The influence of sarcoplasmic reticulum Ca2+ concentration on Ca2+ sparks and spontaneous transient outward currents in single smooth muscle cells

Localized, transient elevations in cytosolic Ca2+, known as Ca2+ sparks, ca used by Ca2+ release from sarcoplasmic reticulum, are thought to trigger th e opening of large conductance Ca2+-activated potassium channels in the pla sma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca2+ concentrat ion within the sarcoplasmic reticulum and a Ca2+ spark and that between a C a2+ spark and a STOC are not well defined or fully understood. To address t hese problems, we have employed two approaches using single patch-clamped s mooth muscle cells freshly dissociated fi-om toad stomach: a high speed, wi de-field imaging system to simultaneously record Ca2+ sparks and STOCs, and a method to simultaneously measure free global Ca2+ concentration in the s arcoplasmic reticulum ([Ca2+](SR)) and in the cytosol ([Ca2+](CYTO)) along with STOCs. At a holding potential of 0 mV, cells displayed Ca2+ sparks and STOCs. Ca2+ sparks were associated with STOCs; the onset of the sparks coi ncided with the upstroke of STOCs, and both had approximately the same deca y time. The mean increase in [Ca2+],,, at the time and location of the spar k peak was similar to 100 nM above a resting concentration of similar to 10 0 nM. The frequency and amplitude of spontaneous Ca2+ sparks recorded at -8 0 mV were unchanged for a period of 10 min after removal of extracellular C a2+ (nominally Ca2+-free solution with 50 mu M EGTA), indicating that Ca2influx is not necessary for Ca2+ sparks. A brief pulse of caffeine (20 mM) elicited a rapid decrease in [Ca2+](SR) in association with a surge in [Ca2 +] err, and a fusion of STOCs, followed by a fast restoration of [Ca2+](CYT O) and a gradual recovery of [Ca2+](SR) and STOCs. The return of global [Ca 2+](CYTO) to rest was an order of magnitude faster than the refilling of th e sarcoplasmic reticulum with Ca2+. After the global [Ca2+](CYTO) was fully restored, recovery of STOC frequency and amplitude were correlated with th e level of [Ca2+](SR), even though the time for refilling varied greatly. S TOC frequency did not recover substantially until the [Ca2+](SR) was restor ed to 60% or more of resting levels. At [Ca2+](SR) levels above 80% of rest , there was a steep relationship between [Ca2+](SR) and STOC frequency. In contrast, the relationship between [Ca2+](SR) and STOC amplitude was linear . The relationship between [Ca2+](SR) and the frequency and amplitude was t he same for Ca2+ sparks as it was for STOCs. The results of this study sugg est that the regulation of [Ca2+](SR) might provide one mechanism whereby a gents could govern Ca2+ sparks and STOCs. The relationship between Ca2+ spa rks and STOCs also implies a close association between a sarcoplasmic retic ulum Ca2+ release site and the Ca2+-activated potassium channels responsibl e for a STOC.