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

T-type calcium channels (Ca(v)3.1 to Ca(v)3.3) 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(v)3.3 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 HISS 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(v)3.3 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(v)3.3-I860N showing the strongest and Ca(v)3.3-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(v)3.3 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(v)3.3 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(v)3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Ca(v)3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Ca(v)3.3 to slow oscillations with Ca(v)3.3-I860N and an intermediate firing mode with Ca(v)3.3-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.

Automated summary

T-type calcium channels play a crucial role in regulating neuronal activity and are associated with various physiological processes. Mutations in CACNA1I gene, encoding the Ca(v)3.3 channel, have been found in patients with neurodevelopmental phenotypes, affecting channel function and leading to neuronal hyper-excitability. The gain-of-function mutations in Ca(v)3.3 channels may contribute to a spectrum of neurodevelopmental disorders, ranging from mild cognitive impairment to severe developmental delay with epilepsy.