Eminence of VSD I in the Voltage-Dependent Inactivation of the Human CaV1.2 Channel

Abstract

In response to prolonged membrane depolarizations, the L-type calcium channel (CaV1.2) enters a non-conducting inactivated state that diminishes Ca2+ influx. Specifically, CaV1.2 channels undergo two types of inactivation: voltage-dependent inactivation (VDI) and a Ca2+-dependent, calmodulin-mediated inactivation (CDI). What is the structural basis for the voltage dependence of VDI? We hypothesize that at least one of the four CaV1.2 homologous but non-identical voltage-sensing domains (VSDs) should drive or regulate VDI. Using the Voltage-Clamp Fluorometry technique, we optically tracked the movement of the individual VSDs in conducting human CaV1.2 channels (α1C/α2δ-1) expressed in Xenopus oocytes under conditions that show a different extent of VDI (300ms depolarizations to 10 mV, using Ba2+ as the charge carrier to prevent CDI): β3 co-expression, VDI∼35%; β2a co-expression, VDI∼15%; Timothy-Syndrome-causing mutation G406R with β3, VDI∼8%. We found that, in all channels, upon repolarization to −90mV, VSD I deactivated with a bi-exponential decay (τ1∼1ms, τ2∼35ms). Channels with attenuated VDI (Timothy-Syndrome-causing mutation and β2a co-expression) exhibited mostly fast VSD I deactivation (Amp2∼6% and 12%, respectively), while a more pronounced VDI (β3 co-expression) correlated with an increase in the fraction of VSD I returning slowly to their resting state (Amp2∼55%). This feature is reminiscent of “charge immobilization”, whereby the inactivation of voltage-gated channels slows down VSD deactivation and produces an apparent reduction of the off gating charge. Our data support the view that VSD I undergoes “immobilization” following CaV1.2 inactivation. On the other hand, VSDs III and IV kinetics were not affected by the time-course of VDI. In conclusion, our findings reveal a strong correlation between the degree of inactivation and VSD I kinetics, suggesting the involvement of VSD I in setting the rate of VDI.