Auxiliary BKβ4 subunits promote nanoscale coupling between BK and voltage-gated calcium channels

Intracellular Ca ²⁺ signals are essential for many cellular processes but must be tightly regulated to ensure precise spatial and temporal control. The large conductance calcium- and voltage-activated K ⁺ channel (BK) integrates intracellular Ca ²⁺ signaling and membrane depolarization to generate hyperpolarizing K ⁺ currents that regulate membrane excitability. In many cell types, BK channels achieve rapid activation through functional coupling with voltage-gated calcium (Ca _V ) channels that provide localized Ca ²⁺ signals. However, the mechanisms that regulate the nanoscale organization of BK+Ca _V channel complexes remain poorly understood. Here we investigated whether the BK auxiliary subunit β ₄ (BKβ ₄ ), which is widely expressed throughout the brain and modulates BK gating properties, influences the spatial organization and functional coupling of BK and Ca _V channels. Using proximity ligation assays, electrophysiology, and superresolution stochastic optical reconstruction microscopy, we show that BKβ ₄ promotes BK+Ca _V nanodomain organization by increasing channel clustering and altering their spatial proximity at the plasma membrane. These structural changes enhance functional coupling between Ca ²⁺ influx and BK channel activation. Notably, the effects of BKβ ₄ on nanodomain organization and coupling depend on the Ca _V subtype involved. Together, our findings reveal that the BKβ ₄ auxiliary subunit regulates the nanoscale architecture of BK+Ca _V channel complexes and thereby modulates their functional interaction.