Alanine-scanning mutagenesis defines a conserved energetic hotspot in the Cavα1 AID-Cavβ interaction site that is critical for channel modulation

Voltage-gated calcium channels (Ca(v)s) are large, multisubunit complexes that control cellular calcium entry. Ca-v pore-forming (Ca-v alpha(1)) and cytoplasmic (Ca-v beta) subunits associate through a high-affinity interaction between the Cava, a interaction domain (AID) and Ca-v beta alpha binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all Ca(v)1 and Ca(v)2 AID peptides bind Ca-v beta with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of Ca(v)1.2 by Ca-v beta. Together, the data support the primacy of the AID-ABP interface for Ca-v alpha(1)-Ca-v beta association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking Ca-v alpha(1)-Ca-v beta complex formation.