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

Intracellular Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 β₄ (BKβ₄), 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 BKβ₄ 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 Ca²⁺ influx and BK channel activation. Notably, the effects of BKβ₄ on nanodomain organization and coupling depend on the Ca_V subtype involved. Together, our findings reveal that the BKβ₄ auxiliary subunit regulates the nanoscale architecture of BK+Ca_V channel complexes and thereby modulates their functional interaction.