Voltage-gated rearrangements associated with differential β-subunit modulation of the L-type Ca2+ channel inactivation

Auxiliary beta-subunits bound to the cytoplasmic alpha(1)-interaction domain of the pore-forming alpha(1C)-subunit are important modulators of voltage-gated Ca2+ channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta(1a)- and beta(2)-subunits and structural responses of the channel to transition into distinct functional states. The NH2-termini of the alpha(1C)- and beta-subunits were fused with cyan or yellow fluorescent proteins, and functionally coexpressed in COS1 cells. Fluorescence resonance energy transfer ( FRET) between them or with membrane-trapped probes was measured in live cells under voltage clamp. It was found that in the resting state, the tagged NH2-termini of the alpha(1C)- and beta-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha(1C) and beta(1a) suggesting mutual reorientation of the NH2-termini, but their distance vis-a-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the b1a- subunit was replaced by beta(2). Differential beta-subunit modulation of inactivation and of FRET between alpha(1C) and beta were eliminated by inhibition of the slow inactivation. Thus, differential beta-subunit modulation of inactivation correlates with the voltage-gated motion between the NH2-termini of alpha(1C)- and beta-subunits and targets the mechanism of slow voltage-dependent inactivation.