In the present study, we have investigated the spatial relationship between RYRs and BK channels that underlies the functional coupling between Ca2+ sparks and STOCs in airway smooth muscle (ASM) cells from mice. To address this question more precisely, we used two complementary approaches: (1) dual immunocytochemistry to visualize the distribution of BKs and RYRs and (2) functional analysis of the quantitative relationship between sparks and STOCs. For the immunocytochemistry, we used isoform-specific RYR antibodies to determine the 3-D cellular distribution of these proteins at high spatial resolution. For the functional analysis, we simultaneously imaged Ca2+ sparks at 333 Hz and recorded corresponding STOCs at 1 KHz so a detailed analysis of their relationship could be performed. With the combination of these approaches, we found that (a) RYR1 and RYR2 localize near the plasma membrane while RYR3 localizes near the nucleus, so only RYR1 and RYR2 appear functionally able to couple with BK channels; (b) both RYRs and BK channels form clusters; and (c) for a given RYR1 or RYR2 cluster, two to three BK clusters on average are randomly
