Voltage‐gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore‐forming α₁ subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice‐variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre‐existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015.

Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson’s disease. The L-type calcium channel Ca_V1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length Ca_V1.3_L and two C-terminally truncated splice variants (Ca_V1.3_42A and Ca_V1.3_43S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged Ca_V1.3_L revealed its localization at the base-, neck- and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in Ca_V1.3_L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with Ca_V1.3_L and correlated with increased Ca_V1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of Ca_V1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of Ca_V1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

Significance: Skeletal muscle contraction is a tightly orchestrated event that starts with the depolarization of the T-tubular membrane. At the center is a functional and mechanical coupling between two membrane proteins: L-type voltage-gated calcium channels, located in the plasma membrane, and ryanodine receptors, located in the membrane of the sarcoplasmic reticulum. How exactly these proteins associate has remained a mystery, but recent reports have highlighted a key role for the STAC3 adaptor protein in this process. Here, we provide structural snapshots of the three STAC isoforms and identify a cytosolic loop of two Ca _V isoforms as a functional interaction site. A mutation linked to Native American myopathy is at the interface and abolishes the interaction.

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.

In neurons L-type calcium currents function in gene regulation and synaptic plasticity, while excessive calcium influx leads to excitotoxicity and neurodegeneration. The major neuronal Ca_V1.2 L-type channels are localized in clusters in dendritic shafts and spines. Whereas Ca_V1.2 clusters remain stable during NMDA-induced synaptic depression, L-type calcium currents are rapidly downregulated during strong excitatory stimulation. Here we used fluorescence recovery after photobleaching (FRAP), live cell-labeling protocols, and single particle tracking (SPT) to analyze the turnover and surface traffic of Ca_V1.2 in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (Ca_V1.2-SEP) demonstrated ∼20% recovery within 2 min without reappearance of clusters. Pulse–chase labeling showed that membrane-expressed Ca_V1.2-HA is not internalized within1 h, while blocking dynamin-dependent endocytosis resulted in increased cluster density after 30 min. Together, these results suggest a turnover rate of clustered Ca_V1.2s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic Ca_V1.2s show highly confined mobility with diffusion coefficients of ∼0.005 μm²s⁻¹. Consistent with the mobile Ca_V1.2 fraction observed in FRAP, a ∼30% subpopulation of channels reversibly exchanged between confined and diffusive states. Remarkably, high potassium depolarization did not alter the recovery rates in FRAP or the diffusion coefficients in SPT analyses. Thus, an equilibrium of clustered and dynamic Ca_V1.2s maintains stable calcium channel complexes involved in activity-dependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.

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.