Activation of voltage-gated calcium channels at presynaptic terminals leads to local increases in calcium and the fusion of synaptic vesicles containing neurotransmitter. Presynaptic output is a function of the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at Caenorhabditis elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters ~250 nm in diameter with the active zone proteins Neurexin, α-Liprin, SYDE, ELKS/CAST, RIM-BP, α-Catulin, and MAGI1. CaV2 channels are colocalized with the priming protein UNC-13L and mediate the fusion of vesicles docked within 33 nm of the dense projection. CaV2 activity is amplified by ryanodine receptor release of calcium from internal stores, triggering fusion up to 165 nm from the dense projection. By contrast, CaV1 channels are dispersed in the synaptic varicosity, and are colocalized with UNC-13S. CaV1 and ryanodine receptors are separated by just 40 nm, and vesicle fusion mediated by CaV1 is completely dependent on the ryanodine receptor. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.

-->Low voltage-activated Cav1.3 L-type Ca2+-channels are key regulators of neuronal excitability controlling neuronal development and different types of learning and memory. Their physiological functions are enabled by their negative activation voltage-range, which allows Cav1.3 to be active at subthreshold voltages. Alternative splicing in the C-terminus of their pore-forming α1-subunits gives rise to C-terminal long (Cav1.3L) and short (Cav1.3S) splice variants allowing Cav1.3S to activate at even more negative voltages than Cav1.3L. We discovered that inclusion of exons 8b, 11, and 32 in Cav1.3S further shifts activation (-3 to -4 mV) and inactivation (-4 to -6 mV) to more negative voltages as revealed by functional characterization in tsA-201 cells. We found transcripts of these exons in mouse chromaffin cells, the cochlea, and the brain. Our data further suggest that Cav1.3-containing exons 11 and 32 constitute a significant part of native channels in the brain. We therefore investigated the effect of these splice variants on human disease variants. Splicing did not prevent the gating defects of the previously reported human pathogenic variant S652L, which further shifted the voltage-dependence of activation of exon 11-containing channels by more than -12 mV. In contrast, we found no evidence for gating changes of the CACNA1D missense variant R498L, located in exon 11, which has recently been identified in a patient with an epileptic syndrome. Our data demonstrate that alternative splicing outside the C-terminus involving exons 11 and 32 contributes to channel fine-tuning by stabilizing negative activation and inactivation gating properties of wild-type and mutant Cav1.3 channels.

Loss of total muscle force during aging has both atrophic and non-atrophic components. The former deficit is a direct consequence of reduced muscle mass while the latter has been attributed to a depression of excitation-contraction (EC) coupling. It is well established that age-onset reductions in sex hormone production regulate the atrophic component in both males and females. However, it is unknown whether the non-atrophic component is influenced by sex hormones. Since the non-atrophic component has been linked mechanistically to reduced expression of the skeletal muscle L-type Ca2+ channel (CaV1.1), we recorded L-type Ca2+ currents, gating charge movements and depolarization-induced changes in myoplasmic Ca2+ from flexor digitorum brevis (FDB) fibers of naïve and gonadectomized mice of both sexes. Our first set of experiments sought to identify any basal differences in EC coupling or L-type Ca2+ flux between the sexes; no detectable differences in any of the aforementioned parameters were observed between FDB harvested from either naïve males or females. In the latter segments of the study, ovariectomy (OVX) and orchiectomy (ORX) models were used to assess the possible influence of sex hormones on EC coupling and/or L-type Ca2+ flux. In these experiments, FDB fibers harvested from OVX and ORX mice both showed no differences in L-type Ca2+ current, gating charge movement or depolarization-induced changes in Ca2+ release from the sarcoplasmic reticulum. Taken together, our results indicate L-type Ca2+ channel function and EC coupling are: 1) equivalent between the sexes, and 2) not significantly regulated by sex hormones. Since recent NIH review guidelines mandate the consideration of sex differences as a criterion for review, our work indicates the suitability of either sex for the study of the fundamental mechanisms of EC coupling. Thus, our findings may accelerate the research process by conserving animals, labor and financial resources.

The primary route of Ca2+ entry into cardiac myocytes is via 1,4-dihydropyridine-sensitive, voltage-gated L-type Ca2+ channels. Ca2+ influx through these channels influences duration of action potential and engages excitation-contraction (EC) coupling in both the atria and the myocardium. Members of the RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTP-binding proteins are potent, endogenously expressed inhibitors of cardiac L-type channels. Although much work has focused on the molecular mechanisms by which RGK proteins inhibit the CaV 1.2 and CaV 1.3 L-type channel isoforms that expressed in the heart, their impact on greater cardiac function is only beginning to come into focus. In this review, we summarize recent findings regarding the influence of RGK proteins on normal cardiac physiology and the pathological consequences of aberrant RGK activity.

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.

BACKGROUND: Although therapeutically beneficial in the treatment of certain diseases, L-type calcium channel antagonism can result in unwanted off-target pharmacology leading to adverse drug reactions and to the termination of the development of otherwise promising compounds. In the present study three marketed calcium channel inhibitors, nifedipine, verapamil and diltiazem were profiled in a series of in vitro and ex-vivo assays in an effort to determine the ability of these assays to discriminate, between dihydropyridine versus non-dihydropyridine-like compounds, and how well they can predict the cardiovascular effects observed in a conscious telemetered rat model.
METHODS: Standard calcium channel antagonists were profiled in radioligand binding, patch clamp and calcium flux assays. In addition, cardiovascular endpoints related to calcium channel activity were also examined in ex vivo tissue bath preparations, including relaxation of pre-constricted rat aorta and the guinea pig Langendorff isolated heart model. The data generated were correlated with in vivo blood pressure and heart rate data from conscious telemetered rats.
RESULTS: Our results show that the binding, FLIPR and aorta assays allow differentiation of the compounds in two distinct classes of L-type calcium channel antagonists, and are good predictors of in vivo outcomes.
CONCLUSIONS: These results suggest that in vitro and ex vivo profiling remains a valuable tool in predicting potential in vivo cardiovascular safety issues, and can aid in the selection of novel development compounds that show inherent inhibitory activity against L-type calcium channels.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3-5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca(2+) channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation.
METHODS: We recorded L-type currents, charge movements, and myoplasmic Ca(2+) transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1(G93A)).
RESULTS: In FDB fibers obtained from \"symptomatic\" global SOD1(G93A) mice, we observed a substantial reduction of SR Ca(2+) release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1(G93A) fibers when compared to control fibers. Ca(2+) transients were not significantly reduced in similar experiments performed with FDB fibers obtained from \"early-symptomatic\" SOD1(G93A) mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca(2+) release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1(G93A) expression was restricted to skeletal muscle.
CONCLUSIONS: We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1(G93A) mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1(G93A) and skeletal muscle autonomous MLC/SOD1(G93A) mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status.

In skeletal muscle, excitation-contraction (EC) coupling relies on the transmission of an intermolecular signal from the voltage-sensing regions of the L-type Ca(2+) channel (Ca(V)1.1) in the plasma membrane to the channel pore of the type 1 ryanodine receptor (RyR1) nearly 10 nm away in the membrane of the sarcoplasmic reticulum (SR). Even though the roles of Ca(V)1.1 and RyR1 as voltage sensor and SR Ca(2+) release channel, respectively, have been established for nearly 25 years, the mechanism underlying communication between these two channels remains undefined. In the course of this article, I will review current viewpoints on this topic with particular emphasis on recent studies.

The modulation and regulation of voltage-gated Ca(2+) 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 Ca(2+) 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 Ca(2+), 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 Ca(2+)-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of CaV1.2 inactivation induced by Ca(2+) entry into the cell.

Ca(2+)-dependent inactivation (CDI) is a negative feedback regulation of voltage-gated Cav1 and Cav2 channels that is mediated by the Ca(2+) sensing protein, calmodulin (CaM), binding to the pore-forming Cav α1 subunit. David Yue and his colleagues made seminal contributions to our understanding of this process, as well as factors that regulate CDI. Important in this regard are members of a family of Ca(2+) binding proteins (CaBPs) that are related to calmodulin. CaBPs are expressed mainly in neural tissues and can antagonize CaM-dependent CDI for Cav1 L-type channels. This review will focus on the roles of CaBPs as Cav1-interacting proteins, and the significance of these interactions for vision, hearing, and neuronal Ca(2+) signaling events.