Modulation of the smooth-muscle L-type Ca2+ channel alpha 1 subunit (alpha1C-b) by the beta 2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha 1 induces functional uncoupling

Modulation of the smooth-muscle Ca2+ channel alpha 1C-b subunit by the auxi liary beta 2a subunit was studied in the HEK 293 (cell line from human embr yonic kidney cells) expression system. In addition, we tested whether the a lpha 1-beta interaction in functional channels is sensitive to an 18-amino- acid synthetic peptide that corresponds to the sequence of the defined majo r interaction domain in the cytoplasmic I-II linker of alpha 1C (AID-peptid e). Ca2+ channels derived by co-expression of alpha 1C-b and beta 2a subuni ts exhibited an about 3-fold higher open probability (P-0) than alpha 1C-b channels. High-P-0 gating of alpha 1C-b.beta 2a channels was associated wit h the occurrence of long-lasting channel openings [mean open time (tau) > 1 0 ms] which were rarely observed in alpha 1C-b channels. Modulation of fast gating by the beta 2a subunit persisted in the cell-free, inside-out recor ding configuration. Biochemical experiments showed that the AID-peptide bin ds with appreciable affinity to beta 2 subunits of native Ca2+ channels. Bi nding of the beta 2 protein to immobilized AID-peptide was specifically inh ibited (K-i of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-pep tide (10 mu M) to the cytoplasmic side of inside-out patches induced a subs tantial reduction of P-0 of alpha 1C-b.beta 2a channels. The scrambled cont rol peptide failed to affect alpha 1C-b.beta 2a channels, and the AID-pepti de (10 mu M) did not modify alpha 1C-b channel function in the absence of e xpressed beta 2a subunit. Our results demonstrate that the beta 2a subunit controls fast gating of alpha 1C-b channels, and suggest the alpha 1-beta i nteraction domain in the cytoplasmic I-II linker of alpha 1C (AID) as a pos sible target of modulation of the channel. Moreover, our data are consisten t with a model of alpha 1-beta interaction that is based on multiple intera ction sites, including AID as a determinant of the affinity of the alpha 1- beta interaction.