Pournejati, R. | bioRxiv Science

bioRxiv2026

Cholesterol, PtdIns(4,5)P2, 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 statementThis 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.

biophysics↗

bioRxiv2025

Functionally-Coupled Ion Channels Begin Co-assembling at the Start of Their Synthesis

Calcium binding to BK channels lowers BK activation threshold, substantiating functional coupling with calcium-permeable channels. This coupling requires close proximity between different channel types, and the formation of BK-CaV1.3 hetero-clusters at nanometer distances exemplifies this unique organization. To investigate the structural basis of this interaction, we tested the hypothesis that BK and CaV1.3 channels assemble before their insertion into the plasma membrane. Our approach incorporated four strategies: (1) detecting interactions between BK and CaV1.3 proteins inside the cell, (2) identifying membrane compartments where intracellular hetero-clusters reside, (3) measuring the proximity of their mRNAs, and (4) assessing protein interactions at the plasma membrane during early translation. These analyses revealed that a subset of BK and CaV1.3 transcripts are spatially close in micro-translational complexes, and their newly synthesized proteins associate within the endoplasmic reticulum (ER) and Golgi. Comparisons with other proteins, transcripts, and randomized localization models support the conclusion that BK and CaV1.3 hetero-clusters form before their insertion at the plasma membrane. Significance statementThis work examines the proximity between BK and CaV1.3 molecules at the level of their mRNAs and newly synthesized proteins to reveal that these channels interact early in their biogenesis. Two cell models were used: a heterologous expression system to investigate the steps of protein trafficking and a pancreatic beta cell line to study the localization of endogenous channel mRNAs. Our findings show that BK and CaV1.3 channels begin assembling intracellularly before reaching the plasma membrane, revealing new aspects of their spatial organization. This intracellular assembly suggests a coordinated process that contributes to functional coupling.

biophysics↗