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.