Local subplasma membrane Ca²⁺ transients in response to plasma membrane depolarization and caffeine in a voltage-clamped single colonic myocyte. Gradual depolarization from −70 to −20 mV (I) elevated [Ca²⁺] in the subplasma membrane space as measured in TIRF (D–F). The [Ca²⁺] increase was more localized and substantially larger in some regions than others (compare E and F). Two example regions (regions 2 and 3) are shown (E and F); localized rises occurred in region 2, but not region 3. Blue arrows in the frames in B (ii–iv) show examples of the localized rises in [Ca²⁺]. The [Ca²⁺] increase that occurred in region 3 (F) was slow and of smaller magnitude than that of region 2 (E). When the membrane potential was restored to −70 mV (I), [Ca²⁺] was returned toward resting levels. A subsequent transient depolarization to 0 mV (I) activated voltage-dependent Ca²⁺ current (I_Ca; H) and increased [Ca²⁺] (D–F). The increase was larger and declined more rapidly in region 2 (E and inset J) than region 3 (F and inset K). Those regions (E, region 2) that showed large responses to depolarization had small responses to RYR activation by caffeine (G). Conversely, a large response (F, region 3) to caffeine (G) occurred in regions with small responses to depolarization. Region 1 (D) is a subplasma membrane average change. Numerals in images (B) correspond to those in C. Changes in the fluorescence ratio with time (D–F) are derived from 1-pixel boxes (regions 2 and 3, shown at 3 × 3 pixels to facilitate visualization) and from a larger region the entire TIRF image (region 1) to obtain an average subplasma membrane [Ca²⁺] change (D). The position of the regions from which the transients (D–F) were obtained are shown in A (right panel). A bright field image of the cell is shown in A (left panel); see also whole cell electrode (right side) and puffer pipette, which contained caffeine (10 mM; left side). The data in this figure is representative of three similar experiments.