Association of native Ca2+ channel beta subunits with the alpha 1 subunit interaction domain.
beta Subunits of voltage-dependent Ca2+ channels play an important role in regulating Ca2+ channel function. The sites of alpha 1-beta subunit interaction have been localized recently to cytoplasmic domains of both subunits. The alpha 1 subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all alpha 1 subunits which is essential for the binding of beta subunits. In order to further study the interaction of beta subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the alpha 1A subunit that contains the AID. Mutant GST fusion proteins that contain a single amino acid change (Y392S, Y392F, and Y392W) in the AIDA along with control GST were coupled to glutathione-Sepharose beads to form affinity beads. Binding assays using these affinity beads with in vitro synthesized 35S-labeled beta 2 and beta 3 subunits demonstrate that the hydroxyl group on tyrosine 392 of AIDA is critical for binding to beta subunits. The affinity bead assay was also used to identify and characterize native beta subunits from detergent extracts of different tissues. The AIDA affinity beads, but not the control or Y392S beads, specifically bind beta subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of beta 1a in skeletal muscle, beta 2 and beta 3 in cardiac muscle, and beta 1b, beta 3, and beta 4 in brain. The assays also demonstrate the AIDA beads bind to beta subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of beta subunits which is not always associated with alpha 1 subunits. Also, beta subunits from solubilized skeletal muscle triads can be affinity-purified using AIDA CNBr-Sepharose. Our data demonstrate that the AID binds to native beta subunits from detergent and non-detergent tissue extracts illustrating that this domain on the alpha 1 subunit is the major anchoring site for the beta subunit.
Witcher
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Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242, USA.
De Waard
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Liu
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Pragnell
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Campbell
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D011817:Rabbits; D014443:Tyrosine / Q000235:genetics
D015220:Calcium Channels; D014443:Tyrosine