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

Abstract

T-type calcium channels (Cav3.1 to Cav3.3) 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 Cav3.3 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 analyzed possible effects on CaV3.3 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 CaV3.3-I860N showing the strongest and CaV3.3-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 CaV3.3 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 CaV3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the CaV3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the CaV3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type CaV3.3 to slow oscillations with CaV3.3-I860N and an intermediate firing mode with CaV3.3-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.

Calcium channelopathies and intellectual disability: a systematic review

“…Epilepsy was reported in 51.6% (82/159) of the cases. Ten calcium channelopathies related to ID/GDD were identified involving the following genes: CACNA1A [ 18 , 19 , 39 – 66 ], CACNA1C [ 67 – 73 ], CACNA1I [ 74 ], CACNA1H [ 75 , 76 ] , CACNA2D2 [ 77 – 80 ], CACNA2D1 [ 20 , 81 ], CACNA1D [ 21 , 22 , 82 – 84 ], CACNA1E [ 85 ], CACNA1F [ 86 ], and CACNA1G [ 23 , 87 ]. The underlying mechanisms included gain- and/ or loss-of-function, alteration in kinetics (activation, inactivation) and dominant-negative effects of truncated forms of alpha1 subunits.…”

“…Induced pluripotent stem cells (iPSCs) and HEK293T cells for spinocerebellar ataxia [ 302 ] Endostatin, zonisamide, clozapine, roscovitine, mibefradil, iron and zinc can block Cav3.1 channel [ 179 – 185 ] Dearomatized isoprenylated acylphloroglucinol and monoterpenoid, hypatone A could rescue pathological gating properties for spinocerebellar ataxia 42 [ 186 ] Autophagic pathway in melanoma cells [ 198 ] Apoptotic pathway in [ 193 – 195 ] Ras-ERK signaling pathway [ 200 , 201 ] CACNA1H Calcium voltage-gated channel subunit alpha1 H Cav3.2 T-type None TsA-201 cell for amyotrophic lateral sclerosis [ 303 ] KYS-05090S can block Cav3.2 current [ 187 ] Apoptotic pathway in myocardial cells [ 155 ]. Autophagy pathway [ 158 ] CACNA1I Calcium Voltage-Gated Channel Subunit Alpha1 I Cav 3.3 T-type HEK293T and mouse chromaffin cells for ID and epilepsy [ 74 ] None Zinc modulates Cav3.3 channel gating [ 181 ] None CACNA2D1 Calcium voltage-gated channel auxiliary subunit alpha2delta 1 Cav1.3 L-type None MKN74 cells (human gastric cancer cell line) [ 190 ] MicroRNA-107 can inhibit expression of Cav1.3 in cancer [ 188 , 189 ] Amlodipine can block Cav1.3 in cancer [ 190 ] CXCR3/ERK1/2 signaling pathway [ 203 ] Ras/Raf/MEK/ERK signaling pathway [ 202 ] …”

“…Upon intensive review of literature, we found that tsA-201, HEK293T and mouse chromaffin cells are being used to study ID, epilepsy and autism [ 74 , 82 , 84 , 85 ]. Besides, SH-SY5Y human neuroblastoma cells was used for familial hemiplegic migraine type 1 and episodic ataxia [ 159 , 160 ] and progressive myoclonic epilepsy [ 161 ].…”

Small Molecules as Modulators of Voltage-Gated Calcium Channels in Neurological Disorders: State of the Art and Perspectives

“…Seizure and neurodevelopmental disorders [74] Note: # indicates the reference number in the "Online Mendelian Inheritance in Man" (OMIM) database for channelopathies.…”

Recurrent de novo single point mutation on the gene encoding Na+/K+ pump results in epilepsy

“…Central to genetic etiology studies, mutations of ion channels such as potassium, sodium, and calcium channels draw the most attention to unravel the underlying mechanisms of epilepsy (Aiba & Noebels, 2021;Djordjevic et al, 2021;El Ghaleb et al, 2021;Oyrer et al, 2018;Shah, 2021). These ion channels affect neuronal physiology by stabilizing and propagating neuronal activity (Oyrer et al, 2018).…”