Voltage-gated Ca Ca channels play crucial roles in regulating gene transcription, neuronal excitability, and synaptic transmission. Natural or pathological variations in Ca channels have yielded rich insights into the molecular determinants controlling channel function. Here, we report the consequences of a natural, putatively disease-associated mutation in the gene encoding the pore-forming Ca1.3 α subunit. The mutation causes a substitution of a glutamine residue that is highly conserved in the extracellular S1-S2 loop of domain II in all Ca channels with a histidine and was identified by whole-exome sequencing of an individual with moderate hearing impairment, developmental delay, and epilepsy. When introduced into the rat Ca1.3 cDNA, Q558H significantly decreased the density of Ca currents in transfected HEK293T cells. Gating current analyses and cell-surface biotinylation experiments suggested that the smaller current amplitudes caused by Q558H were because of decreased numbers of functional Ca1.3 channels at the cell surface. The substitution also produced more sustained Ca currents by weakening voltage-dependent inactivation. When inserted into the corresponding locus of Ca2.1, the substitution had similar effects as in Ca1.3. However, the substitution introduced in Ca3.1 reduced current density, but had no effects on voltage-dependent inactivation. Our results reveal a critical extracellular determinant of current density for all Ca family members and of voltage-dependent inactivation of Ca1.3 and Ca2.1 channels.

Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming a1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ-1-4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47, Cav1.4Δex47, respectively), and the auxiliary subunits, β2x13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2x13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2x13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.

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Caldendrin is a Ca2+ binding protein that interacts with multiple effectors, such as the Cav1 L-type Ca2+ channel, which play a prominent role in regulating the outgrowth of dendrites and axons (i.e., neurites) during development and in response to injury. Here, we investigated the role of caldendrin in Cav1-dependent pathways that impinge upon neurite growth in dorsal root ganglion neurons (DRGNs). By immunofluorescence, caldendrin was localized in medium- and large- diameter DRGNs. Compared to DRGNs cultured from WT mice, DRGNs of caldendrin knockout (KO) mice exhibited enhanced neurite regeneration and outgrowth. Strong depolarization, which normally represses neurite growth through activation of Cav1 channels, had no effect on neurite growth in DRGN cultures from female caldendrin KO mice. Remarkably, DRGNs from caldendrin KO males were no different from those of WT males in terms of depolarization-dependent neurite growth repression. We conclude that caldendrin opposes neurite regeneration and growth, and this involves coupling of Cav1 channels to growth-inhibitory pathways in DRGNs of females but not males.