Role of a conserved glutamine in the function of voltage-gated Ca2+ channels revealed by a mutation in human CACNA1D

Voltage-gated Ca-v Ca2+ channels play crucial roles in regulating gene transcription, neuronal excitability, and synaptic transmission. Natural or pathological variations in Ca-v 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 CACNA1D gene encoding the pore-forming Ca(v)1.3 (1) 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-v 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 Ca(v)1.3 cDNA, Q558H significantly decreased the density of Ca2+ 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 Ca(v)1.3 channels at the cell surface. The substitution also produced more sustained Ca2+ currents by weakening voltage-dependent inactivation. When inserted into the corresponding locus of Ca(v)2.1, the substitution had similar effects as in Ca(v)1.3. However, the substitution introduced in Ca(v)3.1 reduced current density, but had no effects on voltage-dependent inactivation. Our results reveal a critical extracellular determinant of current density for all Ca-v family members and of voltage-dependent inactivation of Ca(v)1.3 and Ca(v)2.1 channels.