Spatial relationship between RYRs and BK channels. (A) A representative immunostaining showing the spatial relationship between RYR2s and BK channels in approximately one third of the length of a cell. The anti-BK antibody was labeled with Alexa Fluor 488 and is pseudocolored green, and the anti-RYR antibody was labeled with Alexa Fluor 594 and is pseudocolored red. The images show the projection of BK channels and RYR2s localized within 720 nm from the surface membrane. The first inset is the expanded view of RYR2 and BK channels in the box region. Green lines connect RYR2 puncta to BK channels within 600 nm. The right inset depicts the relationship between an RYR2 punctum, as marked by the arrow in the first inset, and its nearby BK channel puncta, indicated by numbers. To display this RYR2 punctum and its corresponding BK channels, RYR2s and BK channels above and below were removed. The center to center distances between the RYR2 punctum and the BK channel puncta are 432 nm (1), 560 nm (2), and 598 nm (3). These distances were used to simulate I_BKs shown in Fig. 7 and Fig. S3. (B) Histograms of the center of mass (COM) distance between RYR1 puncta and the closest BK channel puncta (left) and between RYR2 puncta and the closest BK channel puncta (right). Surprisingly, most BK channel puncta do not colocalize with either RYR1 puncta or RYR2 puncta. Dashed lines are the best fits of the probability distribution of distance to the closest random point. The fitting function is y = 2π × D × x × exp(−πDx²), where D is the puncta density (puncta/micrometers squared),and x is the distance (in micrometers; see eq. 8.2.9 in Cressie, 1993). From the fitting, it yields 1.11 puncta/µm² for RYR1 and 1.02 puncta/µm² for RYR2. This is not the density on the entire plasma membrane, but rather the density on the membrane near each RYR.