Voltage-Gated Rearrangements Associated with Differential beta -Subunit Modulation of the L-Type Ca super(2+) Channel Inactivation

Auxiliary beta -subunits bound to the cytoplasmic alpha sub(1)-interaction domain of the pore-forming alpha sub(1C)-subunit are important modulators of voltage-gated Ca super(2+) channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta sub(1a)- and beta sub(2)-subunits and structural responses of the channel to transition into distinct functional states. The NH sub(2)-termini of the alpha sub(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 NH sub(2)-termini of the alpha sub(1C)- and beta -subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha sub(1C) and beta sub(1a) suggesting mutual reorientation of the NH sub(2)-termini, but their distance vis-a-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the beta sub(1a)-subunit was replaced by beta sub(2). Differential beta -subunit modulation of inactivation and of FRET between alpha sub(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 NH sub(2)-termini of alpha sub(1C)- and beta -subunits and targets the mechanism of slow voltage-dependent inactivation.