Journal article
Properties of the α1-β Anchoring Site in Voltage-dependent Ca2+ Channels
The Journal of biological chemistry, Vol.270(20), pp.12056-12064
05/19/1995
DOI: 10.1074/jbc.270.20.12056
PMID: 7744854
Abstract
In voltage-dependent Ca2+ channels, the β subunit interacts with the α1 subunit via a cytoplasmic site. A biochemical assay has been developed to quantitatively describe the interaction between both subunits. In vitro synthesized35S-labeled β subunits specifically bind to a glutathione S -transferase (GST) fusion protein containing the α1ainteraction domain (AIDA, located between the amino-acids 383 and 400 of the cytoplasmic loop between the hydrophobic domains I and II). Kinetic analysis demonstrates that the association of35S-labeled β1bsubunit to the AIDAGST fusion protein occurs with a fast rate constant at 4°C. The binding is almost irreversible as demonstrated by the absence of dissociation observed after an 8-h incubation with an 18-amino acid synthetic AIDApeptide. The α1-β binding site does not seem to be a target for cytoplasmic regulation. The interaction is mostly unaffected by changes in ionic strength, pH, and Ca2+concentration or by protein kinase C phosphorylation. The specificity of subunit interaction in voltage-dependent Ca2+channels was also followed by saturation analyses. The data obtained show that the AIDAGST fusion protein binds to a single site on the β1bwith an apparent Kdof 5 nM. The affinities of AIDAGST fusion protein for various β subunits was measured and demonstrate that β subunits associate with different affinities to each α1interaction domain. The rank order of AIDAaffinity for each β subunit is as follows: β> > β> > β1b»β3. The binding of the β subunit to α1subunit can be inhibited in vitro by the AIDAsynthetic peptide with an apparent Kiof 285 nM. This interaction can also be prevented in heterologous Ca2+channels by the injection of the AIDAGST fusion protein into Xenopus oocytes. Our results demonstrate that the site of interaction between AID and β subunit is responsible for anchoring the β subunit to the α1subunit and thus allowing the β subunit to modify Ca2+channel activity.
Details
- Title: Subtitle
- Properties of the α1-β Anchoring Site in Voltage-dependent Ca2+ Channels
- Creators
- Michel De Waard - Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242Derrick R Witcher - Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242Marlon Pragnell - Program in Neuroscience, University of Iowa College of Medicine, Iowa City, Iowa 52242Hongyan Liu - Program in Neuroscience, University of Iowa College of Medicine, Iowa City, Iowa 52242Kevin P Campbell - Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.270(20), pp.12056-12064
- DOI
- 10.1074/jbc.270.20.12056
- PMID
- 7744854
- NLM abbreviation
- J Biol Chem
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Publisher
- Elsevier Inc
- Grant note
- T32-AI07343 / National Institutes of Health
- Language
- English
- Date published
- 05/19/1995
- Academic Unit
- Neurology; Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984068378702771
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