Structural rigidity of the I-II loop couples CaV β anchoring to CaV2.2 gating modes

Abstract

The auxiliary beta subunits of voltage-gated Ca2+ (Ca-V) channels are fundamental regulators of channel gating and neuronal excitability. While the subcellular localization of beta subunits is known to influence current density and inactivation, the precise kinetic mechanism by which they differentially modulate channel opening and closing remains elusive. Here, we report a kinetic paradox in Ca(V)2.2 channels: membrane-anchored beta subunits decelerate current decay during depolarization yet accelerate tail deactivation upon repolarization, whereas cytosolic beta subunits promote rapid decay but prolong deactivation. Using quantitative kinetic analysis and Markov state modeling, we demonstrate that macroscopic current decay is not solely a monolithic irreversible inactivation process but a composite of irreversible inactivation and a reversible transition to a nonconducting state. We reveal that membrane-anchored beta subunits suppress the transition to this reversible nonconducting state, thereby maintaining the open state, while facilitating a rapid return from the nonconducting state to prevent kinetic trapping. Furthermore, by manipulating the linker length of beta subunits and engineering the I-II loop hinge region (R370), we identify that the physical proximity of the beta subunit to the plasma membrane, coupled with the structural rigidity of the I-II loop, acts as a mechanical determinant that governs this gating pathway selection. Our findings provide a unified gating model in which the beta subunit fine-tunes the dynamic equilibrium between conducting and nonconducting states via mechanical constraint on the channel complex, offering a comprehensive resolution to the distinct regulation of Ca(V)2.2 kinetics.