Alanine-scanning mutagenesis defines a conserved energetic hotspot in the CaValpha1 AID-CaVbeta interaction site that is critical for channel modulation.

Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaValpha1) and cytoplasmic (CaVbeta) subunits associate through a high-affinity interaction between the CaValpha1 alpha interaction domain (AID) and CaVbeta 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 CaV1 and CaV2 AID peptides bind CaVbeta 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 CaV1.2 by CaVbeta. Together, the data support the primacy of the AID-ABP interface for CaValpha1-CaVbeta association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaValpha1-CaVbeta complex formation.