The Influence of Sarcoplasmic Reticulum Ca2+ Concentration on Ca2+ Sparks and Spontaneous Transient Outward Currents in Single Smooth Muscle Cells

Abstract

Localized, transient elevations in cytosolic Ca2+, known as Ca2+ sparks, caused by Ca2+ release from sarcoplasmic reticulum, are thought to trigger the opening of large conductance Ca2+-activated potassium channels in the plasma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca2+ concentration within the sarcoplasmic reticulum and a Ca2+ spark and that between a Ca2+ 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 Ca2+ sparks and STOCs, and a method to simultaneously measure free global Ca2+ concentration in the sarcoplasmic 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 coincided with the upstroke of STOCs, and both had approximately the same decay time. The mean increase in [Ca2+]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 Ca2+ sparks recorded at −80 mV were unchanged for a period of 10 min after removal of extracellular Ca2+ (nominally Ca2+-free solution with 50 μM EGTA), indicating that Ca2+ influx 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+]CYTO and a fusion of STOCs, followed by a fast restoration of [Ca2+]CYTO and a gradual recovery of [Ca2+]SR and STOCs. The return of global [Ca2+]CYTO to rest was an order of magnitude faster than the refilling of the sarcoplasmic reticulum with Ca2+. After the global [Ca2+]CYTO was fully restored, recovery of STOC frequency and amplitude were correlated with the level of [Ca2+]SR, even though the time for refilling varied greatly. STOC frequency did not recover substantially until the [Ca2+]SR was restored 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 the same for Ca2+ sparks as it was for STOCs. The results of this study suggest that the regulation of [Ca2+]SR might provide one mechanism whereby agents could govern Ca2+ sparks and STOCs. The relationship between Ca2+ sparks and STOCs also implies a close association between a sarcoplasmic reticulum Ca2+ release site and the Ca2+-activated potassium channels responsible for a STOC.

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes

“…In smooth muscle sparks may also modulate membrane conductances (for review see Ref. 14), either opening Ca 2ϩ -activated Cl Ϫ channels to cause depolarization and contraction (40) or promoting relaxation by activating large-conductance Ca 2ϩ -activated K ϩ channels and so hyperpolarizing the membrane (24,41). Understanding the control of Ca 2ϩ spark activity is crucial, therefore, to our understanding of smooth muscle function.…”

“…An elevation in [Ca 2ϩ ] i increases the open probability of the RyR channel, increasing the frequency of Ca 2ϩ sparks (7), whereas changes in the SR load can affect both spark frequency and amplitude (41). The filling state of the SR must reflect the balance between the rate of loss through leak pathways and the rate of Ca 2ϩ uptake via Ca 2ϩ -ATPase.…”

Calcium Mobilization via Intracellular Ion Channels, Store Organization and Mitochondria in Smooth Muscle

“…Complex RyR and IP 3 R Regulation Characteristics and Apparent Store Configuration IP 3 R and RyR are each regulated by the Ca 2+ concentration within the lumen of the store ('luminal Ca 2+ regulation') [4,45]. As the luminal Ca 2+ concentration increases so does the activity of the store release channels [3][4][5][6].…”

“…As the luminal Ca 2+ concentration increases so does the activity of the store release channels [3][4][5][6]. Conversely, the activity of RyR and IP 3 R each decrease as the store Ca 2+ content declines.…”

“…caffeine), by Ca 2+ influx from outside the cell in the process of Ca 2+ -induced Ca 2+ release (CICR), or when the stores's Ca 2+ content exceeds normal physiological values, i.e. in 'store overload' [2][3][4][5][6]. Activation of either receptor allows diffusion of Ca 2+ from the store to increase the cytoplasmic Ca 2+ concentration ([Ca 2+ ] c ) from the resting value of ~100 nM to ~1 μM for many seconds throughout the cell and briefly (e.g.…”