title: Modulation of the smooth-muscle L-type Ca2+ channel alpha1 subunit (alpha1C-b) by the beta2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha1 induces functional uncoupling.
abstract: Modulation of the smooth-muscle Ca(2+) channel alpha1C-b subunit by the auxiliary beta2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the alpha1-beta 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 alpha1C (AID-peptide). Ca(2+) channels derived by co-expression of alpha1C-b and beta2a subunits exhibited an about 3-fold higher open probability (P(o)) than alpha1C-b channels. High-P(o) gating of alpha1C-b.beta2a channels was associated with the occurrence of long-lasting channel openings [mean open time (tau)>10 ms] which were rarely observed in alpha1C-b channels. Modulation of fast gating by the beta2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to beta2 subunits of native Ca(2+) channels. Binding of the beta2 protein to immobilized AID-peptide was specifically inhibited (K(i) of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 microM) to the cytoplasmic side of inside-out patches induced a substantial reduction of P(o) of alpha1C-b.beta2a channels. The scrambled control peptide failed to affect alpha1C-b. beta2a channels, and the AID-peptide (10 microM) did not modify alpha1C-b channel function in the absence of expressed beta2a subunit. Our results demonstrate that the beta2a subunit controls fast gating of alpha1C-b channels, and suggest the alpha1-beta interaction domain in the cytoplasmic I-II linker of alpha1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of alpha1-beta interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the alpha1-beta interaction.
authors.affiliation: Max-Delbrück-Centrum für Molekulare Medizin, D-13092 Berlin, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Hohaus
authors.forename: M
authors.initials: M
authors.lastname: Poteser
authors.forename: C
authors.initials: C
authors.lastname: Romanin
authors.forename: N
authors.initials: N
authors.lastname: Klugbauer
authors.forename: F
authors.initials: F
authors.lastname: Hofmann
authors.forename: I
authors.initials: I
authors.lastname: Morano
authors.forename: H
authors.initials: H
authors.lastname: Haase
authors.forename: K
authors.initials: K
authors.lastname: Groschner
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D009130:Muscle, Smooth / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar
chemical_list: D020746:Calcium Channels, L-Type; D010446:Peptide Fragments