A Ca_Vβ SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca²⁺ channels

Auxiliary Ca²⁺ channel β subunits (Ca_Vβ) regulate cellular Ca²⁺ signaling by trafficking pore-forming α₁ subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3-GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the Ca_Vβ SH3-GK module regulates multiple Ca²⁺ channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca_Vβ SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3-GK interaction, and fully reconstitutes Ca _Vβ effects on channel trafficking, activation gating, and increased open probability (P_o). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3-GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3-GK interaction. Remarkably, this pair discriminated between Ca_Vβ trafficking and gating properties: α_1C targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P_o functions were selectively lost. A more extreme case, in which the trans SH3-GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the α_1C (Ca _V1.2) subunit. The results reveal that Ca_Vβ SH3 and GK domains function codependently to tune Ca²⁺ channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca²⁺ channel activity.