Nadine J. ORTNER

Same, yet different: gating-modifying CACNA1D (Cav1.3) variants in human disease

Nadine J. Ortner1, Ferenc Török, Lucia Zanetti, Yuliia Nikonishyna, Nadja T. Hofer, Jörg Striessnig

Background: Tightly regulated Ca²⁺-influx through voltage-gated Cav1.3 L-type Ca²⁺ channels (LTCCs) is indispensable for proper physiological functions. Missense variants of their pore-forming α1 subunit (CACNA1D gene) have been found in patients with neurodevelopmental and endocrine dysfunction and alter channel gating in a complex manner, promoting Ca²⁺-influx at subthreshold potentials. Since the presence and severity of symptoms differs among affected individuals, we here aim at i) elucidating the underlying molecular mechanisms, ii) identifying factors that impact clinical manifestation, and iii) studying variant-specific responsiveness to clinically available dihydropyridine (DHP) LTCC inhibitors.

Method: Different splice variants of wildtype and mutant Cav1.3 α1 subunits (co-expressed with β3 or β2a and α2δ1) were transiently expressed in tsA201 cells. Biophysical properties and pharmacological modulation by the LTCC inhibitor isradipine were determined in whole-cell patch-clamp recordings (15mM Ca²⁺).

Results: All investigated disease-associated Cav1.3 mutant variants showed typical gating changes, in particular a shift of the voltage-dependence of gating towards more hyperpolarized membrane potentials. The extent of this shift differed strongly among variants (voltage of half-maximal activation, V_0.5, shifted by -10 mV up to -30 mV). Moreover, tail currents were prolonged and kinetics of channel inactivation during 5-s long depolarizing stimuli to the voltage of maximal activation were either unaltered or affected differentially (de- or increased inactivation). Interestingly, mutation-induced gating defects were preserved in different splice variants of Cav1.3 channels. Perfusion with different concentrations of the DHP isradipine revealed mutation-specific changes of drug responsiveness, with most variants showing preserved or even increased isradipine sensitivity at negative membrane potentials (IC₅₀ decreased by 2 to 7-fold with β3).

Conclusion: The low number of 13 patients affected by such high-risk CACNA1D variants precludes the identification of a clear genotype-phenotype correlation to date. However, the complex and variant-specific gating changes likely contribute to the observed inter-patient variability, and larger shifts of V_0.5 seem to correlate with disease severity. In the absence of reliable Cav1.3-selective inhibitors, our findings of preserved or even increased DHP sensitivity justify off-label treatment attempts in affected individuals.