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

Abstract

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.