The Influence of Sarcoplasmic Reticulum Ca(2+) Concentration on Ca(2+) Sparks and Spontaneous Transient Outward Currents in Single Smooth Muscle Cells

Localized, transient elevations in cytosolic Ca(2+), known as Ca(2+) sparks, caused by Ca(2+) release from sarcoplasmic reticulum, are thought to trigger the opening of large conductance Ca(2+)-activated potassium channels in the plasma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca(2+) concentration within the sarcoplasmic reticulum and a Ca(2+) spark and that between a Ca(2+) spark and a STOC are not well defined or fully understood. To address these problems, we have employed two approaches using single patch-clamped smooth muscle cells freshly dissociated from toad stomach: a high speed, wide-field imaging system to simultaneously record Ca(2+) sparks and STOCs, and a method to simultaneously measure free global Ca(2+) concentration in the sarcoplasmic reticulum ([Ca(2+)](SR)) and in the cytosol ([Ca(2+)](CYTO)) along with STOCs. At a holding potential of 0 mV, cells displayed Ca(2+) sparks and STOCs. Ca(2+) sparks were associated with STOCs; the onset of the sparks coincided with the upstroke of STOCs, and both had approximately the same decay time. The mean increase in [Ca(2+)](CYTO) at the time and location of the spark peak was ∼100 nM above a resting concentration of ∼100 nM. The frequency and amplitude of spontaneous Ca(2+) sparks recorded at −80 mV were unchanged for a period of 10 min after removal of extracellular Ca(2+) (nominally Ca(2+)-free solution with 50 μM EGTA), indicating that Ca(2+) influx is not necessary for Ca(2+)sparks. A brief pulse of caffeine (20 mM) elicited a rapid decrease in [Ca(2+)](SR) in association with a surge in [Ca(2+)](CYTO) and a fusion of STOCs, followed by a fast restoration of [Ca(2+)](CYTO) and a gradual recovery of [Ca(2+)](SR) and STOCs. The return of global [Ca(2+)](CYTO) to rest was an order of magnitude faster than the refilling of the sarcoplasmic reticulum with Ca(2+). After the global [Ca(2+)](CYTO) was fully restored, recovery of STOC frequency and amplitude were correlated with the level of [Ca(2+)](SR), even though the time for refilling varied greatly. STOC frequency did not recover substantially until the [Ca(2+)](SR) was restored to 60% or more of resting levels. At [Ca(2+)](SR) levels above 80% of rest, there was a steep relationship between [Ca(2+)](SR) and STOC frequency. In contrast, the relationship between [Ca(2+)](SR) and STOC amplitude was linear. The relationship between [Ca(2+)](SR) and the frequency and amplitude was the same for Ca(2+) sparks as it was for STOCs. The results of this study suggest that the regulation of [Ca(2+)](SR )might provide one mechanism whereby agents could govern Ca(2+) sparks and STOCs. The relationship between Ca(2+) sparks and STOCs also implies a close association between a sarcoplasmic reticulum Ca(2+) release site and the Ca(2+)-activated potassium channels responsible for a STOC.