Auxiliary β-subunits bound to the cytoplasmic α1- interaction domain of the pore-forming α1C-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 β1a- and β2-subunits and structural responses of the channel to transition into distinct functional states. The NH2-termini of the α1C- and β-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 α1C- and β-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between α1C and β1a suggesting mutual reorientation of the NH2-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the β1a-subunit was replaced by β2. Differential β-subunit modulation of inactivation and of FRET between α1C and β were eliminated by inhibition of the slow inactivation. Thus, differential β-subunit modulation of inactivation correlates with the voltage-gated motion between the NH2-termini of α1C- and β-subunits and targets the mechanism of slow voltage-dependent inactivation.
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