To study Ca 2؉ fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in ϳ20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe

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.

No abstract

Local changes in cytosolic [Ca2+] were imaged with a wide‐field, high‐speed, digital imaging system while membrane currents were simultaneously recorded using whole‐cell, perforated patch recording in freshly dissociated guinea‐pig tracheal myocytes. Depending on membrane potential, Ca2+ sparks triggered ‘spontaneous’ transient inward currents (STICs), ‘spontaneous’ transient outward currents (STOCs) and biphasic currents in which the outward phase always preceded the inward (STOICs). The outward currents resulted from the opening of large‐conductance Ca2+‐activated K+ (BK) channels and the inward currents from Ca2+‐activated Cl− (ClCa) channels. A single Ca2+ spark elicited both phases of a STOIC, and sparks originating from the same site triggered STOCs, STICs and STOICs, depending on membrane potential. STOCs had a shorter time to peak (TTP) than Ca2+ sparks and a much shorter half‐time of decay. In contrast, STICs had a somewhat longer TTP than sparks but the same half‐time of decay. Thus, the STIC, not the STOC, more closely reflected the time course of cytosolic Ca2+ elevation during a Ca2+ spark. These findings suggest that ClCa channels and BK channels may be organized spatially in quite different ways in relation to points of Ca2+ release from intracellular Ca2+ stores. The results also suggest that Ca2+ sparks may have functions in smooth muscle not previously suggested, such as a stabilizing effect on membrane potential and hence on the contractile state of the cell, or as activators of voltage‐gated Ca2+ channels due to depolarization mediated by STICs.

Arachidonic acid, as well as fatty acids that are not substrates for cyclooxygenase and lipoxygenase enzymes, activated a specific type of potassium channel in freshly dissociated smooth muscle cells. Activation occurred in excised membrane patches in the absence of calcium and all nucleotides. Therefore signal transduction pathways that require such soluble factors, including the NADPH-dependent cytochrome P450 pathway, do not mediate the response. Thus, fatty acids directly activate potassium channels and so may constitute a class of signal molecules that regulate ion channels.