Association of native Ca2+ channel β subunits with the α1 subunit interaction domain

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Abstract

β Subunits of voltage-dependent Ca2+ channels play an important role in regulating Ca2+ channel function. The sites of α1-β subunit interaction have been localized recently to cytoplasmic domains of both subunits. The α1, subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all α1 subunits which is essential for the binding of β sub-units. In order to further study the interaction of β subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the α1A 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 β2 and β3 subunits demonstrate that the hydroxyl group on tyrosine 392 of AIDA is critical for binding to β subunits. The affinity bead assay was also used to identify and characterize native β subunits from detergent extracts of different tissues. The AIDA affinity beads, but not the control or Y392S beads, specifically bind β subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of β1a in skeletal muscle, β2 and β3 in cardiac muscle, and β1b, β3, and β4 in brain. The assays also demonstrate the AIDA beads bind to β subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of β subunits which is not always associated with α1 subunits. Also, β subunits from solubilized skeletal muscle triads can be affinity-purified using AIDA CNBr-Sepharose. Our data demonstrate that the AID binds to native β subunits from detergent and non-detergent tissue extracts illustrating that this domain on the α1 subunit is the major anchoring site for the β subunit.

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Witcher, D. R., De Waard, M., Liu, H., Pragnell, M., & Campbell, K. P. (1995). Association of native Ca2+ channel β subunits with the α1 subunit interaction domain. Journal of Biological Chemistry, 270(30), 18088–18093. https://doi.org/10.1074/jbc.270.30.18088

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