A novel molecular inactivation determinant of voltage-gated CaV1.2 L-type Ca2+ channel

The inactivation of voltage-gated L-type Ca2+ channels (CaV1) regulates Ca2+ entry and controls intracellular Ca2+ levels that are essential for cellular activity. The molecular entities implicated in L-channel (CaV1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802-807; poly ED motif) at the II-III loop of the alpha 1 subunits of rabbit of Cav1.2, α11.2 and α11.2 ΔN60-Δ1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto CaV1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca2+ or Ba2+ and was not altered by the β2A subunit. Missing from the fast inactivating neuronal P/Q (CaV2.1)-, N (CaV2.2)- or R (CaV2.3)-type channels and modulating CaV1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca2+ channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as CaV1.2 specific built-in “stopper.” In this model, CaV1 accomplishes a large Ca2+ influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.