FMRP is localized in nanoscale proximity to BKα channels and alters their gating properties. (A) Representative BKα current recordings from inside-out patches at 0 µM Ca²⁺ in the absence (black traces) and presence (green traces) of FMRP, after applying the voltage protocol shown in the inset (−100 mV to +200 mV in 20-mV increments). (B) G-V curves obtained at various Ca²⁺ concentrations, color-coded as indicated in the graph legend. Empty symbols correspond to cells expressing BKα and full-colored symbols to BKα+FMRP. Solid lines represent Boltzmann fits to the data. (C) Mean V_1/2 values plotted as a function of Ca²⁺ concentration. Error bars represent SEM. (D) Representative dSTORM images (top) and magnified views of areas of interest (bottom) showing the spatial distribution of BKα (green, Alexa Fluor 488) with either FMRP (left panels, red, Alexa Fluor 647) or δENaC (right panels, red, Alexa Fluor 647) proteins. Scale bars represent 5 µm (top) and 0.5 µm (bottom). (E) NND analysis from the corresponding dual-label experiments. (F and G) Histograms representing the distribution of clusters containing BKα alone (green bars), FMRP alone (red bars in F) or δENaC alone (red bars in G), and both proteins (yellow bars). Colored curves outline the histograms to facilitate visualization. (F)n = 25 cells from three different transfection experiments, 2,591 clusters. (G)n = 7 cells, 1,549 clusters). **, P < 0.01.