Inactivation induced by pathogenic Ca1.3 L-type Ca-channel variants enhances sensitivity for dihydropyridine Ca channel blockers.

BACKGROUND AND PURPOSE

Pathogenic gain-of-function mutations in Ca1.3 L-type voltage-gated Ca-channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain-of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca-channel blocker isradipine, a potential therapeutic for affected patients.

EXPERIMENTAL APPROACH

Wildtype and mutant channels were expressed in tsA-201 cells and their gating analysed using whole-cell and single-channel patch-clamp recordings. The voltage-dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations.

KEY RESULTS

Both mutations were confirmed pathogenic due to characteristic shifts of voltage-dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30- to 40-fold). The IC was indistinguishable for wildtype and mutants when >50% of channels were inactivated.

CONCLUSIONS AND IMPLICATIONS

Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage-dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Ca1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.