A Timothy Syndrome Causing Mutation Perturbs Voltage Sensor Operation in Human CaV1.2 Channels

Abstract

The pore-forming α1C subunit of the voltage-gated L-type calcium channel (CaV1.2) consists of four concatenated Repeats (I-IV), each contributing a voltage-sensing domain (VSD, transmembrane helices S1-S4) and a quarter of the central ion-conductive pore domain (S5-S6). In Timothy Syndrome (TS), a rare and severe multi-organ disorder, specific alterations in CaV1.2 voltage-dependent properties lead to autism, immunodeficiency, QT prolongation (LQT8) and lethal arrhythmias. The most frequent TS-causing mutation (G406R) is located at the intracellular flank of S6 in Repeat I and promotes Ca2+ influx by (1) shifting CaV1.2 voltage- dependent activation towards more negative potentials and (2) impairing voltage-dependent inactivation. To gain insight on the molecular basis underlying the TS-causing voltage-sensitivity anomalies, we studied the structural rearrangements of VSDs I and III in human CaV1.2 channels carrying the G406R mutation, using the voltage-clamp fluorometry technique. Briefly, this involves conjugating a small, environment-sensitive fluorophore at a specific VSD that reports local conformational rearrangements as fluorescence changes. Under voltage-clamp, we simultaneously acquired ionic current and fluorescence signals, which corresponded to specific VSD rearrangements during channel activation. We found that the G406R mutation dramatically altered the operation of VSDs I and III compared to wild-type channels, by inducing a hyperpolarizing shift in their activation voltage dependence of ∼80mV and ∼50mV, respectively. These shifts were associated with a significant reduction in the effective valences of VSD I and III by ∼50% and ∼42%, respectively. Moreover, the sign of the fluorescence signals detected from TS channels was opposite to that observed in wild-type CaV1.2 channels. Taken together, these results suggest that the TS-causing mutation causes an overall structural perturbation, manifested as a change in both the fluorophore quenching process reported from VSD I and VSD III and their altered voltage-dependence. Funded by: NIH,AHA,FONDECYT,ACT.