A novel molecular inactivation determinant of voltage-gated Ca_V1.2 L-type Ca²⁺ channel

The inactivation of voltage-gated L-type Ca²⁺ channels (Ca_V1) regulates Ca²⁺ entry and controls intracellular Ca²⁺ levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca_V1.2) 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 Ca_v1.2, 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 Ca_V1.2. 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 (Ca_V2.1)-, N (Ca_V2.2)- or R (Ca_V2.3)-type channels and modulating Ca_V1.2 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 Ca_V1.2 specific built-in "stopper." In this model, Ca_V1 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.