Because RYR1 and RYR2 are the dominant isoforms localized beneath the plasma membrane in a punctuate manner, we determined whether BK channels colocalize with these two types of RYRs by carrying out dual immunocytochemistry. Fig. 5 A displays an example showing the distribution and spatial relationship of RYR2 and BK channels. Two striking features stand out in their distributions. (1) Similar to RYRs, BK channels form puncta along the membrane (Video 4). The size of BK channel puncta was 11 ± 0.7 voxels, which could possess a plasma membrane area of 0.07–0.22 µm2 (see Materials and methods for calculation). (2) Only a small subset of RYRs and BK channels colocalized near the surface membrane. Quantitatively, <0.2% of RYR1 and RYR2 puncta colocalized with BK channels (Fig. 5 B) in the whole cell based on the estimate of the distance between the centers of RYRs and BK channel puncta. (When the nearest voxel distances between these channel puncta were considered, 9% of RYR2 puncta and 20% of RYR1 puncta were colocalized with BK channel puncta.) On average, the center of mass
