Search

α 2 δ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α 2 δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for α 2 δ-1 to -4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in α 2 δ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of α 2 δ-2. Our data show that the corresponding p.R596P mutation in mouse α 2 δ-2 drastically decreases membrane expression and synaptic targeting of α 2 δ-2. This defect correlates with altered biophysical properties of postsynaptic Ca V 1.3 channel but has no effect on presynaptic Ca V 2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α 2 δ-2_R596P in primary cultures of hippocampal neurons affects the ability of α 2 δ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous Ca V 2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans-synaptic recruitment of GABA A receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α 2 δ-2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying α 2 δ mutations not only in the context of channelopathies but also synaptopathies., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Studies in genetically modified animals and human genetics have recently provided new insight into the role of voltage-gated L-type Ca 2+ channels in human disease. Therefore, the inhibition of L-type Ca 2+ channels in vivo in wildtype and mutant mice by potent dihydropyridine (DHP) Ca 2+ channel blockers serves as an important pharmacological tool. These drugs have a short plasma half-life in humans and especially in rodents and show high first-pass metabolism upon oral application. In the vast majority of in vivo studies, they have therefore been delivered through parenteral routes, mostly subcutaneously or intraperitoneally. High peak plasma concentrations of DHPs cause side effects, evident as DHP-induced aversive behaviors confounding the interpretation of behavioral readouts. Nevertheless, pharmacokinetic data measuring the exposure achieved with these applications are sparse. Moreover, parenteral injections require animal handling and can be associated with pain, discomfort and stress which could influence a variety of physiological processes, behavioral and other functional readouts. Here, we describe a noninvasive oral application of the DHP isradipine by training mice to quickly consume small volumes of flavored yogurt that can serve as drug vehicle. This procedure does not require animal handling, allows repeated drug application over several days and reproducibly achieves peak plasma concentrations over a wide range previously shown to be well-tolerated in humans. This protocol should facilitate ongoing nonclinical studies in mice exploring new indications for DHP Ca 2+ channel blockers.

L-type Ca 2+ channels (Ca V 1.2/1.3) convey influx of calcium ions that orchestrate a bevy of biological responses including muscle contraction, neuronal function, and gene transcription. Deficits in Ca V 1 function play a vital role in cardiac and neurodevelopmental disorders. Here, we develop a genetically encoded enhancer of Ca V 1.2/1.3 channels (GeeC L ) to manipulate Ca 2+ entry in distinct physiological settings. We functionalized a nanobody that targets the Ca V complex by attaching a minimal effector domain from an endogenous Ca V modulator-leucine-rich repeat containing protein 10 (Lrrc10). In cardiomyocytes, GeeC L selectively increased L-type current amplitude. In neurons in vitro and in vivo, GeeC L augmented excitation-transcription (E-T) coupling. In all, GeeC L represents a powerful strategy to boost Ca V 1.2/1.3 function and lays the groundwork to illuminate insights on neuronal and cardiac physiology and disease.

High voltage-gated Ca 2+ 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 α 1 and the auxiliary β, α 2 δ and γ subunits. Four genes code for the α 2 δ isoforms. At the mRNA level, mouse chromaffin cells (MCCs) express predominantly the CACNA2D1 gene coding for the α 2 δ-1 isoform. Here we show that α 2 δ-1 deletion led to ∼60% reduced HVCC Ca 2+ influx with slower inactivation kinetics. Pharmacological dissection showed that HVCC composition remained similar in α 2 δ-1 -/- MCCs compared to wild-type (WT), demonstrating that α 2 δ-1 exerts similar functional effects on all HVCC isoforms. Consistent with reduced HVCC Ca 2+ influx, α 2 δ-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 α 2 δ-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 Ca 2+ -dependent K + currents. Additionally, despite the reduced HVCC Ca 2+ influx, the intracellular Ca 2+ transients and vesicle exocytosis or endocytosis were unaltered in α 2 δ-1 -/- MCCs compared to WT during sustained stimulation. In conclusion, our study shows that α 2 δ-1 genetic deletion reduces Ca 2+ influx in cultured MCCs but leads to a paradoxical increase in catecholamine secretion due to increased excitability. KEY POINTS: Deletion of the α 2 δ-1 high voltage-gated Ca 2+ channel (HVCC) subunit reduces mouse chromaffin cell (MCC) Ca 2+ influx by ∼60% but causes a paradoxical increase in induced excitability. MCC intracellular Ca 2+ transients are unaffected by the reduced HVCC Ca 2+ influx. Deletion of α 2 δ-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 α 2 δ-1 loss-of-function mutations., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

In humans, type 2 diabetes mellitus shows a higher prevalence in men compared with women, a phenotype that has been attributed to a lower peripheral insulin sensitivity in men. Whether sex-specific differences in pancreatic β cell function also contribute is largely unknown. Here, we characterized the electrophysiological properties of β cells in intact male and female mouse islets. Elevation of glucose concentration above 5 mM triggered an electrical activity with a similar glucose dependence in β cells of both sexes. However, female β cells had a more depolarized membrane potential and increased firing frequency compared with males. The higher membrane depolarization in female β cells was caused by approximately 50% smaller Kv2.1 K+ currents compared with males but otherwise unchanged KATP, large-conductance and small-conductance Ca2+-activated K+ channels, and background TASK1/TALK1 K+ current densities. In female β cells, the higher depolarization caused a membrane potential-dependent inactivation of the voltage-gated Ca2+ channels (CaV), resulting in reduced Ca2+ entry. Nevertheless, this reduced Ca2+ influx was offset by a higher action potential firing frequency. Because exocytosis of insulin granules does not show a sex-specific difference, we conclude that the higher electrical activity promotes insulin release in females, improving glucose tolerance.

Germline de novo missense variants of the CACNA1D gene, encoding the pore-forming α1 subunit of Cav1.3 L-type Ca2+ channels (LTCCs), have been found in patients with neurodevelopmental and endocrine dysfunction, but their disease-causing potential is unproven. These variants alter channel gating, enabling enhanced Cav1.3 activity, suggesting Cav1.3 inhibition as a potential therapeutic option. Here we provide proof of the disease-causing nature of such gating-modifying CACNA1D variants using mice (Cav1.3AG) containing the A749G variant reported de novo in a patient with autism spectrum disorder (ASD) and intellectual impairment. In heterozygous mutants, native LTCC currents in adrenal chromaffin cells exhibited gating changes as predicted from heterologous expression. The A749G mutation induced aberrant excitability of dorsomedial striatum-projecting substantia nigra dopamine neurons and medium spiny neurons in the dorsal striatum. The phenotype observed in heterozygous mutants reproduced many of the abnormalities described within the human disease spectrum, including developmental delay, social deficit, and pronounced hyperactivity without major changes in gross neuroanatomy. Despite an approximately 7-fold higher sensitivity of A749G-containing channels to the LTCC inhibitor isradipine, oral pretreatment over 2 days did not rescue the hyperlocomotion. Cav1.3AG mice confirm the pathogenicity of the A749G variant and point toward a pathogenetic role of altered signaling in the dopamine midbrain system.

The skeletal muscle Ca V 1.1 channel functions as the voltage-sensor of excitation-contraction (EC) coupling. Recently, the adaptor protein STAC3 was found to be essential for both Ca V 1.1 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 Ca V 1.1, as STAC3 is required for its functional expression. However, upon strong membrane depolarization, Ca V 1.1 conducts calcium currents characterized by very slow kinetics of activation and inactivation. Therefore, we hypothesized that the negligible inactivation observed in Ca V 1.1 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 Ca V 1.3 currents. When coexpressed in Ca V 1.1/STAC3 double knockout myotubes, the mutant STAC3-ETLAAA failed to colocalize with Ca V 1.1 in the sarcoplasmic reticulum/membrane junctions. However, combined patch-clamp and calcium recording experiments revealed that STAC3-ETLAAA supports Ca V 1.1 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 Ca V 1.1 currents, suggesting that STAC3 determines the slow Ca V 1.1 currents kinetics. To examine if STAC3 specifically inhibits the CDI of Ca V 1.1 currents, we performed patch-clamp recordings using calcium and barium as charge carriers in HEK cells. While Ca V 1.1 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 Ca V 1.1 currents. Altogether, these results designate STAC3 as a crucial determinant for the slow activation kinetics of Ca V 1.1 currents and implicate STAC proteins as modulators of both components of inactivation of LTCC., (© 2022 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as a voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming α1S subunit and the auxiliary α2δ-1 and γ1 subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage dependence of activation to positive potentials outside the physiological range. We generated new HEK293 cell lines stably expressing α2δ-1, β3, and STAC3. When the adult (CaV1.1a) and embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further coexpressed the γ1 subunit was the current density of CaV1.1a, but not that of CaV1.1e, reduced by >50%. In addition, γ1 caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of γ1, unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the γ1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the γ1-dependent reduction of current density, suggesting that structural rearrangements in CaV1.1a induced by inclusion of exon 29 may allosterically empower the γ1 subunit to exert its inhibitory action on CaV1.1 calcium currents., (© 2022 El Ghaleb et al.)