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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Research Article|
June 07 2000
Modulation of the smooth-muscle L-type Ca2+ channel α1 subunit (α1C-b) by the β2a subunit: a peptide which inhibits binding of β to the I–II linker of α1 induces functional uncoupling
Annette HOHAUS;
Annette HOHAUS
*Max-Delbrück-Centrum für Molekulare Medizin, D-13092 Berlin, Germany
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Michael POTESER;
Michael POTESER
†Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria
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Christoph ROMANIN;
Christoph ROMANIN
‡Institut für Biophysik, Johannes-Kepler-Universität Linz, A-4040 Linz, Austria
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Norbert KLUGBAUER;
Norbert KLUGBAUER
§Institut für Pharmakologie und Toxikologie, Technische Universität München, D-80802 München, Germany
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Franz HOFMANN;
Franz HOFMANN
§Institut für Pharmakologie und Toxikologie, Technische Universität München, D-80802 München, Germany
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Ingo MORANO;
Ingo MORANO
*Max-Delbrück-Centrum für Molekulare Medizin, D-13092 Berlin, Germany
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Hannelore HAASE;
Hannelore HAASE
*Max-Delbrück-Centrum für Molekulare Medizin, D-13092 Berlin, Germany
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Klaus GROSCHNER
Klaus GROSCHNER
1
†Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria
1To whom correspondence should be sent (e-mail klaus.groschner@;kfunigraz.ac.at).
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Publisher: Portland Press Ltd
Received:
November 16 1999
Revision Received:
March 15 2000
Accepted:
April 07 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 348 (3): 657–665.
Article history
Received:
November 16 1999
Revision Received:
March 15 2000
Accepted:
April 07 2000
Citation
Annette HOHAUS, Michael POTESER, Christoph ROMANIN, Norbert KLUGBAUER, Franz HOFMANN, Ingo MORANO, Hannelore HAASE, Klaus GROSCHNER; Modulation of the smooth-muscle L-type Ca2+ channel α1 subunit (α1C-b) by the β2a subunit: a peptide which inhibits binding of β to the I–II linker of α1 induces functional uncoupling. Biochem J 15 June 2000; 348 (3): 657–665. doi: https://doi.org/10.1042/bj3480657
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