Search

High voltage‐gated Ca2+ channels (HVCCs) shape the electrical activity and control hormone release in most endocrine cells. HVCCs are multi‐subunit protein complexes formed by the pore‐forming α1 and the auxiliary β, α2δ and γ subunits. Four genes code for the α2δ isoforms. At the mRNA level, mouse chromaffin cells (MCCs) express predominantly the CACNA2D1 gene coding for the α2δ‐1 isoform. Here we show that α2δ‐1 deletion led to ∼60% reduced HVCC Ca2+ influx with slower inactivation kinetics. Pharmacological dissection showed that HVCC composition remained similar in α2δ‐1−/− MCCs compared to wild‐type (WT), demonstrating that α2δ‐1 exerts similar functional effects on all HVCC isoforms. Consistent with reduced HVCC Ca2+ influx, α2δ‐1−/− MCCs showed reduced spontaneous electrical activity with action potentials (APs) having a shorter half‐maximal duration caused by faster rising and decay slopes. However, the induced electrical activity showed opposite effects with α2δ‐1−/− MCCs displaying significantly higher AP frequency in the tonic firing mode as well as an increase in the number of cells firing AP bursts compared to WT. This gain‐of‐function phenotype was caused by reduced functional activation of Ca2+‐dependent K+ currents. Additionally, despite the reduced HVCC Ca2+ influx, the intracellular Ca2+ transients and vesicle exocytosis or endocytosis were unaltered in α2δ‐1−/− MCCs compared to WT during sustained stimulation. In conclusion, our study shows that α2δ‐1 genetic deletion reduces Ca2+ influx in cultured MCCs but leads to a paradoxical increase in catecholamine secretion due to increased excitability. Key points: Deletion of the α2δ‐1 high voltage‐gated Ca2+ channel (HVCC) subunit reduces mouse chromaffin cell (MCC) Ca2+ influx by ∼60% but causes a paradoxical increase in induced excitability.MCC intracellular Ca2+ transients are unaffected by the reduced HVCC Ca2+ influx.Deletion of α2δ‐1 reduces the immediately releasable pool vesicle exocytosis but has no effect on catecholamine (CA) release in response to sustained stimuli.The increased electrical activity and CA release from MCCs might contribute to the previously reported cardiovascular phenotype of patients carrying α2δ‐1 loss‐of‐function mutations. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels (CaV) regulate numerous vital functions in nerve and muscle cells. To fulfill their diverse functions, the multiple members of the CaV channel family are activated over a wide range of voltages. Voltage sensing in potassium and sodium channels involves the sequential transition of positively charged amino acids across a ring of residues comprising the charge transfer center. In CaV channels, the precise molecular mechanism underlying voltage sensing remains elusive. Here we combined Rosetta structural modeling with site-directed mutagenesis to identify the molecular mechanism responsible for the specific gating properties of two CaV1.1 splice variants. Our data reveal previously unnoticed interactions of S4 arginines with an aspartate (D1196) outside the charge transfer center of the fourth voltage-sensing domain that are regulated by alternative splicing of the S3-S4 linker. These interactions facilitate the final transition into the activated state and critically determine the voltage sensitivity and current amplitude of these CaV channels.

The skeletal muscle CaV1.1 channel functions as the voltage‐sensor of excitation−contraction (EC) coupling. Recently, the adaptor protein STAC3 was found to be essential for both CaV1.1 functional expression and EC coupling. Interestingly, STAC proteins were also reported to inhibit calcium‐dependent inactivation (CDI) of L‐type calcium channels (LTCC), an important negative feedback mechanism in calcium signaling. The same could not be demonstrated for CaV1.1, as STAC3 is required for its functional expression. However, upon strong membrane depolarization, CaV1.1 conducts calcium currents characterized by very slow kinetics of activation and inactivation. Therefore, we hypothesized that the negligible inactivation observed in CaV1.1 currents reflects the inhibitory effect of STAC3. Here, we inserted a triple mutation in the linker region of STAC3 (ETLAAA), as the analogous mutation abolished the inhibitory effect of STAC2 on CDI of CaV1.3 currents. When coexpressed in CaV1.1/STAC3 double knockout myotubes, the mutant STAC3‐ETLAAA failed to colocalize with CaV1.1 in the sarcoplasmic reticulum/membrane junctions. However, combined patch‐clamp and calcium recording experiments revealed that STAC3‐ETLAAA supports CaV1.1 functional expression and EC coupling, although at a reduced extent compared to wild‐type STAC3. Importantly, STAC3‐ETLAAA coexpression dramatically accelerated the kinetics of activation and inactivation of CaV1.1 currents, suggesting that STAC3 determines the slow CaV1.1 currents kinetics. To examine if STAC3 specifically inhibits the CDI of CaV1.1 currents, we performed patch‐clamp recordings using calcium and barium as charge carriers in HEK cells. While CaV1.1 displayed negligible CDI with STAC3, this did not increase in the presence of STAC3‐ETLAAA. On the contrary, our data demonstrate that STAC3 specifically inhibits the voltage‐dependent inactivation (VDI) of CaV1.1 currents. Altogether, these results designate STAC3 as a crucial determinant for the slow activation kinetics of CaV1.1 currents and implicate STAC proteins as modulators of both components of inactivation of LTCC. [ABSTRACT FROM AUTHOR]

To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent [Ca.sup.2+] channel ([Ca.sub.v]) [[alpha].sub.1S]-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of [Ca.sub.v][[alpha].sub.1S] in all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged [[alpha].sub.1S]T1354S in dysgenic ([[alpha].sub.1S]-null) myotubes. Whole cell patch-clamp analysis revealed that [[alpha].sub.1S]T1354S produced significantly faster activation of L-type [Ca.sup.2+] currents upon 200-ms depolarizing test pulses compared with wild-type GFP-[[alpha].sub.1S] ([[alpha].sub.1S]WT). In addition, [[alpha].sub.1S]T1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced [Ca.sup.2+] release and to larger action-potential-induced intracellular [Ca.sup.2+] transients under low ([less than or equal to]2 mM) caffeine concentrations compared with [[alpha].sub.1S]WT. Thus our data suggest that an additional influx of [Ca.sup.2+] due to faster activation of the [[alpha].sub.1S]T1354S L-type [Ca.sup.2+] current, in concert with higher caffeine sensitivity of [Ca.sup.2+] release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype. excitation-contraction coupling; calcium channels; skeletal muscle disease; caffeine-induced [Ca.sup.2+] release; channelopathy doi: 10.1152/ajpcell.00008.2010.

L-type [Ca.sup.2+] currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic [Ca.sup.2+] signal, which regulates the contraction force. The auxiliary [Ca.sup.2+] channel subunits [[alpha].sub.2][delta]-1 and [[beta].sub.2] are important regulators of membrane expression and current properties of the cardiac [Ca.sup.2+] channel ([Ca.sub.V]1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the [[alpha].sub.2][delta]-1 subunit in a muscle expression system with simulation of APs and [Ca.sup.2+] transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with [Ca.sub.V]1.2 (GFP-[[alpha].sub.1C]) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the [[alpha].sub.2][delta]-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-[alpha].sub.1C] subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, [[alpha].sub.2][delta]-1 depletion shifted the voltage dependence of [Ca.sup.2+] current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic [Ca.sup.2+] concentration during each contraction. Thus, the [Ca.sup.2+] channel [[alpha].sub.2][delta]-1 subunit is not essential for normal [Ca.sup.2+] channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of [[alpha].sub.2][delta]-1 function is predicted to severely perturb normal heart function. calcium | heart | action potential | dysgenic myotube

T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy. [ABSTRACT FROM AUTHOR]