To determine the subcellular localization of the three RYR isoforms, we performed immunocytochemistry with specific anti-RYRs in single isolated ASM cells. Consistent with the low level of mRNA, RYR3 proteins were weakly expressed in these cells. Moreover, most of RYR3 localized near the nuclei, far away from plasma membrane (Fig. 4 B; also see the 3-D Video 1). In contrast, both RYR1 and RYR2 localized at the periphery of the cells, and their quantities decreased further into the center of the cells (Fig. 4 B and Videos 2 and 3). Another feature of these two isoforms is that both of them distribute nonhomogeneously and form puncta. The mean sizes of puncta were 6.5 ± 0.5 voxels for RYR1 and 8.2 ± 0.7 for RYR2 (n = 5 cells for RYR1 and n = 7 cells for RYR2, P < 0.09, RYR1 vs. RYR2). Given the voxel size of the images, the maximal plasma membrane area covered by each punctum on average was 0.05–0.160 µm2 for RYR1 and 0.06–0.18 µm2 for RYR2 (see Materials and methods for calculation). Based on the
