Cholesterol, PtdIns (4,5) P ₂ , and Actin Regulate BK Channel Nanoscale Organization

BK channels form nanoscale clusters, but the membrane and cytoskeletal mechanisms that organize these structures remain poorly understood. Using super-resolution imaging and FRAP, we dissect how cholesterol, PtdIns(4,5)P₂, and actin govern BK channel organization expressed heterologously in tsA-201 cells and expressed endogenously in INS-1 cells. Cholesterol depletion produced opposite outcomes across these systems, enlarging clusters in heterologous cells but reducing both size and density in INS-1 cells. PtdIns(4,5)P₂ displayed a biphasic regulatory profile: both enrichment and depletion enlarged clusters, revealing that BK assemblies are tuned to a narrow PtdIns(4,5)P₂ set point. Actin disruption consistently reduced cluster size and density. Together, these findings show that cholesterol, PtdIns(4,5)P₂, and actin are not only functional regulators of BK channels but also key determinants of their nanoscale organization. Across perturbations, changes in channel density strongly predicted changes in cluster size, identifying density as a central organizing principle. Significance statement: This study shows that the nanoscale organization of BK channels is dynamically controlled by membrane lipids and the cytoskeleton, demonstrating that ion channel organization is not fixed but actively shaped by the local membrane environment. Across perturbations, cluster size scales with channel density, underscoring density as a major driver of BK nanoscale organization.