two equation models: Generic Lengh Scale closures from Umlauf and Burchard, 2003) are able to correctly reproduce the classical test of Kato and Phillips (1969) under favourable numerical conditions while some solutions may diverge depending on the degradation of the spatial and time discretization. The performances of turbulence models were then compared with data measured over a one-year period (mid-2010 to mid-2011) at the PAPA station, located in the North Pacific Ocean. The modelled temperature and salinity were in good agreement with the observations, with a maximum temperature error between -2 and 2 °C during the stratified period (June to October). However the results also depend on the numerical conditions. The vertical RMSE varied, for different turbulent closures, from 0.1 to 0.3 °C during the stratified period and from 0.03 to 0.15 °C during the homogeneous period. This 1-D configuration at the PAPA station (called PAPA1D) is now available in NEMO as a reference configuration including the input files and atmospheric forcing set described in this paper. Thus, all the results described can be recovered by downloading and launching PAPA1D. The configuration is described on the NEMO site (http://www.nemo-ocean.eu/Using-NEMO/Configurations/C1D_PAPA). This package is a good starting point for further investigation of vertical processes.

  19. Exploring triggers for polar tropospheric ODEs, using a 1-D snow photochemistry model (MISTRA-SNOW).

    NASA Astrophysics Data System (ADS)

    Buys, Z.; Jones, A. E.; von Glasow, R.

    2012-04-01

    Tropospheric Ozone Depletion Events (ODEs) have been known to occur in polar regions for over 20 years. During such events, ozone concentrations can fall from background amounts to below instrumental detection limits within a few minutes and remain suppressed for on the order of hours to days. The chemical destruction of ozone is driven by halogens (especially bromine radicals) that have a source associated with the sea ice zone. Although our knowledge of ODEs has increased greatly since their discovery, some of the key processes involved are not yet fully understood. We now know that heterogeneous reactions lead to the activation of Br2 and BrCI, via uptake of HOBr onto aqueous salt solutions /aerosol/ surface snowpack (Fickert et al., 1999), and it is widely accepted that bromine catalytic reaction cycles (the 'bromine explosion') in the gas phase are responsible for surface ozone destruction (Simpson et al., 2007). There is still much debate over the source of bromine in the atmosphere that drives ODEs, but there is strong evidence to suggest a source associated with the sea ice zone. A 1D Marine Boundary Layer (MBL) chemistry model (MISTRA; von Glasow et al., 2002) has been modified to be representative of Antarctic conditions. The chemistry module includes chemical reactions in the gas phase, in and on aerosol particles and takes into account transfer between the gas and aqueous phase. A new snow-photochemistry module has been developed which includes chemistry which takes place in the quasi-liquid layer on aerosol (Thomas et al., 2011), which is of great importance to our understanding of the chemistry which initiates a bromine explosion. Here we use this newly developed 1-D snow photochemistry model (MISTRA-SNOW) to look at some of the suggested triggers for, and the different meteorological conditions required to produce, tropospheric ODEs in polar regions.

  20. Testing the early Mars H2-CO2 greenhouse hypothesis with a 1-D photochemical model

    NASA Astrophysics Data System (ADS)

    Batalha, Natasha; Domagal-Goldman, Shawn D.; Ramirez, Ramses; Kasting, James F.

    2015-09-01

    A recent study by Ramirez et al. (Ramirez, R.M. et al. [2014]. Nat. Geosci. 7(1), 59-63. http://www.nature.com/doifinder/10.1038/ngeo2000 (accessed 16.09.14)) demonstrated that an atmosphere with 1.3-4 bar of CO2 and H2O, in addition to 5-20% H2, could have raised the mean annual and global surface temperature of early Mars above the freezing point of water. Such warm temperatures appear necessary to generate the rainfall (or snowfall) amounts required to carve the ancient martian valleys. Here, we use our best estimates for early martian outgassing rates, along with a 1-D photochemical model, to assess the conversion efficiency of CO, CH4, and H2S to CO2, SO2, and H2. Our outgassing estimates assume that Mars was actively recycling volatiles between its crust and interior, as Earth does today. H2 production from serpentinization and deposition of banded iron-formations is also considered. Under these assumptions, maintaining an H2 concentration of ˜1-2% by volume is achievable, but reaching 5% H2 requires additional H2 sources or a slowing of the hydrogen escape rate below the diffusion limit. If the early martian atmosphere was indeed H2-rich, we might be able to see evidence of this in the rock record. The hypothesis proposed here is consistent with new data from the Curiosity Rover, which show evidence for a long-lived lake in Gale Crater near Mt. Sharp. It is also consistent with measured oxygen fugacities of martian meteorites, which show evidence for progressive mantle oxidation over time.

  1. A 1D P-wave velocity model of the Gargano promontory (south-eastern Italy)

    NASA Astrophysics Data System (ADS)

    de Lorenzo, Salvatore; Michele, Maddalena; Emolo, Antonio; Tallarico, Andrea

    2017-07-01

    We investigate the elastic properties of the crust in the Gargano promontory, located in the northern part of the Apulia region (Southeastern Italy). Starting on April, 2013, a local-scale seismic network, composed of 12 short-period (1 Hz) seismic stations, was deployed on the Gargano promontory. Starting on October, 2013, the network was integrated with the recordings of nine seismic stations managed by the Italian Institute of Geophysics and Volcanology (INGV). The network recorded more than 1200 seismic events in about 15 months of data acquisition, with more than 700 small magnitude events localized in the Gargano promontory and surrounding areas. A Wadati-modified method allowed us to infer VP/VS = 1.73 for the area. A subset of about 400 events having a relatively smaller azimuthal gap (<200°) was selected to calibrate a 1D P-wave velocity model of the area, using the VELEST inversion code. The preferred model was obtained from the average of ten velocity models, each of them representing the inversion result from given initial velocity models, calibrated on previous geological and geophysical studies in the area. The results obtained under the assumption that VP could decrease with depth are unstable, with very different depths of the top of low-velocity layers. Therefore, the velocity model was obtained from the average of the results obtained under the assumption that VP cannot decrease with depth. A strong reduction of both RMS (about 58%) and errors on the location of the events was obtained with respect to the starting model. The final velocity model shows a strong velocity gradient in the upper 5 km of the crust and a small increase (from 6.7 to 7 km) at 30 km of depth. The epicenters of relocated events do not show clear correlations with the surface projection of known seismic faults. A cluster of the epicenters of the relocated events intersects almost perpendicularly the Candelaro fault trace at the surface.

  2. L-type calcium channels may regulate neurite initiation in cultured chick embryo brain neurons and N1E-115 neuroblastoma cells.

    PubMed

    Audesirk, G; Audesirk, T; Ferguson, C; Lomme, M; Shugarts, D; Rosack, J; Caracciolo, P; Gisi, T; Nichols, P

    1990-08-01

    The intracellular free Ca2+ concentration, [Ca2+]i, plays an important role in regulating neurite growth in cultured neurons. Insofar as [Ca2+]i is partly a function of Ca2+ influx through voltage-sensitive calcium channels (VSCC), Ca2+ entry through VSCC should influence neurite growth. Vertebrate neurons may possess several types of VSCC. The most frequently described VSCC types are usually designated L, T and N. In most preparations, these VSCC types respond differently to certain pharmacological agents, including Cd2+, Ni2+, the dihydropyridines nifedipine and BAY K8644, and the aminoglycoside antibiotics. We used these agents to study the role of Ca2+ influx in regulating neurite initiation and length in cultures of chick embryo brain neurons and N1E-115 mouse neuroblastoma cells. In chick neurons, nifedipine and Cd2+ (less than 50 microM), which have been reported to inhibit L-type channels, reduced neurite initiation, but not mean neurite length. Ni2+ (less than 100 microM), reported to inhibit T-type channels, had no effect on either initiation or length. Low concentrations of most aminoglycosides (less than 300 microM), reported to inhibit N-type channels, had no effect on neurite initiation, but high concentrations of streptomycin (great than 300 microM), reported to inhibit both L- and N-type channels, reduced neurite initiation. BAY K8644, which enhances current flow through L-type channels, had no effect except at high concentration (50 microM), which inhibited initiation. N1E-115 neuroblastoma cells have been reported to contain L-type and T-type channels, but thus far no channel similar to the N-type has been described. In cultured N1E-115 cells, nifedipine (5 microM), Cd2+ (5 microM), and streptomycin (200 microM) reduced neurite initiation, while nickel (50 microM) and neomycin (100 microM) did not affect initiation. None of these agents altered neurite length. In N1E-115 cells, whole-cell voltage clamp recordings showed that nifedipine and Cd2

  3. Localization and functional modification of L-type voltage-gated calcium channels in equine spermatozoa from fresh and frozen semen.

    PubMed

    Albrizio, M; Moramarco, A M; Nicassio, M; Micera, E; Zarrilli, A; Lacalandra, G M

    2015-02-01

    It is well known that insemination of cryopreserved semen always results in lower fertility when compared with fresh semen, but there is an increased interest and demand for frozen equine semen by the major breeder associations because of the utility arising from semen already "on hand" at breeding time. In this article, we report that equine sperm cells express L-type voltage-gated calcium channels; their localization is restricted to sperm neck and to the principal piece of the tail in both fresh and frozen-thawed spermatozoa. We also studied the causes of cryoinjury at the membrane level focusing on the function of L-type calcium channels. We report that in cryopreserved spermatozoa the mean basal value of [Ca(2+)]i is higher than that of spermatozoa from fresh semen (447.130 vs. 288.3 nM; P < 0.001) and L-type channels function differently in response to their agonist and antagonist in relation to semen condition (fresh or frozen-thawed). We found that on addition of agonist to the culture medium, the increase in intracellular calcium concentrations ([Ca(2+)]i) was greater in frozen semen than in fresh semen (Δ[Ca(2+)]i = 124.59 vs. 16.04 nM; P < 0.001), whereas after the addition of antagonist the decrease in [Ca(2+)]i was lower in frozen semen than in fresh semen (Δ[Ca(2+)]i = 32.5 vs. 82.5 nM; P < 0.001). In this article, we also discuss the impact of cryopreservation on sperm physiology.

  4. Activation of L-type calcium channel in twitch skeletal muscle fibres of the frog.

    PubMed Central

    Francini, F; Bencini, C; Squecco, R

    1996-01-01

    1. The activation of the L-type calcium current (ICa) was studied in normally polarized (-100 mV) cut skeletal muscle fibres of the frog with the double Vaseline-gap voltage-clamp technique. Both external and internal solutions were Ca2+ buffered. Solutions were made in order to minimize all but the Ca2+ current. 2. The voltage-dependent components of the time course of activation were determined by two procedures: fast and slow components were evaluated by multiexponential fitting to current traces elicited by long voltage pulses (5 s) after removing inactivation; fast components were also determined by short voltage pulses having different duration (0.5-70 ms). 3. The components of deactivation were evaluated after removing the charge-movement current from the total tail current by the difference between two short (50 and 70 ms) voltage pulses to 10 mV, moving the same intramembrane charge. Two exponential components, fast and slow (time constants, 6 +/- 0.3 and 90 +/- 7 ms at -100 mV; n = 26), were found. 4. The time onset of ICa was evaluated either by multiexponential fitting to the ICa activation or by pulses of different duration to test the beginning of the 'on' and 'off' inequality. This was at about 2 ms, denoting that it was very early. 5. The time constant vs. voltage plots indicated the presence of four voltage-dependent components in the activation pathway. Various kinetic models are discussed. Models with independent transitions, like a Hodgkin-Huxley scheme, were excluded. Suitable models were a five-state sequential and a four-state cyclic with a branch scheme. The latter gave the best simulation of the data. 6. The steady-state activation curve saturated at high potentials. It had a half-voltage value of 1 +/- 0.2 mV and the opening probability was only 0.82 +/- 0.2 at 20 mV (n = 32). This result implies a larger number of functional calcium channels than was previously supposed and is in agreement with the number of dihydropyridine (DHP

  5. Parameter sensitivities in a 1-D model for DMS and sulphur cycling in the upper ocean

    NASA Astrophysics Data System (ADS)

    Steiner, N.; Denman, K.

    2008-07-01

    We have developed a marine DMS (dimethylsulfide) module and implemented it in a 1-D coupled atmosphere-ocean-biogeochemical model. In developing the marine sulphur model we have found that several parameters used in the model are not known to even an order of magnitude. Our approach is used to test the model's sensitivity to these parameters. A parameter change of ±25% is applied to test the respective range of changes in the DMS fluxes. The model is run for a 3-year time period as well as for the time period of the Subarctic Ecosystem Response to Iron Enrichment Study (SERIES) in July 2002. The simulated seasonal cycle is in agreement with available observations: Near surface DMS concentrations vary from 1.5nmolL-1 in winter to 13.5nmolL-1 in summer. Simulated DMS production is found to be most sensitive to variations of the S:N ratio and the bacterial consumption rate of DMS. Implementing light or UV limited bacterial activity shows a negligible effect in winter and increases DMS concentrations by 0.2- 0.6nmolL-1 in summer. Similarly a yield increase under UV stress increases summer values by 1- 2nmolL-1. The simulated diel cycle in surface DMS concentration is no more than 2.5nmolL-1, even when light-dependent changes in bacterial activity are considered. Simulating the DMS response to iron fertilization with the standard run leads to overestimation during an initial bloom of small phytoplankton. While implementing light-dependent bacterial activity has a minor effect, the implementation of yields that depend on nutrient availability significantly improves the results. The model confirms earlier results showing the importance of including atmospheric DMS concentrations in gas flux calculations when there are high surface concentrations and small atmospheric boundary layer heights. Simulated summer concentrations in the upper layer can be underestimated by 2nmolL-1 or more if the atmospheric concentration is set to zero. Our study shows that inclusion of

  6. Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type Cav channels.

    PubMed

    Klint, Julie K; Berecki, Géza; Durek, Thomas; Mobli, Mehdi; Knapp, Oliver; King, Glenn F; Adams, David J; Alewood, Paul F; Rash, Lachlan D

    2014-05-15

    Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba(2+) currents (IBa) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited IBa mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1-E5) or C-terminus (ΔW35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Modulation of L-type calcium current by intracellular magnesium in differentiating cardiomyocytes derived from induced pluripotent stem cells.

    PubMed

    Nguemo, Filomain; Semmler, Judith; Reppel, Michael; Hescheler, Jürgen

    2014-06-15

    Intracellular Mg(2+), which is implicated in arrhythmogenesis and transient cardiac ischemia, inhibits L-type Ca(2+) calcium channel current (ICaL) of adult cardiomyocytes (CMs). We take the advantage of an in vitro model of CMs based on induced pluripotent stem cells to investigate the effects of intracellular Mg(2+) on the phosphorylation or dephosphorylation processes of L-type Ca(2+) channels (LTCCs) at early and late stages of cardiac cell differentiation. Using the whole-cell patch-clamp technique, we demonstrate that increasing intracellular Mg(2+) concentration [Mg(2+)]i from 0.2 to 5 mM markedly reduced the peak of ICaL density, showing less effect on both the activation and inactivation properties in the late differentiation stage (LDS) of CMs more so than in the early differentiation stage (EDS). Increasing the [Mg(2+)]i from 0.2 to 2 mM in the presence of cAMP-dependent protein kinase A significantly decreased ICaL in LDS (70%) and in EDS (36%) CMs. In addition, the effect of forskolin was greatly attenuated in the presence of 2 mM [Mg(2+)]i in LDS but not in EDS CMs. The effect of forskolin was enhanced in the presence of ATP-γ-S in LDS CMs compared with EDS CMs. The exposure of both EDS and LDS CMs to 2 mM [Mg(2+)]i considerably reduced the effects of isobutylmethylxanthine (IBMX) and okadaic acid on ICaL. Our results provide evidence for differential regulation of LTCCs activities by cytosolic Mg(2+) concentration in developing cardiac cells and confirm that Mg(2+) acts under conditions that favor opening of the LTCCs caused by channel phosphorylation.

  8. Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.

    PubMed

    Quignard, J F; Mironneau, J; Carricaburu, V; Fournier, B; Babich, A; Nurnberg, B; Mironneau, C; Macrez, N

    2001-08-31

    Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.

  9. Targeting of Protein Phosphatases PP2A and PP2B to the C-terminus of the L-type Calcium Channel Cav1.2†

    PubMed Central

    Xu, Hui; Ginsburg, Kenneth S.; Hall, Duane D.; Zimmermann, Maike; Stein, Ivar S.; Zhang, Mingxu; Tandan, Samvit; Hill, Joseph A.; Horne, Mary C.; Bers, Donald; Hell, Johannes W.

    2010-01-01

    The L-type Ca2+ channel Cav1.2 forms macromolecular signaling complexes that comprise the β2 adrenergic receptor, trimeric Gs protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA1) for efficient signaling in heart and brain. The protein phosphatases PP2A and PP2B are part of this complex. PP2A counteracts increase in Cav1.2 channel activity by PKA and other protein kinases, whereas PP2B can either augment or decrease Cav1.2 currents in cardiomyocytes depending on the precise experimental conditions. We found that PP2A binds to two regions in the C-terminus of the central, pore-forming α1 subunit of Cav1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residue 1971. Injection of a peptide that contained residues 1965-1971 and displaced PP2A but not PP2B from endogenous Cav1.2 increased basal and isoproterenol-stimulated L-type Ca2+ currents in acutely isolated cardiomyocytes. Together with our biochemical data, these physiological results indicate that anchoring of PP2A at this site of Cav1.2 in the heart negatively regulates cardiac L-type currents, likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases. PMID:21053940

  10. Rational design of D-A1-D-A2 conjugated polymers with superior spectral coverage.

    PubMed

    Hedström, Svante; Tao, Qiang; Wang, Ergang; Persson, Petter

    2015-10-28

    The spectral coverage of a light-harvesting polymer largely determines the maximum achievable photocurrent in organic photovoltaics, and therefore constitutes a crucial parameter for improving their performance. The D-A1-D-A2 copolymer motif is a new and promising design strategy for extending the absorption range by incorporating two acceptor units with complementary photoresponses. The fundamental factors that promote an extended absorption are here determined for three prototype D-A1-D-A2 systems through a combination of experimental and computational methods. Systematic quantum chemical calculations are then used to reveal the intrinsic optical properties of ten further D-A1-D-A2 polymer candidates. These investigated polymers are all predicted to exhibit intense primary absorption peaks at 615-954 nm, corresponding to charge-transfer (CT) transitions to the stronger acceptor, as well as secondary absorption features at 444-647 nm that originate from CT transitions to the weaker acceptors. Realization of D-A1-D-A2 polymers with superior spectral coverage is thereby found to depend critically on the spatial and energetic separation between the two distinct acceptor LUMOs. Two promising D-A1-D-A2 copolymer candidates were finally selected for further theoretical and experimental study, and demonstrate superior light-harvesting properties in terms of significantly extended spectral coverage. This demonstrates great potential for enhanced light-harvesting in D-A1-D-A2 polymers via multiple absorption features compared to traditional D-A polymers.

  11. A 1-D radiative conductive model to study the SOIR/VEx thermal profiles

    NASA Astrophysics Data System (ADS)

    Mahieux, Arnaud; Erwin, Justin T.; Chamberlain, Sarah; Robert, Séverine; Carine Vandaele, Ann; Wilquet, Valérie; Thomas, Ian; Yelle, Roger V.; Bertaux, Jean-Loup

    2015-04-01

    SOIR is an infrared spectrometer on board Venus Express that probes the Venus terminator region since 2006. The measurements are taken on the morning and evening sides of the terminator, covering all latitudes from the North Pole to the South Pole. Its wavelength range - 2.2 to 4.3 μm - allows a detailed chemical inventory of the Venus atmosphere [1-5], such as CO2, CO, H2O, HCl, HF, SO2 and aerosols. CO2 is detected from 70 km up to 165 km, CO from 70 km to 140 km, and the minor species typically below 110 km down to 70 km. Number density profiles of these species are computed from the measured spectra. Temperature profiles are obtained while computing the spectral inversion of the CO2 spectra combined with the hydrostatic law [6]. These temperature measurements show a striking permanent temperature minimum (at 125 km) and a weaker temperature maximum (over 100-115 km). The time variability of the CO2 density profiles spans over two orders of magnitude, and a clear trend is seen with latitude. The temperature variations are also important, of the order of 35 K for a given pressure level, but the latitude variation are small. Miss-RT, a 1D radiative transfer model has been developed to reproduce the SOIR terminator profiles, derived from the Mars thermosphere code presented in [7]. This model has been expanded to better account for the CO2, CO, and O non-LTE radiative heating and cooling processes which have to be considered in the dense atmosphere of Venus. Radiative cooling by minor species detected by SOIR (e.g. HCl, SO2, and H2O) are found to be small in comparison to the 15 μm CO2 cooling. Aerosol cooling in the 60-90km altitude range may be important to the thermal balance. There is a good agreement between the 1D model temperature profile and the mean SOIR temperature profile. Further we can suggest parameters that can be adjusted to improve the agreement between the model and measurements. The remaining differences can be attributed to the atmosphere

  12. L-Type Voltage-Dependent Calcium Channel Currents of Cerebral Arterial Smooth Muscle Cells are Increased by 2-Week Hindlimb Unweighting in Rats

    NASA Astrophysics Data System (ADS)

    Tang, Hao; Xue, Jun-Hui; Bai, Yun-Gang; Xie, Man-Jiang; Bao, Jun-Xiang; Ma, Jin

    2008-06-01

    To investigate alterations of L-type voltage-dependent calcium channel (CaL) in cerebral vascular smooth muscle cells isolated from rats subjected to a two-week simulated weightlessness, and influence of Bay K 8644 (an agonist of CaL) to the channel currents. Tail-suspended rat model was used to simulate the effects of microgravity. Whole-cell patch-clamp technique was used to record CaL currents before and after Bay K 8644 treatment, with intracellular Ca2+ concentration maintained physiological level. The corresponding parameters such as steady state activation and inactivation curves were also recorded. Whole-cell CaL current densities increased obviously, and sensitivity of CaL to Bay K 8644 also increased in cerebral vascular smooth muscle cells from suspension group. But membrane capacitance (Cm), access resistance (Ra), and other parameters of CaL such as steady state activation / inactivation curves have no significant changes compared with those of control group. These results suggest that enhanced CaL function of cerebrovascular smooth muscle cells induced by simulated microgravity may be one of the electrophysiological mechanisms that mediate enhanced vasoreactivity of cerebrovascular smooth muscle cells during adaptation to simulated weightlessness in rats.

  13. {beta}1-Adrenergic receptor activation induces mouse cardiac myocyte death through both L-type calcium channel-dependent and -independent pathways.

    PubMed

    Wang, Wei; Zhang, Hongyu; Gao, Hui; Kubo, Hajime; Berretta, Remus M; Chen, Xiongwen; Houser, Steven R

    2010-08-01

    Cardiac diseases persistently increase the contractility demands of cardiac myocytes, which require activation of the sympathetic nervous system and subsequent increases in myocyte Ca(2+) transients. Persistent exposure to sympathetic and/or Ca(2+) stress is associated with myocyte death. This study examined the respective roles of persistent beta-adrenergic receptor (beta-AR) agonist exposure and high Ca(2+) concentration in myocyte death. Ventricular myocytes (VMs) were isolated from transgenic (TG) mice with cardiac-specific and inducible expression of the beta(2a)-subunit of the L-type Ca(2+) channel (LTCC). VMs were cultured, and the rate of myocyte death was measured in the presence of isoproterenol (ISO), other modulators of Ca(2+) handling and the beta-adrenergic system, and inhibitors of caspases and reactive oxygen species generation. The rate of myocyte death was greater in TG vs. wild-type myocytes and accelerated by ISO in both groups, although ISO did not increase LTCC current (I(Ca-L)) in TG-VMs. Nifedipine, an LTCC antagonist, only partially prevented myocyte death. These results suggest both LTCC-dependent and -independent mechanisms in ISO induced myocyte death. ISO increased the contractility of wild type and TG-VMs by enhancing sarcoplasmic reticulum function and inhibiting sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)/Ca(2+) exchanger, and CaMKII partially protected myocyte from death induced by both Ca(2+) and ISO. Caspase and reactive oxygen species inhibitors did not, but beta(2)-AR activation did, reduce myocyte death induced by enhanced I(Ca-L) and ISO stimulation. Our results suggest that catecholamines induce myocyte necrosis primarily through beta(1)-AR-mediated increases in I(Ca-L), but other mechanisms are also involved in rodents.

  14. S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

    PubMed

    Liu, Taiming; Schroeder, Hobe J; Zhang, Meijuan; Wilson, Sean M; Terry, Michael H; Longo, Lawrence D; Power, Gordon G; Blood, Arlin B

    2016-08-31

    S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

  15. 76 FR 49300 - Corporate Reorganizations; Distributions Under Sections 368(a)(1)(D) and 354(b)(1)(B); Correction

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-10

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF THE TREASURY Internal Revenue Service 26 CFR Part 1 RIN 1545-BF83 Corporate Reorganizations; Distributions Under Sections 368(a)(1)(D) and 354(b)(1)(B); Correction AGENCY: Internal Revenue Service (IRS), Treasury....

  16. Allosteric interactions at L-type calcium channels between FPL 64176 and the enantiomers of the dihydropyridine Bay K 8644.

    PubMed

    Usowicz, M M; Gigg, M; Jones, L M; Cheung, C W; Hartley, S A

    1995-11-01

    Functional interactions between the enantiomers of the dihydropyridine 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridi ne carboxylic acid methyl ester (Bay K 8644) and the benzoylpyrrole methyl 2,5-dimethyl-4-[2(phenylmethyl)benzoyl]-H-pyrrole-3-carboxylate (FPL 64176) were investigated on L-type Ca++ channels in guinea pig ileal longitudinal smooth muscle. The effects of these drugs, when applied individually, were as described in earlier studies. For instance, both (-)-(S)-Bay K 8644 and FPL 64176 caused concentration-dependent contraction, which is consistent with Ca++ channel activation, whereas (+)-(R)-Bay K 8644 gave concentration-dependent relaxation, which is consistent with Ca++ channel inhibition. The activities of the different drugs were dependent on the extracellular levels of KCI. When applied in combination, however, the responses evoked were not those predicted from the effects of the drugs applied individually. Contractions produced by FPL 64176 (25 nM to 1 microM) were abolished in the presence of 100 nM (-)-(S)-Bay K 8644 but were potentiated by 10 to 150 nM (+)-(R)-Bay K 8644 and inhibited by 1 microM (+)-(R)-Bay K 8644. Conversely, contractile responses to (-)-(S)-Bay K 8644 were abolished by 100 nM FPL 64176. In the presence of 1 microM FPL 64176, however, (-)-(S)-Bay K 8644 gave concentration-dependent relaxation of the muscle, which is consistent with Ca++ channel inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. New mechanisms of antiplatelet activity of nifedipine, an L-type calcium channel blocker.

    PubMed

    Chou, Tz-Chong

    2014-01-01

    Platelet hyperactivity often occursd in hypertensive patients and is a key factor in the development of cardiovascular diseases including thrombosis and atherosclerosis. Nifedipine, an L-type calcium channel blocker, is widely used for hypertension and coronary heart disease therapy. In addition, nifedipine is known to exhibit an antiplatelet activity, but the underlying mechanisms involved remain unclear. Several transcription factors such as peroxisome proliferator-activated receptors (PPARs) and nuclear factor kappa B (NF-κB) exist in platelets and have an ability to regulate platelet aggregation through a non-genomic mechanism. The present article focuses on describing the mechanisms of the antiplatelet activity of nifedipine via PPAR activation. It has been demonstrated that nifedipine treatment increases the activity and intracellular amount of PPAR-β/-γ in activated platelets. Moreover, the antiplatelet activity of nifedipine is mediated by PPAR-β/-γ-dependent upon the up-regulation of the PI3K/AKT/NO/cyclic GMP/PKG pathway, and inhibition of protein kinase Cα (PKCα) activity via an interaction between PPAR-β/-γ and PKCα. Furthermore, suppressing NF-κB activation by nifedipine through enhanced association of PPAR-β/-γ with NF-κB has also been observed in collagen-stimulated platelets. Blocking PPAR-β/-γ activity or increasing NF-κB activation greatly reverses the antiplatelet activity and inhibition of intracellular Ca(2+) mobilization, PKCα activity, and surface glycoprotein IIb/IIIa expression caused by nifedipine. Thus, PPAR-β/-γ- dependent suppression of NF-κB activation also contributes to the antiplatelet activity of nifedipine. Consistently, administration of nifedipine markedly reduces fluorescein sodium-induced vessel thrombus formation in mice, which is considerably inhibited when the PPAR-β/-γ antagonists are administrated simultaneously. Collectively, these results provide important information regarding the mechanism by

  18. Recombinant human ZP3-induced sperm acrosome reaction: evidence for the involvement of T- and L-type voltage-gated calcium channels.

    PubMed

    José, Omar; Hernández-Hernández, Oscar; Chirinos, Mayel; González-González, María Elena; Larrea, Fernando; Almanza, Angélica; Felix, Ricardo; Darszon, Alberto; Treviño, Claudia L

    2010-05-14

    For successful fertilization mammalian spermatozoa must undergo the acrosome reaction (AR), an exocytotic event that allows this cell to penetrate the outer layer of the oocyte, the zona pellucida (ZP). Four glycoproteins (ZP1-ZP4) compose the human ZP, being ZP3 the physiological inductor of the AR. This process requires changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) involving not fully understood mechanisms. Even in mouse sperm, the pharmacologically documented participation of voltage-gated Ca(2+) (Ca(V)) channels and store-operated channels (SOCs) in the ZP-induced AR is being debated. The situation in human sperm is even less clear due to the limited availability of human ZP. Here, we used recombinant human ZP3 (rhZP3) produced in baculovirus-infected Sf9 cells to investigate the involvement of Ca(V) channels in the human sperm AR. Our findings showed that Ni(2+) and mibefradil at concentrations that block T-type or Ca(V)3 channels, and nimodipine and diltiazem that block L-type or Ca(V)1 channels, significantly inhibited the rhZP3-initiated AR. On the other hand, the AR was insensitive to concentrations of omega-Agatoxin IVA, omega-Conotoxin GVIA and SNX-482 that block P/Q, N and R-type channels, respectively (Ca(V)2 channels). Our overall findings suggest that Ca(V)1 and Ca(V)3 channels participate in human sperm AR. Consistent with this, we detected in human sperm transcripts for the Ca(V)1 auxiliary subunits, alpha(2)delta, beta(1), beta(2) and beta(4), but not the neuronal specific isoforms beta(3) and gamma(2).

  19. N- and L-Type Voltage-Gated Calcium Channels Mediate Fast Calcium Transients in Axonal Shafts of Mouse Peripheral Nerve

    PubMed Central

    Barzan, Ruxandra; Pfeiffer, Friederike; Kukley, Maria

    2016-01-01

    In the peripheral nervous system (PNS) a vast number of axons are accommodated within fiber bundles that constitute peripheral nerves. A major function of peripheral axons is to propagate action potentials along their length, and hence they are equipped with Na+ and K+ channels, which ensure successful generation, conduction and termination of each action potential. However little is known about Ca2+ ion channels expressed along peripheral axons and their possible functional significance. The goal of the present study was to test whether voltage-gated Ca2+ channels (VGCCs) are present along peripheral nerve axons in situ and mediate rapid activity-dependent Ca2+ elevations under physiological circumstances. To address this question we used mouse sciatic nerve slices, Ca2+ indicator Oregon Green BAPTA-1, and 2-photon Ca2+ imaging in fast line scan mode (500 Hz). We report that transient increases in intra-axonal Ca2+ concentration take place along peripheral nerve axons in situ when axons are stimulated electrically with single pulses. Furthermore, we show for the first time that Ca2+ transients in peripheral nerves are fast, i.e., occur in a millisecond time-domain. Combining Ca2+ imaging and pharmacology with specific blockers of different VGCCs subtypes we demonstrate that Ca2+ transients in peripheral nerves are mediated mainly by N-type and L-type VGCCs. Discovery of fast Ca2+ entry into the axonal shafts through VGCCs in peripheral nerves suggests that Ca2+ may be involved in regulation of action potential propagation and/or properties in this system, or mediate neurotransmitter release along peripheral axons as it occurs in the optic nerve and white matter of the central nervous system (CNS). PMID:27313508

  20. MicroRNAs are essential for stretch-induced vascular smooth muscle contractile differentiation via microRNA (miR)-145-dependent expression of L-type calcium channels.

    PubMed

    Turczynska, Karolina M; Sadegh, Mardjaneh Karbalaei; Hellstrand, Per; Swärd, Karl; Albinsson, Sebastian

    2012-06-01

    Stretch of the vascular wall is an important stimulus to maintain smooth muscle contractile differentiation that is known to depend on L-type calcium influx, Rho-activation, and actin polymerization. The role of microRNAs in this response was investigated using tamoxifen-inducible and smooth muscle-specific Dicer KO mice. In the absence of Dicer, which is required for microRNA maturation, smooth muscle microRNAs were completely ablated. Stretch-induced contractile differentiation and Rho-dependent cofilin-2 phosphorylation were dramatically reduced in Dicer KO vessels. On the other hand, acute stretch-sensitive growth signaling, which is independent of influx through L-type calcium channels, was not affected by Dicer KO. Contractile differentiation induced by the actin polymerizing agent jasplakinolide was not altered by deletion of Dicer, suggesting an effect upstream of actin polymerization. Basal and stretch-induced L-type calcium channel expressions were both decreased in Dicer KO portal veins, and inhibition of L-type channels in control vessels mimicked the effects of Dicer deletion. Furthermore, inhibition of miR-145, a highly expressed microRNA in smooth muscle, resulted in a similar reduction of L-type calcium channel expression. This was abolished by the Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN93, suggesting that Ca(2+)/calmodulin-dependent protein kinase IIδ, a target of miR-145 and up-regulated in Dicer KO, plays a role in the regulation of L-type channel expression. These results show that microRNAs play a crucial role in stretch-induced contractile differentiation in the vascular wall in part via miR-145-dependent regulation of L-type calcium channels.

  1. Expression of voltage sensitive calcium channel (VSCC) L-type Cav1.2 (alpha1C) and T-type Cav3.2 (alpha1H) subunits during mouse bone development.

    PubMed

    Shao, Ying; Alicknavitch, Michael; Farach-Carson, Mary C

    2005-09-01

    Voltage-sensitive calcium channels (VSCCs) are key regulators of osteoblast plasma membrane Ca(2+) permeability and are under control of calcitropic hormones. Subtype specific antibodies were used to probe L-type Ca(v)1.2 (alpha(1C)) and T-type Ca(v)3.2 (alpha(1H)) subunit expression during mouse skeletal development. Commencing from E14.5 and continuing through skeletal maturity, immunoreactivity of Ca(v)1.2 (alpha(1C)) subunits was evident in regions of rapid long bone growth, including the perichondrium, periosteum, chondro-osseous junction and trabecular bones. Ca(v)3.2 (alpha(1H)) subunits appeared simultaneously and followed a similar distribution pattern. Both subunits were observed in osteoblasts and chondrocytes under high magnification. Interestingly, Ca(v)3.2 (alpha(1H)) subunits were present, but Ca(v)1.2 (alpha(1C)) subunits were absent from osteocytes. Western Blot and immunohistochemical assessment of in vitro cell culture models of osteogenesis and chondrogenesis confirmed the in vivo observations. We conclude that both L-type Ca(v)1.2 (alpha(1C)) and T-type Ca(v)3.2 (alpha(1H)) VSCCs are dynamically regulated in bones and cartilages during endochondral bone development. Copyright 2005 Wiley-Liss, Inc.

  2. Pharmacology of L-type Calcium Channels: Novel Drugs for Old Targets?

    PubMed Central

    Striessnig, Jörg; Ortner, Nadine J.; Pinggera, Alexandra

    2015-01-01

    Inhibition of voltage-gated L-type calcium channels by organic calcium channel blockers is a well-established pharmacodynamic concept for the treatment of hypertension and cardiac ischemia. Since decades these antihypertensives (such as the dihydropyridines amlodipine, felodipine or nifedipine) belong to the most widely prescribed drugs 
world-wide. Their tolerability is excellent because at therapeutic doses their pharmacological effects in humans are limited to the cardiovascular system. During the last years substantial progress has been made to reveal the physiological role of different L-type calcium channel isoforms in many other tissues, including the brain, endocrine and sensory cells. 
Moreover, there is accumulating evidence about their involvement in various human diseases, such as Parkinson's disease, neuropsychiatric disorders and hyperaldosteronism. In this review we discuss the pathogenetic role of L-type calcium channels, potential new indications for existing or isoform-selective compounds and strategies to minimize potential side effects. PMID:25966690

  3. Potentiation of prolactin secretion following lactotrope escape from dopamine action. II. Phosphorylation of the alpha(1) subunit of L-type, voltage-dependent calcium channels.

    PubMed

    Hernández, M E; del Mar Hernández, M; Díaz-Muñoz, M; Clapp, C; de la Escalera, G M

    1999-07-01

    Modulation of Ca(2+) channels has been shown to alter cellular functions. It can play an important role in the amplification of signals in the endocrine system, including the pleiotropically regulated pituitary lactotropes. Prolactin (PRL) secretion is tonically inhibited by dopamine (DA), the escape from which triggers acute episodes of hormone secretion. The magnitude of these episodes is explained by a potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). While the mechanisms of this potentiation are not fully understood, it is known to be mimicked by the dihydropyridine, L-type Ca(2+) channel agonist Bay K 8644 and blocked by nifedipine and methoxyverapamil. The potentiation is also blocked by inhibitors of cyclic AMP-dependent protein kinase and protein kinase C. We recently described that the escape from tonic actions of DA results in increased macroscopic Ca(2+) currents in GH(4)C(1) lactotropic clonal cells transfected with a cDNA encoding the long form of the human D(2)-DA receptor. Here we show that the withdrawal from DA potentiates the PRL-releasing action of TRH in GH(4)C(1)/D(2)-DAR cells to the same extent as in enriched lactotropes in primary culture. In both experimental models a low density of dihydropyridine receptors was shown by (+)-[(3)H]PN200-110 binding. Photoaffinity labelling with the dihydropyridine [(3)H]azidopine revealed a protein consistent with the alpha(1) subunit of L-type Ca(2+) channels of 165-170 kDa. In both experimental models, the facilitation of TRH action triggered by the escape from DA was correlated with an enhanced rate of phosphorylation of this putative alpha(1) subunit, the nature of which was further supported by immunoprecipitation with selective antibodies directed against the alpha(1C) and alpha(1D) subunit of voltage-gated calcium channels. We propose that PKA- and PKC-dependent phosphorylation of the alpha(1) subunit of high voltage activated, L-type Ca(2

  4. L-type calcium channel gating is modulated by bradykinin with a PKC-dependent mechanism in NG108-15 cells.

    PubMed

    Toselli, Mauro; Taglietti, Vanni

    2005-05-01

    Bradykinin (BK) excites dorsal root ganglion cells, leading to the sensation of pain. The actions of BK are thought to be mediated by heterotrimeric G protein-regulated pathways. Indeed there is strong evidence that in different cell types BK is involved in phosphoinositide breakdown following activation of G(q/11). In the present study we show that the Ca(2+) current flowing through L-type voltage-gated Ca(2+) channels in NG108-15 cells (differentiated in vitro to acquire a neuronal phenotype), measured using the whole-cell patch clamp configuration, is reversibly inhibited by BK in a voltage-independent fashion, suggesting a cascade process where a second messenger system is involved. This inhibitory action of BK is mimicked by the application of 1,2-oleoyl-acetyl glycerol (OAG), an analog of diacylglycerol that activates PKC. Interestingly, OAG occluded the effects of BK and both effects were blocked by selective PKC inhibitors. The down modulation of single L-type Ca(2+) channels by BK and OAG was also investigated in cell-attached patches. Our results indicate that the inhibitory action of BK involves activation of PKC and mainly shows up in a significant reduction of the probability of channel opening, caused by an increase and clustering of null sweeps in response to BK.

  5. Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels.

    PubMed Central

    Nonner, W; Eisenberg, B

    1998-01-01

    L-type Ca channels contain a cluster of four charged glutamate residues (EEEE locus), which seem essential for high Ca specificity. To understand how this highly charged structure might produce the currents and selectivity observed in this channel, a theory is needed that relates charge to current. We use an extended Poisson-Nernst-Planck (PNP2) theory to compute (mean) Coulombic interactions and thus to examine the role of the mean field electrostatic interactions in producing current and selectivity. The pore was modeled as a central cylinder with tapered atria; the cylinder (i.e., "pore proper") contained a uniform volume density of fixed charge equivalent to that of one to four carboxyl groups. The pore proper was assigned ion-specific, but spatially uniform, diffusion coefficients and excess chemical potentials. Thus electrostatic selection by valency was computed self-consistently, and selection by other features was also allowed. The five external parameters needed for a system of four ionic species (Na, Ca, Cl, and H) were determined analytically from published measurements of thre limiting conductances and two critical ion concentrations, while treating the pore as a macroscopic ion-exchange system in equilibrium with a uniform bath solution. The extended PNP equations were solved with these parameters, and the predictions were compared to currents measured in a variety of solutions over a range of transmembrane voltages. The extended PNP theory accurately predicted current-voltage relations, anomalous mole fraction effects in the observed current, saturation effects of varied Ca and Na concentrations, and block by protons. Pore geometry, dielectric permittivity, and the number of carboxyl groups had only weak effects. The successful prediction of Ca fluxes in this paper demonstrates that ad hoc electrostatic parameters, multiple discrete binding sites, and logistic assumptions of single-file movement are all unnecessary for the prediction of permeation in

  6. Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels.

    PubMed

    Nonner, W; Eisenberg, B

    1998-09-01

    L-type Ca channels contain a cluster of four charged glutamate residues (EEEE locus), which seem essential for high Ca specificity. To understand how this highly charged structure might produce the currents and selectivity observed in this channel, a theory is needed that relates charge to current. We use an extended Poisson-Nernst-Planck (PNP2) theory to compute (mean) Coulombic interactions and thus to examine the role of the mean field electrostatic interactions in producing current and selectivity. The pore was modeled as a central cylinder with tapered atria; the cylinder (i.e., "pore proper") contained a uniform volume density of fixed charge equivalent to that of one to four carboxyl groups. The pore proper was assigned ion-specific, but spatially uniform, diffusion coefficients and excess chemical potentials. Thus electrostatic selection by valency was computed self-consistently, and selection by other features was also allowed. The five external parameters needed for a system of four ionic species (Na, Ca, Cl, and H) were determined analytically from published measurements of thre limiting conductances and two critical ion concentrations, while treating the pore as a macroscopic ion-exchange system in equilibrium with a uniform bath solution. The extended PNP equations were solved with these parameters, and the predictions were compared to currents measured in a variety of solutions over a range of transmembrane voltages. The extended PNP theory accurately predicted current-voltage relations, anomalous mole fraction effects in the observed current, saturation effects of varied Ca and Na concentrations, and block by protons. Pore geometry, dielectric permittivity, and the number of carboxyl groups had only weak effects. The successful prediction of Ca fluxes in this paper demonstrates that ad hoc electrostatic parameters, multiple discrete binding sites, and logistic assumptions of single-file movement are all unnecessary for the prediction of permeation in

  7. The role of L-type amino acid transporter 1 in human tumors

    PubMed Central

    Zhao, Yu; Wang, Lin; Pan, Jihong

    2015-01-01

    Summary L-type amino acid transporter 1 (LAT1) is an L-type amino acid transporter and transports large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine. LAT1 was found to be highly expressed especially in human cancer tissues, and up-regulated LAT1 can lead to dysfunction in human tumor cells. These findings suggest that LAT1 plays an important role in human tumors. This review provides an overview of the current understanding of LAT1 expression and its clinical significance and function in tumors. PMID:26668776

  8. GLP-2 potentiates L-type CA2+ channel activity associated with stimulated glucose uptake in hippocampal neurons

    USDA-ARS?s Scientific Manuscript database

    Glucagon-like peptide-2 (GLP-2) is a neuropeptide secreted from endocrine cells in the gut and neurons in the brain. GLP-2 stimulates intestinal crypt cell proliferation and mucosal blood flow while decreasing gastric emptying and gut motility. However, a GLP-2-mediated signaling network has not bee...

  9. Effects of wenxin keli on the action potential and L-type calcium current in rats with transverse aortic constriction-induced heart failure.

    PubMed

    Chen, Yu; Li, Yang; Guo, Lili; Chen, Wen; Zhao, Mingjing; Gao, Yonghong; Wu, Aiming; Lou, Lixia; Wang, Jie; Liu, Xiaoqiu; Xing, Yanwei

    2013-01-01

    Objective. We investigated the effects of WXKL on the action potential (AP) and the L-type calcium current (I Ca-L) in normal and hypertrophied myocytes. Methods. Forty male rats were randomly divided into two groups: the control group and the transverse aortic constriction- (TAC-) induced heart failure group. Cardiac hypertrophy was induced by TAC surgery, whereas the control group underwent a sham operation. Eight weeks after surgery, single cardiac ventricular myocytes were isolated from the hearts of the rats. The APs and I Ca-L were recorded using the whole-cell patch clamp technique. Results. The action potential duration (APD) of the TAC group was prolonged compared with the control group and was markedly shortened by WXKL treatment in a dose-dependent manner. The current densities of the I Ca-L in the TAC group treated with 5 g/L WXKL were significantly decreased compared with the TAC group. We also determined the effect of WXKL on the gating mechanism of the I Ca-L in the TAC group. We found that WXKL decreased the I Ca-L by accelerating the inactivation of the channels and delaying the recovery time from inactivation. Conclusions. The results suggest that WXKL affects the AP and blocked the I Ca-L, which ultimately resulted in the treatment of arrhythmias.

  10. L-type Voltage-Gated Calcium Channels in Conditioned Fear: A Genetic and Pharmacological Analysis

    ERIC Educational Resources Information Center

    McKinney, Brandon C.; Sze, Wilson; White, Jessica A.; Murphy, Geoffrey G.

    2008-01-01

    Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear…

  11. Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle

    PubMed Central

    Iwamaru, Yoshifumi; Imamura, Morikazu; Miyazawa, Kohtaro; Matsuura, Yuichi; Masujin, Kentaro; Murayama, Yuichi; Yokoyama, Takashi

    2017-01-01

    To determine oral transmissibility of the L-type bovine spongiform encephalopathy (BSE) prion, we orally inoculated 16 calves with brain homogenates of the agent. Only 1 animal, given a high dose, showed signs and died at 88 months. These results suggest low risk for oral transmission of the L-BSE agent among cattle. PMID:28098532

  12. Distribution of abnormal prion protein in a sheep affected with L-type bovine spongiform encephalopathy.

    PubMed

    Matsuura, Y; Iwamaru, Y; Masujin, K; Imamura, M; Mohri, S; Yokoyama, T; Okada, H

    2013-07-01

    To investigate the topographical distribution and patterns of deposition of immunolabelled abnormal prion protein (PrP(Sc)), interspecies transmission of atypical L-type bovine spongiform encephalopathy (BSE) to Cheviot ewes (ARQ/ARQ genotype) was performed. L-type BSE was successfully transmitted via the intracerebral route to a ewe, with an incubation period of 1,562 days. Minimal vacuolar change was detected in the basal ganglia, thalamus and brainstem, and PrP(Sc) accumulated throughout the brain. The L-type BSE-affected sheep was characterized by conspicuous fine particulate deposits in the neuropil, particulate and/or granular intraneuronal and intraglial deposits, and the absence of PrP(Sc) plaques or stellate deposits. In addition, immunohistochemical and western blot analyses revealed that PrP(Sc) accumulation was present in peripheral nervous tissues (including the trigeminal ganglia and dorsal root ganglion) and adrenal glands, but was absent in lymphoid tissues. These results suggest that L-type BSE has distinct and distinguishable characteristics as well as PrP(Sc) tissue tropism in sheep. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle.

    PubMed

    Okada, Hiroyuki; Iwamaru, Yoshifumi; Imamura, Morikazu; Miyazawa, Kohtaro; Matsuura, Yuichi; Masujin, Kentaro; Murayama, Yuichi; Yokoyama, Takashi

    2017-02-01

    To determine oral transmissibility of the L-type bovine spongiform encephalopathy (BSE) prion, we orally inoculated 16 calves with brain homogenates of the agent. Only 1 animal, given a high dose, showed signs and died at 88 months. These results suggest low risk for oral transmission of the L-BSE agent among cattle.

  14. L-type Voltage-Gated Calcium Channels in Conditioned Fear: A Genetic and Pharmacological Analysis

    ERIC Educational Resources Information Center

    McKinney, Brandon C.; Sze, Wilson; White, Jessica A.; Murphy, Geoffrey G.

    2008-01-01

    Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear…

  15. L-type VDCCs participate in behavioral-LTP and memory retention.

    PubMed

    Han, Yuan-Yuan; Wang, Xiao-Dong; Liu, Li; Guo, Hong-Mei; Cong, Wei; Yan, Wen-Wen; Huang, Jun-Ni; Xiao, Peng; Li, Chu-Hua

    2017-09-01

    Although L-type voltage-dependent calcium channels (VDCCs) have been reported to display different even contrary actions on cognitive functions and long-term potentiation (LTP) formation, there is little information regarding the role of L-type VDCCs in behavioral LTP, a learning-induced LTP model, in the intact brain of freely behaving animals. Here we investigated the effects of verapamil, a non-selective blocker of L-type VDCCs, on behavioral LTP and cognitive functions. Population spikes (PS) were recorded by using electrophysiological methods to examine the role of verapamil in behavioral LTP in the hippocampal dentate gyrus (DG) region. Y-maze assay was used to evaluate the effects of verapamil on learning and memory. Electron microscope was used to observe the changes on synaptic ultrastructural morphology in hippocampal DG area. We found that intrahippocampal verapamil treatments had no significant changes on the PS amplitude during a 90min recordings period. However, intrahippocampal applications of verapamil, including pre- or post-training, reduced behavioral LTP magnitude and memory retention but did not prevent the induction of behavioral LTP and the acquisition of learning. The saline group with behaving trainings showed obvious increases in the number of smile synapses, the length of active zones and the thickness of postsynaptic density as compared to the baseline group, but verapamil with pre-training treatment almost returned these changes to the baseline levels except for the synaptic interface curvature. In conclusion, our results suggest that L-type VDCCs may only contribute to the magnitude of behavioral LTP and the memory maintenance with an activity-independent relationship. L-type VDCCs may be critical to new information long-term storage rather than acquisition in hippocampus. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Collective Excitations of a Trapped Bose-Einstein Condensate in the Presence of a 1D Optical Lattice

    NASA Astrophysics Data System (ADS)

    Fort, C.; Cataliotti, F. S.; Fallani, L.; Ferlaino, F.; Maddaloni, P.; Inguscio, M.

    2003-04-01

    We study low-lying collective modes of an elongated 87Rb condensate produced in a 3D magnetic harmonic trap with the addition of a 1D periodic potential which is provided by a laser standing wave along the axial direction. While the transverse breathing mode remains unperturbed, quadrupole and dipole oscillations along the optical lattice are strongly modified. Precise measurements of the collective mode frequencies at different heights of the optical barriers provide a stringent test of the theoretical model recently introduced [

    M. Krämer et al., Phys. Rev. Lett.PRLTAO0031-9007 88, 180404 (2002)
    ].

  17. Multiple mobility edges in a 1D Aubry chain with Hubbard interaction in presence of electric field: Controlled electron transport

    NASA Astrophysics Data System (ADS)

    Saha, Srilekha; Maiti, Santanu K.; Karmakar, S. N.

    2016-09-01

    Electronic behavior of a 1D Aubry chain with Hubbard interaction is critically analyzed in presence of electric field. Multiple energy bands are generated as a result of Hubbard correlation and Aubry potential, and, within these bands localized states are developed under the application of electric field. Within a tight-binding framework we compute electronic transmission probability and average density of states using Green's function approach where the interaction parameter is treated under Hartree-Fock mean field scheme. From our analysis we find that selective transmission can be obtained by tuning injecting electron energy, and thus, the present model can be utilized as a controlled switching device.

  18. Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels.

    PubMed

    Guo, Kai; Wang, Xianming; Gao, Guofeng; Huang, Congxin; Elmslie, Keith S; Peterson, Blaise Z

    2010-11-01

    We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149-1159, 2010). The short 17 amino acid extracellular NH(2)-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate Ca(V)1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with Ca(V)1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on Ca(V)1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca(2+) dynamics in the heart.

  19. Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels

    PubMed Central

    Guo, Kai; Wang, Xianming; Gao, Guofeng; Huang, Congxin; Elmslie, Keith S.

    2010-01-01

    We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149–1159, 2010). The short 17 amino acid extracellular NH2-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate CaV1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with CaV1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on CaV1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca2+ dynamics in the heart. PMID:20720179

  20. L-type Calcium Channel Cav1.2 Is Required for Maintenance of Auditory Brainstem Nuclei*

    PubMed Central

    Ebbers, Lena; Satheesh, Somisetty V.; Janz, Katrin; Rüttiger, Lukas; Blosa, Maren; Hofmann, Franz; Morawski, Markus; Griesemer, Désirée; Knipper, Marlies; Friauf, Eckhard; Nothwang, Hans Gerd

    2015-01-01

    Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca2+ channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue. PMID:26242732

  1. Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels

    PubMed Central

    Lee, Moon Young; Park, Chanjae; Ha, Se Eun; Park, Paul J.; Berent, Robyn M.; Jorgensen, Brian G.; Corrigan, Robert D.; Grainger, Nathan; Blair, Peter J.; Slivano, Orazio J.; Miano, Joseph M.; Ward, Sean M.; Smith, Terence K.; Sanders, Kenton M.

    2017-01-01

    Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract. PMID:28152551

  2. Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure

    PubMed Central

    Sanchez-Alonso, Jose L.; Bhargava, Anamika; O’Hara, Thomas; Glukhov, Alexey V.; Schobesberger, Sophie; Bhogal, Navneet; Sikkel, Markus B.; Mansfield, Catherine; Korchev, Yuri E.; Lyon, Alexander R.; Punjabi, Prakash P.; Nikolaev, Viacheslav O.; Trayanova, Natalia A.

    2016-01-01

    Rationale: Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. Objective: To explore microdomain-targeted remodeling of ventricular L-type Ca2+ channels (LTCCs) in HF. Methods and Results: Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore the distribution of single LTCCs in different membrane microdomains of nonfailing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to the redistribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability was dramatically increased (0.034±0.011 versus 0.154±0.027, P<0.001). High open probability was linked to enhance calcium–calmodulin kinase II–mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current, which contributed to the development of early afterdepolarizations. A novel model of LTCC function in HF was developed; after its validation with experimental data, the model was used to ascertain how HF-induced T-tubule loss led to altered LTCC function and early afterdepolarizations. The HF myocyte model was then implemented in a 3-dimensional left ventricle model, demonstrating that such early afterdepolarizations can propagate and initiate reentrant arrhythmias. Conclusions: Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electric remodeling in HF and adds a new dimension to cardiovascular disease. PMID:27572487

  3. L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling

    PubMed Central

    Jacquemet, Guillaume; Baghirov, Habib; Georgiadou, Maria; Sihto, Harri; Peuhu, Emilia; Cettour-Janet, Pierre; He, Tao; Perälä, Merja; Kronqvist, Pauliina; Joensuu, Heikki; Ivaska, Johanna

    2016-01-01

    Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion. PMID:27910855

  4. Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland.

    PubMed

    Barbosa, Roseli; Scialfa, Julieta Helena; Terra, Ilza Mingarini; Cipolla-Neto, José; Simonneaux, Valérie; Afeche, Solange Castro

    2008-02-27

    Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP(3)-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of serotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland.

  5. Synthesis, crystal structure, and properties of a 1-D terbium-substituted monolacunary Keggin-type polyoxotungstate.

    PubMed

    Ma, Pengtao; Si, Yanan; Wan, Rong; Zhang, Shaowei; Wang, Jingping; Niu, Jingyang

    2015-03-05

    A new 1-D linear chainlike terbium-substituted polyoxometalate [Tb(H2O)2(α-PW11O39)](4-) (1) has been synthesized in aqueous solution and characterized by elemental analysis, inductively coupled plasma atomic emission spectrometry (ICP-AES), X-ray powder diffraction (XRPD), IR spectrum, thermal analysis, electrospray ionization mass spectrometry (ESI-MS), and X-ray single-crystal diffraction. X-ray structural analysis reveals that 1 displays a 1-D linear chain containing [Tb(H2O)2(α-PW11O39)](4-) moieties. The Tb(III) cation incorporated into the monolacunary Keggin-type [α-PW11O39](7-) unit resides in a distorted monocapped triangular prismatic geometry and acts as a linker to join two adjacent [α-PW11O39](7-) units to form a 1-D chain structure. Solid-state photoluminescent property of 1 has been investigated at room temperature and the photoluminescent emission mainly results from the synergistic effect of the Tb(III) cation and the Na7[α-PW11O39] precursor. The ESI-MS spectrum of 1 confirms that the polyanion [Tb(H2O)(HPW11O39)](3-) is stable in aqueous solution.

  6. Synthesis, crystal structure, and properties of a 1-D terbium-substituted monolacunary Keggin-type polyoxotungstate

    NASA Astrophysics Data System (ADS)

    Ma, Pengtao; Si, Yanan; Wan, Rong; Zhang, Shaowei; Wang, Jingping; Niu, Jingyang

    2015-03-01

    A new 1-D linear chainlike terbium-substituted polyoxometalate [Tb(H2O)2(α-PW11O39)]4- (1) has been synthesized in aqueous solution and characterized by elemental analysis, inductively coupled plasma atomic emission spectrometry (ICP-AES), X-ray powder diffraction (XRPD), IR spectrum, thermal analysis, electrospray ionization mass spectrometry (ESI-MS), and X-ray single-crystal diffraction. X-ray structural analysis reveals that 1 displays a 1-D linear chain containing [Tb(H2O)2(α-PW11O39)]4- moieties. The Tb(III) cation incorporated into the monolacunary Keggin-type [α-PW11O39]7- unit resides in a distorted monocapped triangular prismatic geometry and acts as a linker to join two adjacent [α-PW11O39]7- units to form a 1-D chain structure. Solid-state photoluminescent property of 1 has been investigated at room temperature and the photoluminescent emission mainly results from the synergistic effect of the TbIII cation and the Na7[α-PW11O39] precursor. The ESI-MS spectrum of 1 confirms that the polyanion [Tb(H2O)(HPW11O39)]3- is stable in aqueous solution.

  7. A 1D model for tides waves and fine sediment in short tidal basins—Application to the Wadden Sea

    NASA Astrophysics Data System (ADS)

    van Prooijen, Bram Christiaan; Wang, Zheng Bing

    2013-12-01

    In order to simulate the dynamics of fine sediments in short tidal basins, like the Wadden Sea basins, a 1D cross-sectional averaged model is constructed to simulate tidal flow, depth-limited waves, and fine sediment transport. The key for this 1D model lies in the definition of the geometry (width and depth as function of the streamwise coordinate). The geometry is computed by implementing the water level and flow data, from a 2D flow simulation, and the hypsometric curve in the continuity equation. By means of a finite volume method, the shallow-water equations and sediment transport equations are solved. The bed shear stress consists of the sum of shear stresses by waves and flow, in which the waves are computed with a depth-limited growth equation for wave height and wave frequency. A new formulation for erosion of fines from a sandy bed is proposed in the transport equation for fine sediment. It is shown by comparison with 2D simulations and field measurements that a 1D schematization gives a proper representation of the dynamics in short tidal basins.

  8. Inflammatory cytokine signaling in insulin producing beta-cells enhances the colocalization correlation coefficient between L-type voltage-dependent calcium channel and calcium-sensing receptor.

    PubMed

    Parkash, Jai

    2008-08-01

    The immunological processes in type 1 diabetes and metabolic/inflammatory disorder in type 2 diabetes converge on common signaling pathway(s) leading to beta-cell death in these two diseases. The cytokine-mediated beta-cell death seems to be dependent on voltage-dependent calcium channel (VDCC)-mediated Ca2+ entry. The Ca2+ handling molecular networks control the homeostasis of [Ca2+]i in the beta-cell. The activity and membrane density of VDCC are regulated by several mechanisms including G protein-coupled receptors (GPCRs). CaR is a 123-kDa seven transmembrane extracellular Ca2+ sensing protein that belongs to GPCR family C. Tumor necrosis factor-alpha (TNF-alpha), is a cytokine widely known to activate nuclear factor-kappaB (NF-kappaB) transcription in beta-cells. To obtain a better understanding of TNF-alpha-induced molecular interactions between CaR and VDCC, confocal fluorescence measurements were performed on insulin-producing beta-cells exposed to varying concentrations of TNF-alpha and the results are discussed in the light of increased colocalization correlation coefficient. The insulin producing beta-cells were exposed to 5, 10, 20, 30, and 50 ng/ml TNF-alpha for 24 h at 37 degrees . The cells were then immunolabelled with antibodies directed against CaR, VDCC, and NF-kappaB. The confocal fluorescence imaging data showed enhancement in the colocalization correlation coefficient between CaR and VDCC in beta-cells exposed to TNF-alpha thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. TNF-alpha-induced colocalization of VDCC with CaR was inhibited by nimodipine, an inhibitor of L-type VDCC thereby suggesting that VDCC activity is required for spatial interactions with CaR. The 3-D confocal fluorescence imaging data also demonstrated that addition of TNF-alpha to RIN cells led to the translocation of NF-kappaB from the cytoplasm to the nucleus. Such molecular interactions between CaR and VDCC in tissues

  9. Enhanced contractility in pregnancy is associated with augmented TRPC3, L-type, and T-type voltage-dependent calcium channel function in rat uterine radial artery.

    PubMed

    Senadheera, Sevvandi; Bertrand, Paul P; Grayson, T Hilton; Leader, Leo; Tare, Marianne; Murphy, Timothy V; Sandow, Shaun L

    2013-10-15

    In pregnancy, α-adrenoceptor-mediated vasoconstriction is augmented in uterine radial arteries and is accompanied by underlying changes in smooth muscle (SM) Ca(2+) activity. This study aims to determine the Ca(2+) entry channels associated with altered vasoconstriction in pregnancy, with the hypothesis that augmented vasoconstriction involves transient receptor potential canonical type-3 (TRPC3) and L- and T-type voltage-dependent Ca(2+) channels. Immunohistochemistry showed TRPC3, L-type Cav1.2 (as the α1C subunit), T-type Cav3.1 (α1G), and Cav3.2 (α1H) localization to the uterine radial artery SM. Fluorescence intensity of TRPC3, Cav1.2, and Cav3.2 was increased, and Cav3.1 decreased in radial artery SM from pregnant rats. Western blot analysis confirmed increased TRPC3 protein expression in the radial artery from pregnant rats. Pressure myography incorporating pharmacological intervention to examine the role of these channels in uterine radial arteries showed an attenuation of phenylephrine (PE)-induced constriction with Pyr3 {1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid}-mediated TRPC3 inhibition or with nifedipine-mediated L-type channel block alone in vessels from pregnant rats; both effects of which were diminished in radial arteries from nonpregnant rats. Combined TRPC3 and L-type inhibition attenuated PE-induced constriction in radial arteries, and the residual vasoconstriction was reduced and abolished with T-type channel block with NNC 55-0396 in arteries from nonpregnant and pregnant rats, respectively. With SM Ca(2+) stores depleted and in the presence of PE, nifedipine, and NNC 55-0396, blockade of TRPC3 reversed PE-induced constriction. These data suggest that TRPC3 channels act synergistically with L- and T-type channels to modulate radial artery vasoconstriction, with the mechanism being augmented in pregnancy.

  10. Effects of the removal of extracellular Ca2+ on [Ca2+]i responses to FCCP and acetate in carotid body glomus cells of adult rabbits.

    PubMed

    Sato, M

    1997-09-12

    The effects of the removal of extracellular Ca2+ on the responses of cytosolic concentrations of Ca2+ ([Ca2+]i) to acidic stimuli, a protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and an organic acid acetate, were examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2 microfluorometry. Application of FCCP (1 microM) induced an increase in [Ca2+]i (mean +/- S.E.M., 108 +/- 14%). After withdrawal of the protonophore the increased [Ca2+]i returned slowly to a resting level. The [Ca2+]i response was attenuated by an inorganic Ca2+ channel antagonist Ni2+ (2 mM) by 81 +/- 4%, and by an L-type voltage-gated Ca2+ channel antagonist D600 (10 microM) by 53 +/- 13%. The removal of extracellular Ca2+ eliminated the [Ca2+]i response in 71% of the tested cells (n = 17), and depressed it by 68 +/- 6% in the rest. Recovery following stimulation with FCCP in the absence of Ca2+ reversibly produced a rapid and large rise in [Ca2+]i, referred to as a [Ca2+]i rise after Ca2+-free/FCCP. The magnitude of a [Ca2+]i rise after Ca2+-free/FCCP (285 +/- 28%, P < 0.05) was larger than that of an increase in [Ca2+]i induced by FCCP in the presence of Ca2+ and had a correlation with the intensity of the suppression of the [Ca2+]i response by Ca2+ removal. A [Ca2+]i rise after Ca2+-free/FCCP was inhibited mostly by D600. Similarly, recovery following exposure to acetate in the absence of Ca2+ caused a rise in [Ca2+]i, referred to as a [Ca2+]i rise after Ca2+-free/acetate which was sensitive to D600. The magnitude of the [Ca2+]i rise was larger than that of a change in [Ca2+]i caused by acetate in the presence of Ca2+. These results suggest that FCCP-induced increase in [Ca2+]i was, in most cells, due to Ca2+ influx via L-type voltage-gated Ca2+ channels and, in some cells, due to both Ca2+ influx and Ca2+ release from internal Ca2+ pool. The removal of extracellular Ca2+ might modify [Ca2+]i responses to acidic stimuli, causing [Ca2+]i

  11. Effects of total flavones from Acanthopanax senticosus on L-type calcium channels, calcium transient and contractility in rat ventricular myocytes.

    PubMed

    Guan, Shengjiang; Ma, Juanjuan; Chu, Xi; Gao, Yonggang; Zhang, Ying; Zhang, Xuan; Zhang, Fenghua; Liu, Zhenyi; Zhang, Jianping; Chu, Li

    2015-04-01

    Acanthopanax senticosus (Rupr. et Maxim.) Harms (AS), a traditional herbal medicine, has been widely used to treat ischemic heart disease. However, the underlying cellular mechanisms of its benefits to cardiac function remain unclear. The present study examined the effects of total flavones from AS (TFAS) on L-type Ca(2+) channel currents (ICa-L ) using the whole cell patch-clamp technique and on intracellular calcium ([Ca(2+) ]i ) handling and cell contractility in rat ventricular myocytes with the aid of a video-based edge-detection system. Exposure to TFAS resulted in a concentration- and voltage-dependent blockade of ICa-L , with the half-maximal inhibitory concentration (IC50 ) of 283.12 µg/mL and the maximal inhibitory effect of 36.49 ± 1.95%. Moreover, TFAS not only increased the maximum current in the current-voltage relationship but also shifted the activation and inactivation curves of ICa-L toward the hyperpolarizing direction. Meanwhile, TFAS significantly reduced amplitudes of myocyte shortening and [Ca(2+) ]i with an increase in the time to 10% of the peak (Tp) and a decrease in the time to 10% of the baseline (Tr). Thus, the cardioprotective effects of TFAS may be attributed mainly to the attenuation of [Ca(2+) ]i through the direct inhibition of ICa-L in rat ventricular myocytes and consequent negative effect on myocardial contractility.

  12. Force on a slow moving impurity due to thermal and quantum fluctuations in a 1D Bose-Einstein condensate

    SciTech Connect

    Roberts, David; Sykes, Andrew

    2009-01-01

    We study the drag force acting on an impurity moving through a 1D Bose-Einstein condensate in the presence of both quantum and thermal fluctuations. We are able to find exact analytical solutions of the partial differential equations to the level of the Bogoliubov approximation. At zero temperature, we find a nonzero force is exerted on the impurity at subcritical velocities, due to the scattering of quantum fluctuations. We make the following explicit assumptions: far from the impurity the system is in a quantum state given by that of a zero (or finite) temperature Bose-Einstein condensate, and the scattering process generates only causally related reflection/transmission. The results raise unanswered questions in the quantum dynamics associated with the formation of persistent currents.

  13. Effect of Ca2+ Efflux Pathway Distribution and Exogenous Ca2+ Buffers on Intracellular Ca2+ Dynamics in the Rat Ventricular Myocyte: A Simulation Study

    PubMed Central

    Šimurda, Jiří; Orchard, Clive H.

    2014-01-01

    We have used a previously published computer model of the rat cardiac ventricular myocyte to investigate the effect of changing the distribution of Ca2+ efflux pathways (SERCA, Na+/Ca2+ exchange, and sarcolemmal Ca2+ ATPase) between the dyad and bulk cytoplasm and the effect of adding exogenous Ca2+ buffers (BAPTA or EGTA), which are used experimentally to differentially buffer Ca2+ in the dyad and bulk cytoplasm, on cellular Ca2+ cycling. Increasing the dyadic fraction of a particular Ca2+ efflux pathway increases the amount of Ca2+ removed by that pathway, with corresponding changes in Ca2+ efflux from the bulk cytoplasm. The magnitude of these effects varies with the proportion of the total Ca2+ removed from the cytoplasm by that pathway. Differences in the response to EGTA and BAPTA, including changes in Ca2+-dependent inactivation of the L-type Ca2+ current, resulted from the buffers acting as slow and fast “shuttles,” respectively, removing Ca2+ from the dyadic space. The data suggest that complex changes in dyadic Ca2+ and cellular Ca2+ cycling occur as a result of changes in the location of Ca2+ removal pathways or the presence of exogenous Ca2+ buffers, although changing the distribution of Ca2+ efflux pathways has relatively small effects on the systolic Ca2+ transient. PMID:24971358

  14. Effect of Ca2+ efflux pathway distribution and exogenous Ca2+ buffers on intracellular Ca2+ dynamics in the rat ventricular myocyte: a simulation study.

    PubMed

    Pásek, Michal; Simurda, Jiří; Orchard, Clive H

    2014-01-01

    We have used a previously published computer model of the rat cardiac ventricular myocyte to investigate the effect of changing the distribution of Ca(2+) efflux pathways (SERCA, Na(+)/Ca(2+) exchange, and sarcolemmal Ca(2+) ATPase) between the dyad and bulk cytoplasm and the effect of adding exogenous Ca(2+) buffers (BAPTA or EGTA), which are used experimentally to differentially buffer Ca(2+) in the dyad and bulk cytoplasm, on cellular Ca(2+) cycling. Increasing the dyadic fraction of a particular Ca(2+) efflux pathway increases the amount of Ca(2+) removed by that pathway, with corresponding changes in Ca(2+) efflux from the bulk cytoplasm. The magnitude of these effects varies with the proportion of the total Ca(2+) removed from the cytoplasm by that pathway. Differences in the response to EGTA and BAPTA, including changes in Ca(2+)-dependent inactivation of the L-type Ca(2+) current, resulted from the buffers acting as slow and fast "shuttles," respectively, removing Ca(2+) from the dyadic space. The data suggest that complex changes in dyadic Ca(2+) and cellular Ca(2+) cycling occur as a result of changes in the location of Ca(2+) removal pathways or the presence of exogenous Ca(2+) buffers, although changing the distribution of Ca(2+) efflux pathways has relatively small effects on the systolic Ca(2+) transient.

  15. L-Type Calcium Channel blockers and Parkinson’s Disease in Denmark

    PubMed Central

    Ritz, Beate; Rhodes, Shannon L.; Qian, Lei; Schernhammer, Eva; Olsen, Jorgen; Friis, Soren

    2010-01-01

    Objective Investigate L-type calcium channel blockers of the dihydropyridine class for association with Parkinson’s disease because these drugs traverse the blood brain barrier, are potentially neuroprotective, and have previously been evaluated for impact on PD risk. Methods We identified 1,931 patients with a first time diagnosis for PD between 2001 and 2006 as reported in the Danish national hospital/outpatient database and density matched them by birth year and sex to 9,651 controls from the population register. Index date for cases and their corresponding controls was advanced to date of first recorded prescription for anti-Parkinson drugs, if prior to first PD diagnosis in the hospital records. Prescriptions were determined from the national pharmacy database. In our primary analyses, we excluded all calcium channel blockers prescriptions 2-years before index date/PD diagnosis. Results Employing logistic regression analysis adjusting for age, sex, diagnosis of chronic pulmonary obstructive disorder, and Charlson co-morbidity score we found that subjects prescribed centrally acting calcium channel blockers (excludes amlodipine) between 1995 and two years prior to the index date were less likely to develop Parkinson’s disease (Odds Ratio 0.73; 95% Confidence Interval 0.54-0.97); this 27% risk reduction did not differ with length or intensity of use. Risk estimates were close to null for the peripherally acting drug amlodipine and for other antihypertensive medications. Interpretation Our data suggest a potential neuroprotective role for centrally acting L-type calcium channel blockers of the dihydropyridine class in PD that should be further investigated in studies that can distinguish between types of L-Type channel blockers. PMID:20437557

  16. T-type Ca2+ channel modulation by otilonium bromide

    PubMed Central

    Strege, Peter R.; Sha, Lei; Beyder, Arthur; Bernard, Cheryl E.; Perez-Reyes, Edward; Evangelista, Stefano; Gibbons, Simon J.; Szurszewski, Joseph H.

    2010-01-01

    Antispasmodics are used clinically to treat a variety of gastrointestinal disorders by inhibition of smooth muscle contraction. The main pathway for smooth muscle Ca2+ entry is through L-type channels; however, there is increasing evidence that T-type Ca2+ channels also play a role in regulating contractility. Otilonium bromide, an antispasmodic, has previously been shown to inhibit L-type Ca2+ channels and colonic contractile activity. The objective of this study was to determine whether otilonium bromide also inhibits T-type Ca2+ channels. Whole cell currents were recorded by patch-clamp technique from HEK293 cells transfected with cDNAs encoding the T-type Ca2+ channels, CaV3.1 (α1G), CaV3.2 (α1H), or CaV3.3 (α1I) alpha subunits. Extracellular solution was exchanged with otilonium bromide (10−8 to 10−5 M). Otilonium bromide reversibly blocked all T-type Ca2+ channels with a significantly greater affinity for CaV3.3 than CaV3.1 or CaV3.2. Additionally, the drug slowed inactivation in CaV3.1 and CaV3.3. Inhibition of T-type Ca2+ channels may contribute to inhibition of contractility by otilonium bromide. This may represent a new mechanism of action for antispasmodics and may contribute to the observed increased clinical effectiveness of antispasmodics compared with selective L-type Ca2+ channel blockers. PMID:20203058

  17. T-type Ca(2+) channel modulation by otilonium bromide.

    PubMed

    Strege, Peter R; Sha, Lei; Beyder, Arthur; Bernard, Cheryl E; Perez-Reyes, Edward; Evangelista, Stefano; Gibbons, Simon J; Szurszewski, Joseph H; Farrugia, Gianrico

    2010-05-01

    Antispasmodics are used clinically to treat a variety of gastrointestinal disorders by inhibition of smooth muscle contraction. The main pathway for smooth muscle Ca(2+) entry is through L-type channels; however, there is increasing evidence that T-type Ca(2+) channels also play a role in regulating contractility. Otilonium bromide, an antispasmodic, has previously been shown to inhibit L-type Ca(2+) channels and colonic contractile activity. The objective of this study was to determine whether otilonium bromide also inhibits T-type Ca(2+) channels. Whole cell currents were recorded by patch-clamp technique from HEK293 cells transfected with cDNAs encoding the T-type Ca(2+) channels, Ca(V)3.1 (alpha1G), Ca(V)3.2 (alpha1H), or Ca(V)3.3 (alpha1I) alpha subunits. Extracellular solution was exchanged with otilonium bromide (10(-8) to 10(-5) M). Otilonium bromide reversibly blocked all T-type Ca(2+) channels with a significantly greater affinity for Ca(V)3.3 than Ca(V)3.1 or Ca(V)3.2. Additionally, the drug slowed inactivation in Ca(V)3.1 and Ca(V)3.3. Inhibition of T-type Ca(2+) channels may contribute to inhibition of contractility by otilonium bromide. This may represent a new mechanism of action for antispasmodics and may contribute to the observed increased clinical effectiveness of antispasmodics compared with selective L-type Ca(2+) channel blockers.

  18. Novel therapeutic approaches targeting L-type amino acid transporters for cancer treatment

    PubMed Central

    Hayashi, Keitaro; Anzai, Naohiko

    2017-01-01

    L-type amino acid transporters (LATs) mainly assist the uptake of neutral amino acids into cells. Four LATs (LAT1, LAT2, LAT3 and LAT4) have so far been identified. LAT1 (SLC7A5) has been attracting much attention in the field of cancer research since it is commonly up-regulated in various cancers. Basic research has made it increasingly clear that LAT1 plays a predominant role in malignancy. The functional significance of LAT1 in cancer and the potential therapeutic application of the features of LAT1 to cancer management are described in this review. PMID:28144396

  19. Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

    Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.

  20. Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium Channels

    PubMed Central

    Chaplin, Nathan L.; Nieves-Cintrón, Madeline; Fresquez, Adriana M.; Navedo, Manuel F.; Amberg, Gregory C.

    2015-01-01

    Rationale Mitochondria are key integrators of convergent intracellular signaling pathways. Two important second messengers modulated by mitochondria are calcium and reactive oxygen species. To date, coherent mechanisms describing mitochondrial integration of calcium and oxidative signaling in arterial smooth muscle are incomplete. Objective To address and add clarity to this issue we tested the hypothesis that mitochondria regulate subplasmalemmal calcium and hydrogen peroxide microdomain signaling in cerebral arterial smooth muscle. Methods and Results Using an image-based approach we investigated the impact of mitochondrial regulation of L-type calcium channels on subcellular calcium and ROS signaling microdomains in isolated arterial smooth muscle cells. Our single cell observations were then related experimentally to intact arterial segments and to living animals. We found that subplasmalemmal mitochondrial amplification of hydrogen peroxide microdomain signaling stimulates L-type calcium channels and that this mechanism strongly impacts the functional capacity of the vasoconstrictor angiotensin II. Importantly, we also found that disrupting this mitochondrial amplification mechanism in vivo normalized arterial function and attenuated the hypertensive response to systemic endothelial dysfunction. Conclusions From these observations we conclude that mitochondrial amplification of subplasmalemmal calcium and hydrogen peroxide microdomain signaling is a fundamental mechanism regulating arterial smooth muscle function. As the principle components involved are fairly ubiquitous and positioning of mitochondria near the plasma membrane is not restricted to arterial smooth muscle, this mechanism could occur in many cell types and contribute to pathological elevations of intracellular calcium and increased oxidative stress associated with many diseases. PMID:26390880

  1. Phenotype-dependent role of the L-type calcium current in embryonic stem cell derived cardiomyocytes

    PubMed Central

    Dan, Pauline; Zeng, Zheng; Li, Ying; Qu, Yang; Hove-Madsen, Leif; Tibbits, Glen F

    2014-01-01

    Although the L-type Ca2+ current (ICa,L) plays an important role in cardiac contractility and pacemaking, its role in embryonic stem-cell derived cardiomyocytes (ESC-CMs) has not yet been explored in detail. We used patch-clamp techniques to characterize ICa,L, action potential properties, and nifedipine (an ICa,L blocker) sensitivity on spontaneously contracting embryoid bodies (EBs) or isolated ESC-CMs. Cellular preparations exhibited differential sensitivity to nifedipine, with substantial variation in the dose required to abolish automaticity. Isolated ESC-CMs expressing nodal-like action potentials were highly sensitive to nifedipine; 1 nM significantly decreased firing rate, diastolic depolarization rate (DDR), and upstroke velocity, and 10 nM completely abolished spontaneous activity. In contrast, ESC-CMs expressing atrial-like action potentials were relatively nifedipine-resistant, requiring 10 μM to arrest automaticity; 1 μM significantly decreased upstroke velocity while the firing rate and DDR were unaffected. Nodal-like cells exhibited a more negative voltage for half-maximal ICa activation (-30 ± 1 mV vs. -20 ± 3 mV; p<0.05) and slower inactivation (71 ± 10 ms vs. 43 ± 3 ms; p<0.05) than atrial-like cells. Our data indicate that ICa,L differentially regulates automaticity and chronotropy in nodal-like ESC-CMs, and primarily links excitation to contraction in atrial-like ESC-CMs by contributing to the upstroke phase of the action potential. PMID:24660113

  2. Accelerated Inactivation of the L-type Calcium Current Due to a Mutation in CACNB2b Underlies Brugada Syndrome

    PubMed Central

    Cordeiro, Jonathan M; Marieb, Mark; Pfeiffer, Ryan; Calloe, Kirstine; Burashnikov, Elena; Antzelevitch, Charles

    2009-01-01

    Background Recent studies have demonstrated an association between mutations in CACNA1c or CACNB2b and Brugada syndrome (BrS). Previously described mutations all caused a loss of function secondary to a reduction of peak calcium current (ICa). We describe a novel CACNB2b mutation associated with BrS in which loss of function is caused by accelerated inactivation of ICa. Methods and Results The proband, a 32 yo male, displayed a Type I ST segment elevation in two right precordial ECG leads following a procainamide challenge. EP study was positive with induction of polymorphic VT/VF. Interrogation of implanted ICD revealed brief episodes of very rapid ventricular tachycardia. He was also diagnosed with vasovagal syncope. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 15 ion channel genes were amplified and sequenced. The only mutation uncovered was a missense mutation (T11I) in CACNB2b. We expressed WT or T11I CACNB2b in TSA201 cells co-transfected with WT CACNA1c and CACNA2d. Patch clamp analysis showed no significant difference between WT and T11I in peak ICa density, steady-state inactivation or recovery from inactivation. However, both fast and slow decay of ICa were significantly faster in mutant channels between 0 and +20 mV. Action potential voltage clamp experiments showed that total charge was reduced by almost half compared to WT. Conclusions We report the first BrS mutation in CaCNB2b resulting in accelerated inactivation of L-type calcium channel current. Our results suggest that the faster current decay results in a loss-of-function responsible for the Brugada phenotype. PMID:19358333

  3. Expression of the alpha(2)delta subunit interferes with prepulse facilitation in cardiac L-type calcium channels.

    PubMed Central

    Platano, D; Qin, N; Noceti, F; Birnbaumer, L; Stefani, E; Olcese, R

    2000-01-01

    We investigated the role of the accessory alpha(2)delta subunit on the voltage-dependent facilitation of cardiac L-type Ca(2+) channels (alpha(1C)). alpha(1C) Channels were coexpressed in Xenopus oocytes with beta(3) and alpha(2)delta calcium channel subunits. In alpha(1C) + beta(3), the amplitude of the ionic current (measured during pulses to 10 mV) was in average approximately 1.9-fold larger after the application of a 200-ms prepulse to +80 mV. This phenomenon, commonly referred to as voltage-dependent facilitation, was not observed when alpha(2)delta was coexpressed with alpha(1C) + beta(3). In alpha(1C) + beta(3), the prepulse produced a left shift ( approximately 40 mV) of the activation curve. Instead, the activation curve for alpha(1C) + beta(3) + alpha(2)delta was minimally affected by the prepulse and had a voltage dependence very similar to the G-V curve of the alpha(1C) + beta(3) channel facilitated by the prepulse. Coexpression of alpha(2)delta with alpha(1C) + beta(3) seems to mimic the prepulse effect by shifting the activation curve toward more negative potentials, leaving little room for facilitation. The facilitation of alpha(1C) + beta(3) was associated with an increase of the charge movement. In the presence of alpha(2)delta, the charge remained unaffected after the prepulse. Coexpression of alpha(2)delta seems to set all the channels in a conformational state from where the open state can be easily reached, even without prepulse. PMID:10827975

  4. An L-type calcium channel agonist, bay K8644, extends the window of intervention against ischemic neuronal injury.

    PubMed

    Hu, Hong-hai; Li, Shu-ji; Wang, Pu; Yan, Hua-cheng; Cao, Xiong; Hou, Feng-qin; Fang, Ying-ying; Zhu, Xin-hong; Gao, Tian-ming

    2013-02-01

    Our previous data indicate that the inhibition of L-type calcium channels (LTCCs) might be the cause of post-ischemic neuronal injury and that the activation of LTCCs can give rise to neuroprotection. In the present study, we aimed to profile the intervention window of Bay K8644, an LTCC agonist, and determine the involved mechanisms. The four vessel occlusion and oxygen-glucose deprivation models were employed to mimic ischemia/reperfusion damage in vivo and in vitro. Neuronal injury was analyzed using Nissl and Fluoro-Jade B staining in vivo and Hoechst 33342 and propidium iodide staining in vitro. The behavioral effects were tested using the Morris water maze. The phosphorylation of P38, Jun N-terminal kinase, and extracellular-regulated kinase (ERK) was detected by Western blotting. Our results show that Bay K8644 administered as late as 24 h after reperfusion prevented CA1 neuronal death and ameliorated the deficiencies in spatial learning performance induced by global ischemia. In oxygen-glucose deprivation (OGD), Bay K8644 delivered from 1 to 12 h after re-oxygenation reduced neuronal death. The decrease in p-ERK1/2 that was observed at 1 h after OGD was reversed by Bay K8644, and the effect of Bay K8644 was blocked by treatment with U0126 and MEK kinase dead transfection. Moreover, similar to Bay K8644, FPL 64176, another potent LTCC agonist, extends the window of intervention against neuronal injury in an in vitro model of ischemia. In conclusion, our data suggest that opening LTCCs may be a practicable approach for stroke therapy.

  5. Ontogeny of Ca2+-induced Ca2+ release in rabbit ventricular myocytes.

    PubMed

    Huang, Jingbo; Hove-Madsen, Leif; Tibbits, Glen F

    2008-02-01

    It is commonly accepted that L-type Ca(2+) channel-mediated Ca(2+)-induced Ca(2+) release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the adult mammalian heart and that there is no appreciable CICR in neonates. However, we have observed that cell contraction in the neonatal heart was significantly decreased after sarcoplasmic reticulum (SR) Ca(2+) depletion with caffeine. Therefore, the present study investigated the developmental changes of CICR in rabbit ventricular myocytes at 3, 10, 20, and 56 days of age. We found that the inhibitory effect of the L-type Ca(2+) current (I(Ca)) inhibitor nifedipine (Nif; 15 microM) caused an increasingly larger reduction of Ca(2+) transients on depolarization in older age groups [from approximately 15% in 3-day-old (3d) myocytes to approximately 90% in 56-day-old (56d) myocytes]. The remaining Ca(2+) transient in the presence of Nif in younger age groups was eliminated by the inhibition of Na(+)/Ca(2+) exchanger (NCX) with the subsequent addition of 10 microM KB-R7943 (KB-R). Furthermore, Ca(2+) transients were significantly reduced in magnitude after the depletion of SR Ca(2+) with caffeine in all age groups, although the effect was significantly greater in the older age groups (from approximately 40% in 3d myocytes up to approximately 70% in 56d myocytes). This SR Ca(2+)-sensitive Ca(2+) transient in the earliest developmental stage was insensitive to Nif but was sensitive to the subsequent addition of KB-R, indicating the presence of NCX-mediated CICR that decreased significantly with age (from approximately 37% in 3d myocytes to approximately 0.5% in 56d myocytes). In contrast, the I(Ca)-mediated CICR increased significantly with age (from approximately 10% in 3d myocytes to approximately 70% in 56d myocytes). The CICR gain as estimated by the integral of the CICR Ca(2+) transient divided by the integral of its Ca(2+) transient trigger was smaller when mediated by NCX ( approximately 1.0 for 3d

  6. Energy logistics in an all-optical adder based on a 1D porous silicon photonic crystal

    NASA Astrophysics Data System (ADS)

    Glushko, E. Ya.

    2011-09-01

    The ideology of a photonic crystal resonator covered with optically nonlinear layers is proposed for binary adder and logic gates of various kinds. The all-optical way to transform a physically added sequence of signals into the logical sequence with corresponding shift of digital units is based on the nonlinear band shift effect. In this work, the electromagnetic field structure for optically linear 1D porous silicon photonic crystal is investigated. The optical parameters of a 1D photonic crystal resonator built on layered porous silicon covered with a nonlinear layer are calculated for various nonlinear materials. An approximate design of an all-optical adder based on 1D porous silicon resonator is considered. The adder heating by powered optical pulses and energy distribution inside the device are analyzed and the problem solution with the use of special semitransparent redirecting mirrors is proposed. It was found that from the point of view of heating the R-scheme of signal processing is more optimal.

  7. Sensitivity of transpiration to subsurface properties: Exploration with a 1-D model: ET SENSITIVITY TO SUBSURFACE PROPERTIES

    DOE PAGES

    Vrettas, Michail D.; Fung, Inez Y.

    2017-05-04

    The amount of moisture transpired by vegetation is critically tied to the moisture supply accessible to the root zone. In a Mediterranean climate, integrated evapotranspiration (ET) is typically greater in the dry summer when there is an uninterrupted period of high insolation. We present a 1-D model to explore the subsurface factors that may sustain ET through the dry season. The model includes a stochastic parameterization of hydraulic conductivity, root water uptake efficiency, and hydraulic redistribution by plant roots. Model experiments vary the precipitation, the magnitude and seasonality of ET demand, as well as rooting profiles and rooting depths ofmore » the vegetation. The results show that the amount of subsurface moisture remaining at the end of the wet winter is determined by the competition among abundant precipitation input, fast infiltration, and winter ET demand. The weathered bedrock retains math formula of the winter rain and provides a substantial moisture reservoir that may sustain ET of deep-rooted (>8 m) trees through the dry season. A small negative feedback exists in the root zone, where the depletion of moisture by ET decreases hydraulic conductivity and enhances the retention of moisture. Hence, hydraulic redistribution by plant roots is impactful in a dry season, or with a less conductive subsurface. Suggestions for implementing the model in the CESM are discussed.« less

  8. Impact of sea spray on upper ocean temperature during typhoon passage: simulation with a 1-D turbulent model

    NASA Astrophysics Data System (ADS)

    Zhang, Lianxin; Zhang, Xuefeng; Han, Guijun; Wu, Xinrong; Cui, Xiaojian; Shao, Caixia; Sun, Chunjian; Zhang, Xiaoshuang; Wang, Xidong; Fu, Hongli

    2015-09-01

    At the interface between the lower atmosphere and sea surface, sea spray might significantly influence air-sea heat fluxes and subsequently, modulate upper ocean temperature during a typhoon passage. The effects of sea spray were introduced into the parameterization of sea surface roughness in a 1-D turbulent model, to investigate the effects of sea spray on upper ocean temperature in the Kuroshio Extension area, for the cases of two real typhoons from 2006, Yagi and Soulik. Model output was compared with data from the Kuroshio Extension Observatory (KEO), and Reynolds and AMSRE satellite remote sensing sea surface temperatures. The results indicate drag coefficients that include the spray effect are closer to observations than those without, and that sea spray can enhance the heat fluxes (especially latent heat flux) considerably during a typhoon passage. Consequently, the model results with heat fluxes enhanced by sea spray simulate better the cooling process of the SST and upper-layer temperature profiles. Additionally, results from the simulation of the passage of typhoon Soulik (that passed KEO quickly), which included the sea spray effect, were better than for the simulated passage of typhoon Yagi (that crossed KEO slowly). These promising 1-D results could provide insight into the application of sea spray in general circulation models for typhoon studies.

  9. Study of proton conductivity of a 2D flexible MOF and a 1D coordination polymer at higher temperature.

    PubMed

    Sanda, Suresh; Biswas, Soumava; Konar, Sanjit

    2015-02-16

    We report the proton conduction properties of a 2D flexible MOF and a 1D coordination polymer having the molecular formulas {[Zn(C10H2O8)0.5(C10S2N2H8)]·5H2O]}n (1) and {[Zn(C10H2O8)0.5(C10S2N2H8)]·2H2O]}n (2), respectively. Compounds 1 and 2 show high conductivity values of 2.55 × 10(-7) and 4.39 × 10(-4) S cm(-1) at 80 °C and 95% RH. The conductivity value of compound 1 is in the range of those for previously reported flexible MOFs, and compound 2 shows the highest proton conductivity among the carboxylate-based 1D CPs. The dimensionality and the internal hydrogen bonding connectivity play a vital role in the resultant conductivity. Variable-temperature experiments of both compounds at high humidity reveal that the conductivity values increase with increasing temperature, whereas the variable humidity studies signify the influence of relative humidity on high-temperature proton conductivity. The time-dependent measurements for both compounds demonstrate their ability to retain conductivity up to 10 h.

  10. A 1D-2D Shallow Water Equations solver for discontinuous porosity field based on a Generalized Riemann Problem

    NASA Astrophysics Data System (ADS)

    Ferrari, Alessia; Vacondio, Renato; Dazzi, Susanna; Mignosa, Paolo

    2017-09-01

    A novel augmented Riemann Solver capable of handling porosity discontinuities in 1D and 2D Shallow Water Equation (SWE) models is presented. With the aim of accurately approximating the porosity source term, a Generalized Riemann Problem is derived by adding an additional fictitious equation to the SWEs system and imposing mass and momentum conservation across the porosity discontinuity. The modified Shallow Water Equations are theoretically investigated, and the implementation of an augmented Roe Solver in a 1D Godunov-type finite volume scheme is presented. Robust treatment of transonic flows is ensured by introducing an entropy fix based on the wave pattern of the Generalized Riemann Problem. An Exact Riemann Solver is also derived in order to validate the numerical model. As an extension of the 1D scheme, an analogous 2D numerical model is also derived and validated through test cases with radial symmetry. The capability of the 1D and 2D numerical models to capture different wave patterns is assessed against several Riemann Problems with different wave patterns.

  11. Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer’s disease

    PubMed Central

    Thibault, Olivier; Pancani, Tristano; Landfield, Philip W.; Norris, Christopher M.

    2012-01-01

    Increased function of neuronal L-type voltage-sensitive Ca2+ channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer’s disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal “zipper” slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca2+ dysregulation can differ substantially between animal models of normal aging and models of pathological aging. PMID:22265986

  12. New Findings on the Effects of Tannic Acid: Inhibition of L-Type Calcium Channels, Calcium Transient and Contractility in Rat Ventricular Myocytes.

    PubMed

    Zhu, Fengli; Chu, Xi; Wang, Hua; Zhang, Xuan; Zhang, Yuanyuan; Liu, Zhenyi; Guo, Hui; Liu, Hongying; Liu, Yang; Chu, Li; Zhang, Jianping

    2016-03-01

    Tannic acid (TA) is a group of water-soluble polyphenolic compounds that occur mainly in plant-derived feeds, food grains and fruits. Many studies have explored its biomedical properties, such as anticancer, antibacterial, antimutagenic, antioxidant, antidiabetic, antiinflammatory and antihypertensive activities. However, the effects of TA on the L-type Ca(2+) current (ICa-L) of cardiomyocytes remain undefined. The present study examined the effects of TA on ICa-L using the whole-cell patch-clamp technique and on intracellular Ca(2+) handling and cell contractility in rat ventricular myocytes with the aid of a video-based edge detection system. Exposure to TA resulted in a concentration- and voltage-dependent blockade of ICa-L, with the half maximal inhibitory concentration of 1.69 μM and the maximal inhibitory effect of 46.15%. Moreover, TA significantly inhibited the amplitude of myocyte shortening and peak value of Ca(2+) transient and increased the time to 10% of the peak. These findings provide new experimental evidence for the cellular mechanism of action of TA and may help to expand clinical treatments for cardiovascular disease.

  13. Interactions between calcium channels and SK channels in midbrain dopamine neurons and their impact on pacemaker regularity: Contrasting roles of N- and L-type channels.

    PubMed

    de Vrind, Veronne; Scuvée-Moreau, Jacqueline; Drion, Guillaume; Hmaied, Cyrine; Philippart, Fabian; Engel, Dominique; Seutin, Vincent

    2016-10-05

    Although small-conductance Ca(2+)-activated K(+) (SK) channels and various types of voltage-gated Ca(2+) (Cav) channels have been described in midbrain dopaminergic neurons, the nature of their interactions is unclear. More particularly, the role of various Cav channel types in either promoting irregularity of firing (by generating an inward current during SK channel blockade) or promoting regularity of firing (by providing the source of Ca(2+) for the activation of SK channels) has not been systematically explored. We addressed this question using intracellular and extracellular recordings from substantia nigra, pars compacta (SNc), dopaminergic neurons in rat midbrain slices. Neurons were pharmacologically isolated from their differences. When examining the ability of various Cav channel blockers to inhibit the SK-mediated afterhyperpolarization (AHP), we found that only the N-type Cav channel blocker ω-conotoxin-GVIA was able to reduce the apamin-sensitive AHP, but only partially (~40%). Specific blockers of L, P/Q, T or R channels had no effect on this AHP. Combining ω-conotoxin-GVIA and other specific blockers did not yield greater block and even the broad Cav blocker Cd(2+) induced a submaximal (~75%) effect. Extracellular recordings examining firing regularity yielded congruent results: none of the specific blockers was able to increase firing irregularity to the extent that the specific SK blocker apamin did. The irregularity of firing observed with apamin could only be reversed by blocking L-type Ca(2+) channels. Thus various sources of Ca(2+) appear to be required for SK channel activation in SNc neurons (some of them still unidentified), ensuring robustness of pacemaking regularity. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. ABSTRACTION OF INFORMATION FROM 2- AND 3-DIMENSIONAL PORFLOW MODELS INTO A 1-D GOLDSIM MODEL - 11404

    SciTech Connect

    Taylor, G.; Hiergesell, R.

    2010-11-16

    The Savannah River National Laboratory has developed a 'hybrid' approach to Performance Assessment modeling which has been used for a number of Performance Assessments. This hybrid approach uses a multi-dimensional modeling platform (PorFlow) to develop deterministic flow fields and perform contaminant transport. The GoldSim modeling platform is used to develop the Sensitivity and Uncertainty analyses. Because these codes are performing complementary tasks, it is incumbent upon them that for the deterministic cases they produce very similar results. This paper discusses two very different waste forms, one with no engineered barriers and one with engineered barriers, each of which present different challenges to the abstraction of data. The hybrid approach to Performance Assessment modeling used at the SRNL uses a 2-D unsaturated zone (UZ) and a 3-D saturated zone (SZ) model in the PorFlow modeling platform. The UZ model consists of the waste zone and the unsaturated zoned between the waste zone and the water table. The SZ model consists of source cells beneath the waste form to the points of interest. Both models contain 'buffer' cells so that modeling domain boundaries do not adversely affect the calculation. The information pipeline between the two models is the contaminant flux. The domain contaminant flux, typically in units of moles (or Curies) per year from the UZ model is used as a boundary condition for the source cells in the SZ. The GoldSim modeling component of the hybrid approach is an integrated UZ-SZ model. The model is a 1-D representation of the SZ, typically 1-D in the UZ, but as discussed below, depending on the waste form being analyzed may contain pseudo-2-D elements. A waste form at the Savannah River Site (SRS) which has no engineered barriers is commonly referred to as a slit trench. A slit trench, as its name implies, is an unlined trench, typically 6 m deep, 6 m wide, and 200 m long. Low level waste consisting of soil, debris, rubble, wood

  15. A 1D Model of Radial Ion Motion Interrupted by Ion-Neutral Interactions in a Cometary Coma

    NASA Astrophysics Data System (ADS)

    Vigren, E.; Eriksson, A. I.

    2017-04-01

    Because ion-neutral reaction cross sections are energy dependent, the distance from a cometary nucleus within which ions remain collisionally coupled to the neutrals is dictated not only by the comet’s activity level but also by the electromagnetic fields in the coma. Here we present a 1D model simulating the outward radial motion of water group ions with radial acceleration by an ambipolar electric field interrupted primarily by charge transfer processes with H2O. We also discuss the impact of plasma waves. For a given electric field profile, the model calculates key parameters, including the total ion density, n I , the H3O+/H2O+ number density and flux ratios, R dens and R flux, and the mean ion drift speed, < {u}I> , as a function of cometocentric distance. We focus primarily on a coma roughly resembling that of the ESA Rosetta mission target comet 67P/Churyumov-Gerasimenko near its perihelion in 2015 August. In the presence of a weak ambipolar electric field in the radial direction the model results suggest that the neutral coma is not sufficiently dense to keep the mean ion flow speed close to that of the neutrals by the spacecraft location (˜200 km from the nucleus). In addition, for electric field profiles giving n I and < {u}I> within limits constrained by measurements, the R dens values are significantly higher than values typically observed. However, when including the ion motion in large-amplitude plasma waves in the model, results more compatible with observations are obtained. We suggest that the variable and often low H3O+/H2O+ number density ratios observed may reflect nonradial ion trajectories strongly influenced by electromagnetic forces and/or plasma instabilities, with energization of the ion population by plasma waves.

  16. A 1-D variational retrieval of temperature, humidity, and liquid cloud properties: Performance under idealized and real conditions

    NASA Astrophysics Data System (ADS)

    Ebell, K.; Löhnert, U.; Päschke, E.; Orlandi, E.; Schween, J. H.; Crewell, S.

    2017-02-01

    An extended version of the Integrated Profiling Technique (IPT) is presented. The IPT combines measurements from cloud radar and microwave radiometer (MWR) with prior information in a 1D-Var approach in order to retrieve physically consistent atmospheric profiles of temperature, absolute humidity, liquid water content (LWC), and recently also cloud droplet effective radius (REF). Physical consistency implies the reproducibility of the measurements within the uncertainties. Results based on synthetic observations revealed a good retrieval performance with a high convergence rate of 98%. Retrieval uncertainties are typically around 0.06 g m-3 for LWC and 0.6 μm for REF. For the application to real measurements, quality-controlled, bias-free observations are crucial. A newly developed MWR spectral consistency check, which was applied to the measurements at the Jülich Observatory for Cloud Evolution (JOYCE), revealed strongly bias-affected channels. The IPT itself can serve as a further quality check: particularly in clear-sky cases, nonconvergence or physically inconsistent solutions may hint at measurement offset errors. Based on sensitivity studies, the final set of MWR frequencies was identified and the retrieval applied to 1 year of data. Physically consistent solutions could be found in 62% of all processed cases. A focus was put on the analysis of nondrizzling single-layer water clouds which typically have small geometrical thicknesses (<300 m), low liquid water paths (<50 g m-2), and small REF (<5 μm). The retrieved data product contains a high potential for the analysis of warm cloud characteristics and, in combination with auxiliary information from the JOYCE instrumentation, of associated boundary layer processes.

  17. CaV1.1: The atypical prototypical voltage-gated Ca2+ channel

    PubMed Central

    Bannister, Roger A.; Beam, Kurt G.

    2012-01-01

    CaV1.1 is the prototype for the other nine known CaV channel isoforms, yet it has functional properties that make it truly atypical of this group. Specifically, CaV1.1 is expressed solely in skeletal muscle where it serves multiple purposes; it is the voltage sensor for excitation-contraction (EC) coupling and it is an L-type Ca2+ channel which contributes to a form of activity-dependent Ca2+ entry that has been termed Excitation-Coupled Ca2+ Entry (ECCE). The ability of CaV1.1 to serve as voltage-sensor for EC coupling appears to be unique amongst CaV channels, whereas the physiological role of its more conventional function as a Ca2+ channel has been a matter of uncertainty for nearly 50 years. In this chapter, we discuss how CaV1.1 supports EC coupling, the possible relevance of Ca2+ entry through CaV1.1 and how alterations of CaV1.1 function can have pathophysiological consequences. PMID:22982493

  18. Role of sarcoplasmic reticulum Ca2+ content in Ca2+ entry of bovine airway smooth muscle cells.

    PubMed

    Bazán-Perkins, Blanca; Flores-Soto, Edgar; Barajas-López, Carlos; Montaño, Luis M

    2003-10-01

    Depletion of intracellular Ca(2+) stores induces the opening of an unknown Ca(2+ )entry pathway to the cell. We measured the intracellular free-Ca(2+) concentration ([Ca(2+)]i) at different sarcoplasmic reticulum (SR) Ca(2+) content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca(2+) in the extracellular medium generated a time-dependent decrement in [Ca(2+)]i which was proportional to the reduction in the SR-Ca(2+) content. This SR-Ca(2+) level was indirectly determined by measuring the amount of Ca(2+) released by caffeine. Ca(2+) restoration at different times after Ca(2+)-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca(2+)]i. This increase in [Ca(2+)]i was considered as Ca(2+) entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca(2+) content was higher than 50% (2 and 4 min in Ca(2+)-free medium), and significantly ( p<0.01) accelerated (>1.0 nM/s) when SR-Ca(2+) content was lower than 50% (6 and 10 min in Ca(2+)-free medium). Thapsigargin significantly induced a higher rate of this Ca(2+) entry ( p<0.01). Variations in Ca(2+) influx after SR-Ca(2+) depletion were estimated more directly by a Mn(2+) quench approach. Ca(2+) restoration to the medium 4 min after Ca(2+) removal did not modify the Mn(2+) influx. However, when Ca(2+) was added after 10 min in Ca(2+)-free medium, an increment of Mn(2+) influx was observed, corroborating an increase in Ca(2+) entry. The fast Ca(2+) influx was Ni(2+) sensitive but was not affected by other known capacitative Ca(2+) entry blockers such as La(3+), Mg(2+), SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2(+) channels with methoxyverapamil or by the sustained K(+)-induced depolarisation. The slow Ca(2+) influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca(2+) influx after SR-Ca(2+) depletion has two rates: A) The slow Ca(2+) influx, which occurred in cells

  19. Specific inhibition of long-lasting, L-type calcium channels by synthetic parathyroid hormone

    SciTech Connect

    Pang, P.K.T.; Wang, R.; Shan, J.; Karpinski, E.; Benishin, C.G. )

    1990-01-01

    The effect of an active synthetic N-terminal fragment of bovine parathyroid hormone (bPTH), bPTH-(1-34), on Ca{sup 2+} channels was studied in mouse neuroblastoma cells (N1E-115). With the whole-cell variation of the patch-clamp technique, T (transient) and L (long-lasting) types of Ca{sup 2+} currents were identified. Pharmacological characterization showed that the L current was amplified by the Ca{sup 2+} channel stimulator BAY K-8644, but the T current was unaffected. The administration of bPTH-(1-34) produced dose-related inhibition of the L current, which could be reversed by BAY K-8644. The peptide had no effect on the T current. In addition, use of the fluorescent indicator fura-2 showed that bPTH-(1-34) inhibited the KCl-stimulated increase in intracellular free Ca{sup 2+} in neuroblastoma cells with L channels but not in cells with T channels. An inactivated (oxidized) preparation of bPTH-(1-34) failed to affect the L current. High-affinity binding of labeled PTH analog to these neuroblastoma cells was also demonstrated. In addition, bPTH-(1-34) inhibited the L current in cultured vascular smooth muscle cells from rat tail artery. These data indicate that, in some tissues PTH can act as an endogenous blocker of Ca{sup 2+} entry.

  20. Broad-spectrum antiemetic potential of the L-type calcium channel antagonist nifedipine and evidence for its additive antiemetic interaction with the 5-HT(3) receptor antagonist palonosetron in the least shrew (Cryptotis parva).

    PubMed

    Darmani, Nissar A; Zhong, Weixia; Chebolu, Seetha; Vaezi, Mariam; Alkam, Tursun

    2014-01-05

    Cisplatin-like chemotherapeutics cause vomiting via release of multiple neurotransmitters (dopamine, serotonin (5-HT), or substance P (SP)) from the gastrointestinal enterochromaffin cells and/or the brainstem via a calcium dependent process. Diverse channels in the plasma membrane allow extracellular Ca(2+) entry into cells for the transmitter release process. Agonists of 5-HT3 receptors increase calcium influx through both 5-HT3 receptors and L-type Ca(2+) channels. We envisaged that L-type calcium agonists such as FPL 64176 should cause vomiting and corresponding antagonists such as nifedipine would behave as broad-spectrum antiemetics. Administration of FPL 64176 did cause vomiting in the least shrew in a dose-dependent fashion. Nifedipine and the 5-HT3 receptor antagonist palonosetron, potently suppressed FPL 64176-induced vomiting, while a combination of ineffective doses of these antagonists was more efficacious. Subsequently, we investigated the broad-spectrum antiemetic potential of nifedipine against diverse emetogens including agonists of serotonergic 5-HT3- (e.g. 5-HT or 2-Me-5-HT), SP tachykinin NK1- (GR73632), dopamine D2- (apomorphine or quinpirole), and cholinergic M1- (McN-A-343) receptors, as well as the non-specific emetogen, cisplatin. Nifedipine by itself suppressed vomiting in a potent and dose-dependent manner caused by the above emetogens except cisplatin. Moreover, low doses of nifedipine potentiated the antiemetic efficacy of non-effective or semi-effective doses of palonosetron against vomiting caused by either 2-Me-5-HT or cisplatin. Thus, our findings demonstrate that activation of L-type calcium channels causes vomiting, whereas blockade of these ion channels by nifedipine-like antagonists not only provides broad-spectrum antiemetic activity but can also potentiate the antiemetic efficacy of well-established antiemetics such as palonosetron. L-type calcium channel antagonists should also provide antiemetic activity against drug

  1. Novel HIV-1 Recombinants Spreading across Multiple Risk Groups in the United Kingdom: The Identification and Phylogeography of Circulating Recombinant Form (CRF) 50_A1D

    PubMed Central

    Foster, Geraldine M.; Ambrose, John C.; Hué, Stéphane; Delpech, Valerie C.; Fearnhill, Esther; Abecasis, Ana B.; Leigh Brown, Andrew J.; Geretti, Anna Maria

    2014-01-01

    Background An increase in non-B HIV-1 infections among men who have sex with men (MSM) in the United Kingdom (UK) has created opportunities for novel recombinants to arise and become established. We used molecular mapping to characterize the importance of such recombinants to the UK HIV epidemic, in order to gain insights into transmission dynamics that can inform control strategies. Methods and Results A total of 55,556 pol (reverse transcriptase and protease) sequences in the UK HIV Drug Resistance Database were analyzed using Subtype Classification Using Evolutionary Algorithms (SCUEAL). Overall 72 patients shared the same A1/D recombination breakpoint in pol, comprising predominantly MSM but also heterosexuals and injecting drug users (IDUs). In six MSM, full-length single genome amplification of plasma HIV-1 RNA was performed in order to characterize the A1/D recombinant. Subtypes and recombination breakpoints were identified using sliding window and jumping profile hidden markov model approaches. Global maximum likelihood trees of gag, pol and env genes were drawn using FastTree version 2.1. Five of the six strains showed the same novel A1/D recombinant (8 breakpoints), which has been classified as CRF50_A1D. The sixth strain showed a complex CRF50_A1D/B/U structure. Divergence dates and phylogeographic inferences were determined using Bayesian Evolutionary Analysis using Sampling Trees (BEAST). This estimated that CRF50_A1D emerged in the UK around 1992 in MSM, with subsequent transmissions to heterosexuals and IDUs. Analysis of CRF50_A1D/B/U demonstrated that around the year 2000 CRF50_A1D underwent recombination with a subtype B strain. Conclusions We report the identification of CRF50_A1D, a novel circulating recombinant that emerged in UK MSM around 1992, with subsequent onward transmission to heterosexuals and IDUs, and more recent recombination with subtype B. These findings highlight the changing dynamics of HIV transmission in the UK and the

  2. Two-step spin transition in a 1D Fe(II) 1,2,4-triazole chain compound.

    PubMed

    Dîrtu, Marinela M; Schmit, France; Naik, Anil D; Rusu, Ionela; Rotaru, Aurelian; Rackwitz, Sergej; Wolny, Juliusz A; Schünemann, Volker; Spinu, Leonard; Garcia, Yann

    2015-04-07

    A thermochromic 1D spin crossover coordination (SCO) polymer [Fe(βAlatrz)3](BF4)2⋅2 H2O (1⋅2 H2O), whose precursor βAlatrz, (1,2,4-triazol-4-yl-propionate) has been tailored from a β-amino acid ester is investigated in detail by a set of superconducting quantum interference device (SQUID), (57)Fe Mössbauer, differential scanning calorimetry, infrared, and Raman measurements. An hysteretic abrupt two-step spin crossover (T1/2(↓) = 230 K and T1/2(↑) = 235 K, and T1/2(↓) = 172 K and T1/2(↑) = 188 K, respectively) is registered for the first time for a 1,2,4-triazole-based Fe(II) 1D coordination polymer. The two-step SCO configuration is observed in a 1:2 ratio of low-spin/high-spin in the intermediate phase for a 1D chain. The origin of the stepwise transition was attributed to a distribution of chains of different lengths in 1⋅2 H2O after First Order Reversal Curves (FORC) analyses. A detailed DFT analysis allowed us to propose the normal mode assignment of the Raman peaks in the low-spin and high-spin states of 1⋅2 H2O. Vibrational spectra of 1⋅2 H2O reveal that the BF4(-) anions and water molecules play no significant role on the vibrational properties of the [Fe(βAlatrz)3](2+) polymeric chains, although non-coordinated water molecules have a dramatic influence on the emergence of a step in the spin transition curve. The dehydrated material [Fe(βAlatrz)3](BF4)2 (1) reveals indeed a significantly different magnetic behavior with a one-step SCO which was also investigated.

  3. A Computational Model of Cytosolic and Mitochondrial [Ca2+] in Paced Rat Ventricular Myocytes

    PubMed Central

    Choi, Seong Woo; Jang, Chang Han; Kim, Hyoung Kyu; Leem, Chae Hun; Kim, Nari; Han, Jin

    2011-01-01

    We carried out a series of experiment demonstrating the role of mitochondria in the cytosolic and mitochondrial Ca2+ transients and compared the results with those from computer simulation. In rat ventricular myocytes, increasing the rate of stimulation (1~3 Hz) made both the diastolic and systolic [Ca2+] bigger in mitochondria as well as in cytosol. As L-type Ca2+ channel has key influence on the amplitude of Ca2+-induced Ca2+ release, the relation between stimulus frequency and the amplitude of Ca2+ transients was examined under the low density (1/10 of control) of L-type Ca2+ channel in model simulation, where the relation was reversed. In experiment, block of Ca2+ uniporter on mitochondrial inner membrane significantly reduced the amplitude of mitochondrial Ca2+ transients, while it failed to affect the cytosolic Ca2+ transients. In computer simulation, the amplitude of cytosolic Ca2+ transients was not affected by removal of Ca2+ uniporter. The application of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) known as a protonophore on mitochondrial membrane to rat ventricular myocytes gradually increased the diastolic [Ca2+] in cytosol and eventually abolished the Ca2+ transients, which was similarly reproduced in computer simulation. The model study suggests that the relative contribution of L-type Ca2+ channel to total transsarcolemmal Ca2+ flux could determine whether the cytosolic Ca2+ transients become bigger or smaller with higher stimulus frequency. The present study also suggests that cytosolic Ca2+ affects mitochondrial Ca2+ in a beat-to-beat manner, however, removal of Ca2+ influx mechanism into mitochondria does not affect the amplitude of cytosolic Ca2+ transients. PMID:21994480

  4. Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes

    PubMed Central

    Glukhov, Alexey V.; Balycheva, Marina; Sanchez-Alonso, Jose L.; Ilkan, Zeki; Alvarez-Laviada, Anita; Bhogal, Navneet; Diakonov, Ivan; Schobesberger, Sophie; Sikkel, Markus B.; Bhargava, Anamika; Faggian, Giuseppe; Punjabi, Prakash P.; Houser, Steven R.

    2015-01-01

    Background— Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). Methods and Results— Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈40% greater current. Optical mapping of Ca2+ transients revealed that rat AMs presented ≈3-fold as many spontaneous Ca2+ release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca2+ transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-β-cyclodextrin, with an associated ≈30% whole-cell ICa,L reduction. Heart failure (16 weeks post–myocardial infarction) in rats resulted in a T-tubule degradation (by ≈40%) and significant elevation of spontaneous Ca2+ release events. Although heart failure did not affect LTCC occurrence, it led to ≈25% decrease in T-tubule LTCC amplitude. Conclusions— We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner. PMID:26450916

  5. Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model

    PubMed Central

    Bencsik, Anna; Biacabe, Anne-Gaëlle; Morignat, Eric; Bessen, Richard A.

    2007-01-01

    Transmissible mink encepholapathy (TME) is a foodborne transmissible spongiform encephalopathy (TSE) of ranch-raised mink; infection with a ruminant TSE has been proposed as the cause, but the precise origin of TME is unknown. To compare the phenotypes of each TSE, bovine-passaged TME isolate and 3 distinct natural bovine spongiform encephalopathy (BSE) agents (typical BSE, H-type BSE, and L-type BSE) were inoculated into an ovine transgenic mouse line (TgOvPrP4). Transgenic mice were susceptible to infection with bovine-passaged TME, typical BSE, and L-type BSE but not to H-type BSE. Based on survival periods, brain lesions profiles, disease-associated prion protein brain distribution, and biochemical properties of protease-resistant prion protein, typical BSE had a distint phenotype in ovine transgenic mice compared to L-type BSE and bovine TME. The similar phenotypic properties of L-type BSE and bovine TME in TgOvPrP4 mice suggest that L-type BSE is a much more likely candidate for the origin of TME than is typical BSE. PMID:18258040

  6. Phenotypic similarity of transmissible mink encephalopathy in cattle and L-type bovine spongiform encephalopathy in a mouse model.

    PubMed

    Baron, Thierry; Bencsik, Anna; Biacabe, Anne-Gaëlle; Morignat, Eric; Bessen, Richard A

    2007-12-01

    Transmissible mink encepholapathy (TME) is a foodborne transmissible spongiform encephalopathy (TSE) of ranch-raised mink; infection with a ruminant TSE has been proposed as the cause, but the precise origin of TME is unknown. To compare the phenotypes of each TSE, bovine-passaged TME isolate and 3 distinct natural bovine spongiform encephalopathy (BSE) agents (typical BSE, H-type BSE, and L-type BSE) were inoculated into an ovine transgenic mouse line (TgOvPrP4). Transgenic mice were susceptible to infection with bovine-passaged TME, typical BSE, and L-type BSE but not to H-type BSE. Based on survival periods, brain lesions profiles, disease-associated prion protein brain distribution, and biochemical properties of protease-resistant prion protein, typical BSE had a distint phenotype in ovine transgenic mice compared to L-type BSE and bovine TME. The similar phenotypic properties of L-type BSE and bovine TME in TgOvPrP4 mice suggest that L-type BSE is a much more likely candidate for the origin of TME than is typical BSE.

  7. In vitro characterization of L-type calcium channels and their contribution to firing behavior in invertebrate respiratory neurons.

    PubMed

    Spafford, J David; Dunn, Tyler; Smit, August B; Syed, Naweed I; Zamponi, Gerald W

    2006-01-01

    L-type calcium channel activity has been associated with a number of cytoplasmic responses, including gene transcription and activation of calcium-dependent enzymes, yet their direct contribution to the electrical activities of neurons has remained largely unexplored. Here we report the cloning and functional characterization of a molluscan L-type calcium channel homologue, LCa(v)1, and investigate its role in coordinating neuronal firing patterns. The LCav1 channel exhibits many hallmarks of vertebrate L-type channels in that it is high-voltage activated, slowly inactivating, and dihydropyridine sensitive and displays calcium-dependent inactivation in recording solutions with standard EGTA concentrations. We show that despite comprising less than approximately 20% of the total whole cell current in identified Lymnaea respiratory network neurons, the L-type channels are essential for maintaining rhythmic action potential discharges without being involved in synaptic release. Our data therefore suggest an important role of L-type calcium channels in maintaining rhythmical pattern activity underlying breathing behavior in Lymnaea.

  8. Properties of L-type bovine spongiform encephalopathy in intraspecies passages.

    PubMed

    Okada, H; Iwamaru, Y; Kakizaki, M; Masujin, K; Imamura, M; Fukuda, S; Matsuura, Y; Shimizu, Y; Kasai, K; Mohri, S; Yokoyama, T

    2012-09-01

    The origin and transmission routes of atypical bovine spongiform encephalopathy (BSE) remain unclear. To assess whether the biological and biochemical characteristics of atypical L-type BSE detected in Japanese cattle (BSE/JP24) are conserved during serial passages within a single host, 3 calves were inoculated intracerebrally with a brain homogenate prepared from first-passaged BSE/JP24-affected cattle. Detailed immunohistochemical and neuropathologic analysis of the brains of second-passaged animals, which had developed the disease and survived for an average of 16 months after inoculation, revealed distribution of spongiform changes and disease-associated prion protein (PrP(Sc)) throughout the brain. Although immunolabeled PrP(Sc) obtained from brain tissue was characterized by the presence of PrP plaques and diffuse synaptic granular accumulations, no stellate-type deposits were detected. Western blot analysis suggested no obvious differences in PrP(Sc) molecular mass or glycoform pattern in the brains of first- and second-passaged cattle. These findings suggest failures to identify differences in mean incubation period and biochemical and neuropathologic properties of the BSE/JP24 prion between the first and second passages in cattle.

  9. The role of Ca(2+)-activated Cl(-) current in tone generation in the rabbit corpus cavernosum.

    PubMed

    Hannigan, Karen I; Griffin, Caoimhin S; Large, Roddy L; Sergeant, Gerard P; Hollywood, Mark A; McHale, Noel G; Thornbury, Keith D

    2017-08-23

    Rabbit corpus cavernosum smooth muscle (RCCSM) cells express channels that produce Ca(2+)-activated Cl(-) (IClCa) current, but low sensitivity to conventional antagonists have made its role in tone generation difficult to evaluate. We have re-examined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed both L-type Ca(2+) current and IClCa T16Ainh-A01 and CaCCinh-A01 (10 M) reduced IClCa by ~85%, while 30 M abolished it. L-type Ca(2+) current was unaffected by 10 M CaCCinh-A01, but was reduced by 50% at 30 M CaCCinh-A01, 46% at 10 M T16Ainh-A01 and 78% at 30 M T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60-70% at 10 M and >90% at 30 M. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 μM, CaCCinh-A01; 14 μM, T16Ainh-A01). CaCCinh-A01 10 M had little effect on 60 mM KCl contractures, though they were reduced by 30 M CaCCinh-A01 and T16Ainh-A01 (10 M & 30 M) consistent with their effects on L-type Ca(2+) current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca(2+) waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. Although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca(2+) current preclude its use for evaluating the role of IClCa in tone generation. However, 10 μM CaCCinh-A01 selectively blocked IClCa vs L-type Ca(2+) current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence. Copyright © 2017, American Journal of Physiology-Cell Physiology.

  10. The role of L-type calcium channels in the development and expression of behavioral sensitization to ethanol.

    PubMed

    Broadbent, Julie

    2013-10-11

    Behavioral sensitization is thought to play a significant role in drug addiction. L-type calcium channels have been implicated in sensitization to stimulant and opiate drugs but it is unclear if these channels also contribute to sensitization to ethanol. The effects of three L-type calcium channel blockers, nifedipine (1-7.5 mg/kg), diltiazem (12.5-50 mg/kg), and verapamil (12.5 and 25 mg/kg), on sensitization to ethanol (2 g/kg) were examined in DBA/2J mice. All three blockers reduced but did not prevent expression of sensitization. Only nifedipine blocked acquisition of sensitization. Nifedipine and verapamil decreased blood ethanol levels. The current findings suggest L-type calcium channels do not play a substantial role in sensitization to ethanol and that the neural mechanisms underlying sensitization to ethanol are distinct from those mediating sensitization to stimulants and opiates.

  11. The Role of L-Type Calcium Channels in the Development and Expression of Behavioral Sensitization to Ethanol

    PubMed Central

    Broadbent, Julie

    2013-01-01

    Behavioral sensitization is thought to play a significant role in drug addiction. L-type calcium channels have been implicated in sensitization to stimulant and opiate drugs but it is unclear if these channels also contribute to sensitization to ethanol. The effects of three L-type calcium channel blockers, nifedipine (1 – 7.5 mg/kg), diltiazem (12.5 – 50 mg/kg), and verapamil (12.5 and 25 mg/kg), on sensitization to ethanol (2 g/kg) were examined in DBA/2J mice. All three blockers reduced but did not prevent expression of sensitization. Only nifedipine blocked acquisition of sensitization. Nifedipine and verapamil decreased blood ethanol levels. The current findings suggest L-type calcium channels do not play a substantial role in sensitization to ethanol and that the neural mechanisms underlying sensitization to ethanol are distinct from those mediating sensitization to stimulants and opiates. PMID:23994059

  12. Regulation of Ca2+ and electrical alternans in cardiac myocytes: Role of CaMKII and repolarizing currents

    PubMed Central

    Livshitz, Leonid M.; Rudy, Yoram

    2007-01-01

    Alternans of cardiac repolarization is associated with arrhythmias and sudden death. At the cellular level, alternans involves beat-to-beat oscillation of the action potential (AP) and possibly Ca2+ transient (CaT). Because of experimental difficulty in independently controlling the Ca2+ and electrical subsystems, mathematical modelling provides additional insights into mechanisms and causality. Pacing protocols were conducted in a canine ventricular myocyte model with the following results: 1. (I) CaT alternans results from refractoriness of the SR Ca2+ release system; alternation of the L-type calcium current (ICa(L)) has a negligible effect; (II) CaT-AP coupling during late AP occurs through the sodium-calcium exchanger (INaCa) and underlies APD alternans; (III) Increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity extends the range of CaT and APD alternans to slower frequencies and increases alternans magnitude; its decrease suppresses CaT and APD alternans, exerting an antiarrhythmic effect; (IV). Increase of the rapid delayed rectifier current (IKr) also suppresses APD alternans, but without suppressing CaT alternans. Thus, CaMKII inhibition eliminates APD alternans by eliminating its cause (CaT alternans), while IKr enhancement does so by weakening CaT-APD coupling. The simulations identify combined CaMKII inhibition and IKr enhancement as a possible antiar-rhythmic intervention. PMID:17277017

  13. Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts.

    PubMed

    Zhang, Xuemei; Li, Fangping; Guo, Lin; Hei, Hongya; Tian, Lulu; Peng, Wen; Cai, Hui

    2015-01-01

    Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.

  14. Cloning and characterization of two different L-type lectin genes from the Chinese mitten crab Eriocheir sinensis.

    PubMed

    Huang, Ying; Tan, Jing-Min; Wang, Zheng; Yin, Shao-Wu; Huang, Xin; Wang, Wen; Ren, Qian

    2014-10-01

    L-type lectins contain a leguminous lectin domain and bind to high-mannose type oligosaccharides. In the secretory pathway, L-type lectins play crucial functions in the trafficking, sorting, and targeting of maturing glycoproteins. This study identified two novel L-type lectins, designated as EsERGIC-53 and EsVIP36, from the Chinese mitten crab Eriocheir sinensis. The complete nucleotide sequence of ERGIC-53 cDNA was 1955 bp, containing a 1506 bp open reading frame (ORF) encoding a putative protein of 501 deduced amino acids. The full-length cDNA of VIP36 was 3474 bp with a 984 bp ORF encoding a 327-amino acid peptide. The deduced ERGIC-53 and VIP36 proteins contained a putative signal peptide and an L-type lectin-like domain. Phylogenetic analysis showed that ERGIC-53 and VIP36 belonged to different clades of L-type lectin family. Reverse transcription PCR showed that ERGIC-53 and VIP36 were expressed in all tested tissues. Quantitative real-time RT-PCR analysis revealed that ERGIC-53 and VIP36 transcripts in hepatopancreas were significantly induced at various time points after infection with lipopolysaccharide (LPS), peptidoglycan (PGN), Staphylococcus aureus, Vibrio parahaemolyticus, and Aeromonas hydrophila. A bacterium-binding experiment showed that both ERGIC-53 and VIP36 could bind to different microbes. Sugar binding assay revealed that these lectins could also bind to the glycoconjugates of bacteria surface, such as LPS, PGN, d-Mannose, and N-Acetyl-d-mannosamine. Moreover, these two L-type lectins agglutinated bacteria in a calcium-dependent manner, and both exerted the ability of facilitating the clearance of injected bacteria V. parahaemolyticus in the crab. Our results suggested that ERGIC-53 and VIP36 functioned as pattern recognition receptors in the immune system of E. sinensis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. L-type voltage-gated calcium channels in conditioned fear: A genetic and pharmacological analysis

    PubMed Central

    McKinney, Brandon C.; Sze, Wilson; White, Jessica A.; Murphy, Geoffrey G.

    2008-01-01

    Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear using a molecular genetics approach. Previously, we used this approach to demonstrate that the Cav1.3 isoform mediates consolidation, but not extinction, of contextually conditioned fear. Here, we used mice in which the gene for the L-VGCC pore-forming subunit Cav1.2 was conditionally deleted in forebrain excitatory neurons (Cav1.2cKO mice) to address the role of Cav1.2 in consolidation and extinction of conditioned fear. We demonstrate that Cav1.2cKO mice consolidate and extinguish conditioned fear as well as control littermates. These data suggest that Cav1.2 is not critical for these processes and together with our previous data argue against a role for either of the brain-expressed L-VGCCs (Cav1.2 or Cav1.3) in extinction of conditioned fear. Additionally, we present data demonstrating that the L-VGCC antagonist nifedipine, which has been used in previous conditioned fear extinction studies, impairs locomotion, and induces an aversive state. We further demonstrate that this aversive state can enter into associations with conditioned stimuli that are present at the time that it is experienced, suggesting that previous studies using nifedipine were likely confounded by drug toxicity. Taken together, our genetic and pharmacological data argue against a role for Cav1.2 in consolidation of conditioned fear as well as a role for L-VGCCs in extinction of conditioned fear. PMID:18441291

  16. Verapamil - L type voltage gated calcium channel inhibitor diminishes aggressive behavior in male Siamese fighting fish.

    PubMed

    Kania, B F; Dębski, B; Wrońska, D; Zawadzka, E

    2015-01-01

    Verapamil is a L-type voltage gated calcium channels inhibitor (VGCCI), which is a highly prescribed drug used in the treatment of hypertension, angina pectoris, cardiac arrhythmia and cluster headaches. Its common use caused its appearance in water environment. VGCC inhibit epinephrine release and cause many neuro-hormonal changes influencing also fish behavior. Siamese fighting fish was chosen to study the influence of verapamil given to the water on the beginning of experiment in 3 different concentrations of 0 (control), 8 and 160 μg · L-1, on aggressive behavior in these fish. The experimental fish were placed in individual glass containers for 3 weeks and the mirror test was used. The highest concentration led to a significant modulation of fish behavior after 1 week and the lower dose caused statistically significant behavioral changes after 2 weeks of verapamil treatment. Siamese fighting fish males exposed to verapamil had longer latencies to the first chase - 12.6 s (8 μg · L-1 of verapamil) and 18.8 s (160 μg · L-1 of verapamil) compared to 5.6 s in the control group, decreased attack frequency and shorter duration of these attacks. The number of attacks within 10 min was decreased from 38.3 in the control group to 27.1 and 16.1, respectively. Also the total duration of these attacks decreased from 354.8 (control) to 326.4 (decrease statistically insignificant) and to 194.8 s in verapamil treated groups. It was shown, that even relatively low concentrations of verapamil in water may have adverse effects on fish and probably other living organisms.

  17. Insights into the Ropy Phenotype of the Exopolysaccharide-Producing Strain Bifidobacterium animalis subsp. lactis A1dOxR

    PubMed Central

    Hidalgo-Cantabrana, Claudio; Sánchez, Borja; Moine, Deborah; Berger, Bernard; de los Reyes-Gavilán, Clara G.; Gueimonde, Miguel; Margolles, Abelardo

    2013-01-01

    The proteome of the ropy strain Bifidobacterium animalis subsp. lactis A1dOxR, compared to that of its nonropy isogenic strain, showed an overproduction of a protein involved in rhamnose biosynthesis. Results were confirmed by gene expression analysis, and this fact agreed with the high rhamnose content of the ropy exopolysaccharide. PMID:23584772

  18. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

    PubMed

    Wen, Li; Wang, Yu; Wang, Huan; Kong, Lingmin; Zhang, Liang; Chen, Xin; Ding, Yin

    2012-08-03

    L-type voltage-dependent Ca(2+) channels (VDCC(L)) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCC(L) expression was reported in mesenchymal stem cells (MSCs), but the role of VDCC(L) in MSCs is still undetermined. The aim of this study was to determine whether VDCC(L) may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca(2+) currents (I(Ca)) and mRNA expression of VDCC(L) in rMSCs, and then suppressed VDCC(L) using nifedipine (Nif), a VDCC(L) blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected α1C-siRNA into the cells to further confirm the role of VDCC(L) in rMSCs, and a similar effect on osteogenesis was found. These results suggest that VDCC(L) plays a crucial role in the proliferation and osteogenic differentiation of rMSCs. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Effects of the T/L-type calcium channel blocker benidipine on albuminuria and plasma aldosterone concentration. A pilot study involving switching from L-type calcium channel blockers to benidipine.

    PubMed

    Tani, Shigemasa; Takahashi, Atsuhiko; Nagao, Ken; Hirayama, Atsushi

    2014-01-01

    Albuminuria and a high plasma aldosterone concentration (PAC) are prognosis factors predicting a poor outcome for cardiovascular disease. We examined here the effects of benidipine, a T/L-type calcium channel blocker (CCB), on albuminuria and PAC.Thirty-one patients with essential hypertension who received an L-type CCB and achieved the target blood pressure (BP) indicated by the Treatment Guidelines of the Japan Society of Hypertension (JSH2009) were investigated. The Ltype CCB under treatment was switched to benidipine at a dose in which equivalent BP reduction was expected. BP and estimated glomerular filtration rate at 6 months after switching to benidipine were not significantly different from those at baseline. The urinary-albumin-creatinine ratio (UACR) decreased significantly by 36.9% (P = 0.001). No significant change was observed in plasma renin activity (P = 0.063). The PAC of all patients decreased significantly by 11.8% (P = 0.002). When analyzed by daily doses of benidipine, the PAC appeared to have decreased in patients who received 4 mg per day of benidipine (n = 14), although statistical significance was not reached (P = 0.096). The PAC in patients who received 8 mg per day of benidipine (n =17) was significantly reduced by 13.2% (P = 0.017).In hypertensive patients whose BP is controlled by L-type CCB, switching to the T/L-type CCB benidipine maintained BP control and reduced UACR. In addition, the high dose of benidipine reduced the PAC independent of BP control. These results suggest the T/L-type CCB benidipine may contribute to cardio-renal protection in addition to lowering BP.

  20. Protein kinase A modulation of CaV1.4 calcium channels

    PubMed Central

    Sang, Lingjie; Dick, Ivy E.; Yue, David T.

    2016-01-01

    The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family. PMID:27456671

  1. Protein kinase A modulation of CaV1.4 calcium channels

    NASA Astrophysics Data System (ADS)

    Sang, Lingjie; Dick, Ivy E.; Yue, David T.

    2016-07-01

    The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family.

  2. Modeling Ca(2+) currents and buffered diffusion of Ca(2+) in human β-cells during voltage clamp experiments.

    PubMed

    Félix-Martínez, Gerardo J; Godínez-Fernández, J Rafael

    2015-12-01

    Macroscopic Ca(2+) currents of the human β-cells were characterized using the Hodgkin-Huxley formalism. Expressions describing the Ca(2+)-dependent inactivation process of the L-type Ca(2+) channels in terms of the concentration of Ca(2+) were obtained. By coupling the modeled Ca(2+) currents to a three-dimensional model of buffered diffusion of Ca(2+), we simulated the Ca(2+) transients formed in the immediate vicinity of the cell membrane during voltage clamp experiments performed in high buffering conditions. Our modeling approach allowed us to consider the distribution of the Ca(2+) sources over the cell membrane. The effect of exogenous (EGTA) and endogenous Ca(2+) buffers on the temporal course of the Ca(2+) transients was evaluated. We show that despite the high Ca(2+) buffering capacity, nanodomains are formed in the submembrane space, where a peak Ca(2+) concentration between ∼76 and 143 µM was estimated from our simulations. In addition, the contribution of each Ca(2+) current to the formation of the Ca(2+) nanodomains was also addressed. Here we provide a general framework to incorporate the spatial aspects to the models of the pancreatic β-cell, such as a more detailed and realistic description of Ca(2+) dynamics in response to electrical activity in physiological conditions can be provided by future models.

  3. Molecular identification of Ca2+ channels in human sperm.

    PubMed

    Park, Jin-Yong; Ahn, Hyun-Jong; Gu, Jung-Gyu; Lee, Keun-Ho; Kim, Jin-Sung; Kang, Ho-Won; Lee, Jung-Ha

    2003-08-31

    The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+) channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+) channels has been limited. Here we identified Ca(2+) channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non- L-type, and T-type Ca(2+) channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H sequences. RT-PCR using specific primers repeatedly detected alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H mRNAs, and additionally alpha1I mRNA. But alpha1A and alpha1D messages were not detected. Relative expression levels of the detected Ca(2+) channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H alpha1G alpha1E alpha1B alpha1C alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+) channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.

  4. Simulated GABA synaptic input and L-type calcium channels form functional microdomains in hypothalamic gonadotropin-releasing hormone neurons.

    PubMed

    Hemond, Peter J; O'Boyle, Michael P; Roberts, Carson B; Delgado-Reyes, Alfonso; Hemond, Zoe; Suter, Kelly J

    2012-06-27

    Hypothalamic gonadotropin-releasing hormone (GnRH) neurons integrate the multiple internal and external cues that regulate sexual reproduction. In contrast to other neurons that exhibit extensive dendritic arbors, GnRH neurons usually have a single dendrite with relatively little branching. This largely precludes the integration strategy in which a single dendritic branch serves as a unit of integration. In the present study, we identify a gradient in L-type calcium channels in dendrites of mouse GnRH neurons and its interaction with GABAergic and glutamatergic inputs. Higher levels of L-type calcium channels are in somata/proximal dendrites (i.e., 0-26 μm) and distal dendrites (∼130 μm dendrite length), but intervening midlengths of dendrite (∼27-130 μm) have reduced L-type calcium channels. Using uncaging of GABA, there is a decreasing GABAergic influence along the dendrite and the impact of GABA(A) receptors is dependent on activation of L-type calcium channels. This results in amplification of proximal GABAergic signals and attenuation of distal dendritic signals. Most interestingly, the intervening dendritic regions create a filter through which only relatively high-amplitude, low-frequency GABAergic signaling to dendrites elicits action potentials. The findings of the present study suggest that GnRH dendrites adopt an integration strategy whereby segments of single nonbranching GnRH dendrites create functional microdomains and thus serve as units of integration.

  5. Characterization of L-type calcium channel activity in atrioventricular nodal myocytes from rats with streptozotocin-induced Diabetes mellitus.

    PubMed

    Yuill, Kathryn H; Al Kury, Lina T; Howarth, Frank Christopher

    2015-11-01

    Cardiovascular complications are common in patients with Diabetes mellitus (DM). In addition to changes in cardiac muscle inotropy, electrical abnormalities are also commonly observed in these patients. We have previously shown that spontaneous cellular electrical activity is altered in atrioventricular nodal (AVN) myocytes, isolated from the streptozotocin (STZ) rat model of type-1 DM. In this study, utilizing the same model, we have characterized the changes in L-type calcium channel activity in single AVN myocytes. Ionic currents were recorded from AVN myocytes isolated from the hearts of control rats and from those with STZ-induced diabetes. Patch-clamp recordings were used to assess the changes in cellular electrical activity in individual myocytes. Type-1 DM significantly altered the cellular characteristics of L-type calcium current. A reduction in peak ICaL density was observed, with no corresponding changes in the activation parameters of the current. L-type calcium channel current also exhibited faster time-dependent inactivation in AVN myocytes from diabetic rats. A negative shift in the voltage dependence of inactivation was also evident, and a slowing of restitution parameters. These findings demonstrate that experimentally induced type-1 DM significantly alters AVN L-type calcium channel cellular electrophysiology. These changes in ion channel activity may contribute to the abnormalities in cardiac electrical function that are associated with high mortality levels in patients with DM.

  6. The L-Type Calcium Channel Blocker Nifedipine Impairs Extinction, but Not Reduced Contingency Effects, in Mice

    ERIC Educational Resources Information Center

    Jami, Shekib; Barad, Mark; Cain, Christopher K.; Godsil, Bill P.

    2005-01-01

    We recently reported that fear extinction, a form of inhibitory learning, is selectively blocked by systemic administration of L-type voltage-gated calcium channel (LVGCC) antagonists, including nifedipine, in mice. We here replicate this finding and examine three reduced contingency effects after vehicle or nifedipine (40 mg/kg) administration.…

  7. The L-Type Calcium Channel Blocker Nifedipine Impairs Extinction, but Not Reduced Contingency Effects, in Mice

    ERIC Educational Resources Information Center

    Jami, Shekib; Barad, Mark; Cain, Christopher K.; Godsil, Bill P.

    2005-01-01

    We recently reported that fear extinction, a form of inhibitory learning, is selectively blocked by systemic administration of L-type voltage-gated calcium channel (LVGCC) antagonists, including nifedipine, in mice. We here replicate this finding and examine three reduced contingency effects after vehicle or nifedipine (40 mg/kg) administration.…

  8. Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes

    PubMed Central

    Bett, Glenna C L; Dai, Shuiping; Campbell, Donald L

    2002-01-01

    The effects of the cholinergic muscarinic agonist carbachol (CCh) on the basal L-type calcium current, ICa,L, in ferret right ventricular (RV) myocytes were studied using whole cell patch clamp. CCh produced two major effects: (i) in all myocytes, extracellular application of CCh inhibited ICa,L in a reversible concentration-dependent manner; and (ii) in many (but not all) myocytes, upon washout CCh produced a significant transient stimulation of ICa,L (‘rebound stimulation’). Inhibitory effects could be observed at 1 × 10−10m CCh. The mean steady-state inhibitory concentration-response relationship was shallow and could be described with a single Hill equation (maximum inhibition = 34.5 %, IC50 = 4 × 10−8m, Hill coefficient n = 0.60). Steady-state inhibition (1 or 10 μM CCh) had no significant effect on ICa,L selectivity or macroscopic (i) activation characteristics, (ii) inactivation kinetics, (iii) steady-state inactivation or (iv) kinetics of recovery from inactivation. Maximal inhibition of nitric oxide synthase (NOS) activity (preincubation of myocytes in 1 mm l-NMMA (NG-monomethyl-l-arginine) + 1 mm l-NNA (NG-nitro-l-arginine) for 2–3 h plus inclusion of 1 mm l-NMMA + 1 mm l-NNA in the patch pipette solution) produced no significant attenuation of the CCh-mediated inhibition of ICa,L. Protocols involving (i) the nitric oxide (NO) scavenger PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide; 200 μM), (ii) imposition of a ‘cGMP clamp’ (100 μM 8-Bromo-cGMP), and (iii) inhibition of soluble guanylyl cyclase (ODQ (1H-[1,2,4,]oxadiazolo(4,3,-a)quinoxalin-1-one), 50 μM) all failed to attenuate CCh-mediated inhibition of Ica,L. While CCh consistently inhibited basal ICa,L in all RV myocytes studied, not all myocytes displayed rebound stimulation upon CCh washout. However, there was no difference between CCh-mediated inhibition of ICa,L between these two RV myocyte types, and in myocytes displaying rebound stimulation neither ODQ nor 8

  9. Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

    PubMed Central

    Shi, Liheng; Ko, Michael L.; Ko, Gladys Y.-P.

    2017-01-01

    Modulation of ion channels by extracellular proteins plays critical roles in shaping synaptic plasticity. Retinoschisin (RS1) is an extracellular adhesive protein secreted from photoreceptors and bipolar cells, and it plays an important role during retinal development, as well as in maintaining the stability of retinal layers. RS1 is known to form homologous octamers and interact with molecules on the plasma membrane including phosphatidylserine, sodium-potassium exchanger complex, and L-type voltage-gated calcium channels (LTCCs). However, how this physical interaction between RS1 and ion channels might affect the channel gating properties is unclear. In retinal photoreceptors, two major LTCCs are Cav1.3 (α1D) and Cav1.4 (α1F) with distinct biophysical properties, functions and distributions. Cav1.3 is distributed from the inner segment (IS) to the synaptic terminal and is responsible for calcium influx to the photoreceptors and overall calcium homeostasis. Cav1.4 is only expressed at the synaptic terminal and is responsible for neurotransmitter release. Mutations of the gene encoding Cav1.4 cause X-linked incomplete congenital stationary night blindness type 2 (CSNB2), while null mutations of Cav1.3 cause a mild decrease of retinal light responses in mice. Even though RS1 is known to maintain retinal architecture, in this study, we present that RS1 interacts with both Cav1.3 and Cav1.4 and regulates their activations. RS1 was able to co-immunoprecipitate with Cav1.3 and Cav1.4 from porcine retinas, and it increased the LTCC currents and facilitated voltage-dependent activation in HEK cells co-transfected with RS1 and Cav1.3 or Cav1.4, thus providing evidence of a functional interaction between RS1 and LTCCs. The interaction between RS1 and Cav1.3 did not change the calcium-dependent inactivation of Cav1.3. In mice lacking RS1, the expression of Cav1.3 and Cav1.4 in the retina decreased, while in mice with Cav1.4 deletion, the retinal level of RS1 decreased

  10. Retinoschisin, a New Binding Partner for L-type Voltage-gated Calcium Channels in the Retina*

    PubMed Central

    Shi, Liheng; Jian, Kuihuan; Ko, Michael L.; Trump, Dorothy; Ko, Gladys Y.-P.

    2009-01-01

    The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCCα1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCCα1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCCα1 subunits and retinoschisin play an important role in the membrane retention of L-VGCCα1 subunits and photoreceptor-bipolar synaptic transmission. PMID:19074145

  11. The CaV2.3 R-type voltage-gated Ca2+ channel in mouse sleep architecture.

    PubMed

    Siwek, Magdalena Elisabeth; Müller, Ralf; Henseler, Christina; Broich, Karl; Papazoglou, Anna; Weiergräber, Marco

    2014-05-01

    Voltage-gated Ca(2+) channels (VGCCs) are key elements in mediating thalamocortical rhythmicity. Low-voltage activated (LVA) CaV 3 T-type Ca(2+) channels have been related to thalamic rebound burst firing and to generation of non-rapid eye movement (NREM) sleep. High-voltage activated (HVA) CaV 1 L-type Ca(2+) channels, on the opposite, favor the tonic mode of action associated with higher levels of vigilance. However, the role of the HVA Non-L-type CaV2.3 Ca(2+) channels, which are predominantly expressed in the reticular thalamic nucleus (RTN), still remains unclear. Recently, CaV2.3(-/-) mice were reported to exhibit altered spike-wave discharge (SWD)/absence seizure susceptibility supported by the observation that CaV2.3 mediated Ca(2+) influx into RTN neurons can trigger small-conductance Ca(2+)-activated K(+)-channel type 2 (SK2) currents capable of maintaining thalamic burst activity. Based on these studies we investigated the role of CaV2.3 R-type Ca(2+) channels in rodent sleep. The role of CaV2.3 Ca(2+) channels was analyzed in CaV2.3(-/-) mice and controls in both spontaneous and artificial urethane-induced sleep, using implantable video-EEG radiotelemetry. Data were analyzed for alterations in sleep architecture using sleep staging software and time-frequency analysis. CaV2.3 deficient mice exhibited reduced wake duration and increased slow-wave sleep (SWS). Whereas mean sleep stage durations remained unchanged, the total number of SWS epochs was increased in CaV2.3(-/-) mice. Additional changes were observed for sleep stage transitions and EEG amplitudes. Furthermore, urethane-induced SWS mimicked spontaneous sleep results obtained from CaV2.3 deficient mice. Quantitative Real-time PCR did not reveal changes in thalamic CaV3 T-type Ca(2+) channel expression. The detailed mechanisms of SWS increase in CaV2.3(-/-) mice remain to be determined. Low-voltage activated CaV2.3 R-type Ca(2+) channels in the thalamocortical loop and extra

  12. Phosphorylation of the Ca2+-Binding Protein CaBP4 by Protein Kinase C ζ in Photoreceptors

    PubMed Central

    Lee, Amy; Jimenez, Amber; Cui, Guiying; Haeseleer, Françoise

    2009-01-01

    CaBP4 is a calmodulin-like neuronal calcium-binding protein that is crucial for the development and/or maintenance of the cone and rod photoreceptor synapse. Previously, we showed that CaBP4 directly regulates Cav1 L-type Ca2+ channels, which are essential for normal photoreceptor synaptic transmission. Here, we show that the function of CaBP4 is regulated by phosphorylation. CaBP4 is phosphorylated by protein kinase C ζ (PKCζ) at serine 37 both in vitro and in the retina and colocalizes with PKCζ in photoreceptors. CaBP4 phosphorylation is greater in light-adapted than dark-adapted mouse retinas. In electrophysiological recordings of cells transfected with Cav1.3 and CaBP4, mutation of the serine 37 to alanine abolished the effect of CaBP4 in prolonging the Ca2+ current through Cav1.3 channel, whereas inactivating mutations in the CaBP4 Ca2+-binding sites strengthened Cav1.3 modulation. These findings demonstrate how light-stimulated changes in CaBP4 phosphorylation and Ca2+ binding may regulate presynaptic Ca2+ signals in photoreceptors. PMID:18003854