Properties of the α1-β anchoring site in voltage-dependent Ca2+ channels

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 synthesized 35S-labeled β subunits specifically bind to a glutathione S-transferase (GST) fusion protein containing the α1A interaction 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 of 35S-labeled β1b subunit to the AIDA GST 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 AIDA peptide. 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 AIDA GST fusion protein binds to a single site on the β1b with an apparent Kd of 5 nM. The affinities of AIDA GST fusion protein for various β subunits was measured and demonstrate that β subunits associate with different affinities to each α1 interaction domain. The rank order of AIDA affinity for each β subunit is as follows: β4 > β2a > β1b ≫ β3. The binding of the β subunit to α1 subunit can be inhibited in vitro by the AIDA synthetic peptide with an apparent Ki of 285 nM. This interaction can also be prevented in heterologous Ca2+ channels by the injection of the AIDA GST 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 α1 subunit and thus allowing the β subunit to modify Ca2+ channel activity.