Depletion of the SR of Ca²⁺ changes does not reduce the [Ca²⁺] increases in subplasma membrane space or bulk cytoplasm in voltage-clamped single colonic myocytes. Depolarization (−70 to +10 mV; G) activated a voltage-dependent Ca²⁺ current (I_Ca; F) to evoke a rise in [Ca²⁺] (C and D). The rise in [Ca²⁺], which occurred in some regions (B, left panels) of the subplasma membrane space (measured by TIRF; red line in inset in C), were more rapid in both onset and decline than that seen in average measured in the subplasma membrane space (black red line in inset in C; B and E). The same regions in the bulk cytoplasm (measured by wide-field epi-florescence; B, left panels) had approximately comparable rates of rise and decline (D, inset). 10 mM caffeine (E) applied by pressure ejection from a puffer pipette (A, left side; see also whole cell patch electrode [right side]) evoked increases in [Ca²⁺] (C and D). 50 µM ryanodine (C, open bar above the trace) after repeated application of caffeine to open RYR abolished the [Ca²⁺] increase to caffeine presumably because of SR store depletion. In contrast, the depolarization-evoked [Ca²⁺] increase was not reduced in either subplasma membrane space or bulk cytoplasm. After inhibition of the caffeine-evoked [Ca²⁺] increase, the depolarization-evoked [Ca²⁺]_PM or [Ca²⁺]_c increase were not reduced. Furthermore, localized increases in regions of the subplasma membrane space measured by TIRF (red line in inset in C) were more rapid in onset and decline than those seen in average measured in the subplasma membrane space (black red line in inset in C; B and E). This result suggests that SR Ca²⁺ release does not contribute substantially to the depolarization-evoked [Ca²⁺]_PM or [Ca²⁺]_c increases. Changes in the fluorescence ratio with time (C and D) are derived from 1-pixel boxes (B) and from a larger region encompassing the entire TIRF region (B). The latter was used to obtain an average subplasma membrane and bulk average [Ca²⁺] increases (C and D). (A; left) A bright field image of the cell; see also whole cell electrode (right side). B shows an expanded view of A (middle and right panels) to illustrate the regions of measurement. Note the pixel position is in a different position after SR depletion. The cell moved slightly during solution exchanges, and a new voltage-dependent Ca²⁺ channel cluster may have emerged.