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

Item Type:	Article
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
Creators Name:	Hohaus, A. and Poteser, M. and Romanin, C. and Klugbauer, N. and Hofmann, F. and Morano, I. and Haase, H. and Groschner, K.
Abstract:	Modulation of the smooth-muscle Ca2+ channel {alpha}1C-b subunit by the auxiliary {beta}2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the {alpha}1-{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 {alpha}1C (AID-peptide). Ca2+ channels derived by co-expression of {alpha}1C-b and {beta}2a subunits exhibited an about 3-fold higher open probability (P(o)) than {alpha}1C-b channels. High-P(o) gating of {alpha}1C-b·{beta}2a channels was associated with the occurrence of long-lasting channel openings [mean open time (τ) > 10 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 recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to {beta}2 subunits of native Ca2+ channels. Binding of the β2 protein to immobilized AID-peptide was specifically inhibited (K1 of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 muM) to the cytoplasmic side of inside-out patches induced a substantial reduction of P(o) of {alpha}1C-b·{beta}2a channels. The scrambled control peptide failed to affect {alpha}1C-b·{beta}2a channels, and the AID-peptide (10 μM) did not modify {alpha}1C-b channel function in the absence of expressed {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} 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 {alpha}1-{beta} interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the {alpha}1-{beta} interaction.
Keywords:	L-Type Calcium Channels, Cell Line, Cytoplasm, Ion Channel Gating, Peptide Fragments, Protein Binding, Smooth Muscle, Wistar Rats, Animals, Rats
Source:	Biochemical Journal
ISSN:	0264-6021
Publisher:	Portland Press
Volume:	348
Number:	3
Page Range:	657-665
Date:	15 June 2000
Official Publication:	http://www.biochemj.org/bj/348/bj3480657.htm
PubMed:	View item in PubMed

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