Alanine-Scanning Mutagenesis Defines a Conserved Energetic Hotspot in the Ca_Vα₁ AID-Ca_Vβ Interaction Site that Is Critical for Channel Modulation

Voltage-gated calcium channels (Ca_Vs) are large, multisubunit complexes that control cellular calcium entry. Ca_V pore-forming (Ca_Vα₁) and cytoplasmic (Ca_Vβ) subunits associate through a high-affinity interaction between the Ca_Vα₁ α interaction domain (AID) and Ca_Vβ α 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_V1 and Ca_V2 AID peptides bind Ca_Vβ 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_V1.2 by Ca_Vβ. Together, the data support the primacy of the AID-ABP interface for Ca_Vα₁-Ca_Vβ association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking Ca_Vα₁-Ca_Vβ complex formation.