Molecular mechanism responsible for sex differences in electrical activity of mouse pancreatic ß cells.

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 ß cell function also contribute is largely unknown. Here, we characterized the electrophysiological properties of ß 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 ß cells of both sexes. However, female ß cells had a more depolarized membrane potential and increased firing frequency compared with males. The higher membrane depolarization in female ß 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 ß 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.

α2δ-4 and Cachd1 Proteins Are Regulators of Presynaptic Functions.

The α2δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α2δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α2δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α2δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α2δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α2δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α2δ-1-3 isoforms (α2δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α2δ proteins. In contrast to this redundant presynaptic function, α2δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α1-α2δ protein-protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α2δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α2δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α2δ and Cachd1.

Role of High Voltage-Gated Ca2+ Channel Subunits in Pancreatic ß-Cell Insulin Release. From Structure to Function.

The pancreatic islets of Langerhans secrete several hormones critical for glucose homeostasis. The ß-cells, the major cellular component of the pancreatic islets, secrete insulin, the only hormone capable of lowering the plasma glucose concentration. The counter-regulatory hormone glucagon is secreted by the α-cells while δ-cells secrete somatostatin that via paracrine mechanisms regulates the α- and ß-cell activity. These three peptide hormones are packed into secretory granules that are released through exocytosis following a local increase in intracellular Ca2+ concentration. The high voltage-gated Ca2+ channels (HVCCs) occupy a central role in pancreatic hormone release both as a source of Ca2+ required for excitation-secretion coupling as well as a scaffold for the release machinery. HVCCs are multi-protein complexes composed of the main pore-forming transmembrane α1 and the auxiliary intracellular ß, extracellular α2δ, and transmembrane γ subunits. Here, we review the current understanding regarding the role of all HVCC subunits expressed in pancreatic ß-cell on electrical activity, excitation-secretion coupling, and ß-cell mass. The evidence we review was obtained from many seminal studies employing pharmacological approaches as well as genetically modified mouse models. The significance for diabetes in humans is discussed in the context of genetic variations in the genes encoding for the HVCC subunits.

Presynaptic α2δ subunits are key organizers of glutamatergic synapses.

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

CACNA1I gain-of-function mutations differentially affect channel gating and cause neurodevelopmental disorders.

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.

Phenotypic Characterization and Brain Structure Analysis of Calcium Channel Subunit α2δ-2 Mutant (Ducky) and α2δ Double Knockout Mice.

Auxiliary α2δ subunits of voltage-gated calcium channels modulate channel trafficking, current properties, and synapse formation. Three of the four isoforms (α2δ-1, α2δ-2, and α2δ-3) are abundantly expressed in the brain; however, of the available knockout models, only α2δ-2 knockout or mutant mice display an obvious abnormal neurological phenotype. Thus, we hypothesize that the neuronal α2δ isoforms may have partially specific as well as redundant functions. To address this, we generated three distinct α2δ double knockout mouse models by crossbreeding single knockout (α2δ-1 and -3) or mutant (α2δ-2/ducky) mice. Here, we provide a first phenotypic description and brain structure analysis. We found that genotypic distribution of neonatal litters in distinct α2δ-1/-2, α2δ-1/-3, and α2δ-2/-3 breeding combinations did not conform to Mendel's law, suggesting premature lethality of single and double knockout mice. Notably, high occurrences of infant mortality correlated with the absence of specific α2δ isoforms (α2Δ-2 > α2δ-1 > α2δ-3), and was particularly observed in cages with behaviorally abnormal parenting animals of α2δ-2/-3 cross-breedings. Juvenile α2δ-1/-2 and α2δ-2/-3 double knockout mice displayed a waddling gate similar to ducky mice. However, in contrast to ducky and α2δ-1/-3 double knockout animals, α2δ-1/-2 and α2δ-2/-3 double knockout mice showed a more severe disease progression and highly impaired development. The observed phenotypes within the individual mouse lines may be linked to differences in the volume of specific brain regions. Reduced cortical volume in ducky mice, for example, was associated with a progressively decreased space between neurons, suggesting a reduction of total synaptic connections. Taken together, our findings show that α2δ subunits differentially regulate premature survival, postnatal growth, brain development, and behavior, suggesting specific neuronal functions in health and disease.

Neuronal α2δ proteins and brain disorders.

α2δ proteins are membrane-anchored extracellular glycoproteins which are abundantly expressed in the brain and the peripheral nervous system. They serve as regulatory subunits of voltage-gated calcium channels and, particularly in nerve cells, regulate presynaptic and postsynaptic functions independently from their role as channel subunits. α2δ proteins are the targets of the widely prescribed anti-epileptic and anti-allodynic drugs gabapentin and pregabalin, particularly for the treatment of neuropathic pain conditions. Recently, the human genes (CACNA2D1-4) encoding for the four known α2δ proteins (isoforms α2δ-1 to α2δ-4) have been linked to a large variety of neurological and neuropsychiatric disorders including epilepsy, autism spectrum disorders, bipolar disorders, schizophrenia, and depressive disorders. Here, we provide an overview of the hitherto identified disease associations of all known α2δ genes, hypothesize on the pathophysiological mechanisms considering their known physiological roles, and discuss the most immanent future research questions. Elucidating their specific physiological and pathophysiological mechanisms may open the way for developing entirely novel therapeutic paradigms for treating brain disorders.