Modulation of the smooth-muscle Ca2+ channel α1C-b subunit by the auxiliary β2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the α1-β interaction in functional channels is sensitive to an 18-amino-acid synthetic peptide that corresponds to the sequence of the defined major interaction domain in the cytoplasmic I-II linker of α1C (AID-peptide). Ca2+ channels derived by co-expression of α1C-b and β2a subunits exhibited an about 3-fold higher open probability (Po) than α1C-b channels. High-Po gating of α1C-b·β2a channels was associated with the occurrence of long-lasting channel openings [mean open time (τ) > 10 ms] which were rarely observed in α1C-b channels. Modulation of fast gating by the β2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to β2 subunits of native Ca2+ channels. Binding of the β2 protein to immobilized AID-peptide was specifically inhibited (Ki of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 μM) to the cytoplasmic side of inside-out patches induced a substantial reduction of Po of α1C-b·β2a channels. The scrambled control peptide failed to affect α1C-b·β2a channels, and the AID-peptide (10 μM) did not modify α1C-b channel function in the absence of expressed β2a subunit. Our results demonstrate that the β2a subunit controls fast gating of α1C-b channels, and suggest the α1-β interaction domain in the cytoplasmic I-II linker of α1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of α1-β interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the α1-β interaction.

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