T-type calcium channels (Ca_v3.1 to Ca_v3.3) 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 Ca_v3.3 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 Ca_v3.3 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 Ca_v3.3-I860N showing the strongest and Ca_v3.3-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 Ca_v3.3 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 Ca_v3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Ca_v3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Ca_v3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Ca_v3.3 to slow oscillations with Ca_v3.3-I860N and an intermediate firing mode with Ca_v3.3-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.

Emerging evidence for specific neuronal functions of auxiliary calcium channel α2δ subunits

Significance: Voltage-gated calcium channels are important regulators of neuronal functions, as for example synaptic transmission. Their auxiliary α ₂ δ subunits are modulating the calcium currents. Beyond that they have emerged as modulators of synaptic functions. Here, we established a cellular triple knockout/knockdown model in cultured hippocampal neurons by knocking out or knocking down the expression of all three α ₂ δ subunits expressed in brain. Our experiments demonstrate that the presynaptic loss of α ₂ δ proteins leads to a severe defect in glutamatergic synapse formation, which could be rescued by reintroducing any of the three neuronal α ₂ δ isoforms. Thus, our study suggests that α ₂ δ proteins are critical regulators of excitatory synapse formation and thereby contributes to the understanding of basic nerve cell functions.

Presynaptic α₂δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α₂δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α₂δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABA_ARs. Strikingly, presynaptic α₂δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABA_ARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α₂δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α₂δ-2 can modulate postsynaptic GABA_AR abundance. Finally, exclusion of the alternatively spliced exon 23 of α₂δ-2 is essential for the trans-synaptic mechanism. The novel function of α₂δ-2 identified here may explain how abnormal α₂δ subunit expression can cause excitatory–inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENTVoltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α₂δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α₂δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α₂δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.

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.

The pancreatic islets of Langerhans secrete several hormones critical for glucose homeostasis. The β-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 β-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 β, extracellular α₂δ, and transmembrane γ subunits. Here, we review the current understanding regarding the role of all HVCC subunits expressed in pancreatic β-cell on electrical activity, excitation-secretion coupling, and β-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.