Amplitude Of Spontaneous Transient Outward Currents Research Articles

Aging influences cerebrovascular function via BK channel in a sex‐specific manner

The myogenic response of middle cerebral arteries (MCA) protects the brain by altering the diameter in response to lumen pressure to maintain constant cerebral blood flow. Large conductance potassium (BK) channels are known to regulate the myogenic response. However, it is not clear how aging will influence the myogenic response via altering BK channel function in males and females. Here, using vascular reactivity, patch‐clamp and biochemical methods, we found that age‐associated changes in BK channel subunit expression and function alter myogenic response in a sex‐specific manner. Aging exaggerated the myogenic response of MCA of females but did not change the myogenic response in males. Aging abolished differences in myogenic tone of MCA (MT as determined from active and passive diameters) in males and females. Aging, while decreased wall to lumen ratio in females, it increased in males revealing a sex‐specific difference in MCA remodeling. Aging while decreased total K channel and BK channel currents in vascular smooth muscle cells (VSMCs) isolated from female vessels, it did not affect males. Spontaneous transient outward currents (STOC) that represent BK function are also decreased with aging in both males and females. However, the magnitude of the decrease in STOC amplitude and frequency is much greater in aged females than aged males. While aging increased BKα subunit, it decreased β1 subunit protein in cerebral vessels of both males and females. Age‐associated increase in BKα subunit is similar between males and females. However, there is a greater and significant reduction of BKβ1 subunit protein in aged females than aged males. Activation of BKα and β1 subunits either individually or both failed to decrease MT of MCA isolated from aged males and females at physiological lumen pressures. Besides, aging diminished acute estradiol (17βE)‐mediated decrease in MT either alone or combined with BKβ1 agonist in females. However, this combination significantly decreased MT in ovariectomy females that are chronically supplemented with 17βE via increasing BKβ1 protein content. Overall our results suggest that age‐associated changes in cerebral vascular structure and function and that of BK channel subunit expression and function are sex‐specific. Chronic supplementation of 17βE increases BKβ1 protein content, and its activation by agonist combined with acute 17βE attenuates MT in aged females.Support or Funding InformationSupported by AHA SDG (13SDG14000006) and IRSP from UMMC to Mallikarjuna R. Pabbidi.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional molecular complexes of junctophilin‐2 and caveolin‐1 provide a structural/functional basis for Ca2+‐microdomain formation between BKCa channels and RyRs in vascular smooth muscle cells

Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in plasma membrane (PM) and ryanodine receptors (RyRs) in sarcoplasmic reticulum (SR) is an essential mechanism for mechanical force regulation in smooth muscle cells (SMCs). Spontaneous Ca2+ release through RyRs (knows as Ca2+ sparks) and subsequent BKCa channels activation detected as spontaneous transient outward currents (STOCs) occur within PM-SR junctional sites, often called Ca2+ microdomains. We have previously reported that Caveolae, W-shaped invaginations of PM, accumulate BKCa channels within the structure and enhance coupling efficiency between Ca2+ sparks and BKCa channels/STOC activity in mouse mesenteric artery SMCs (mMASMCs). However, the molecular basis between caveolae and RyRs in SR membrane is unclear. In the present study, we demonstrated that both caveolin-1 (Cav1), a caveola forming protein, and junctophilin-2 (JP2), a bridging protein between PM and SR, reciprocally contribute to Ca2+microdomain formation. Co-immunoprecipitation using rat mesenteric arterial tissues and double-immunocytochemical staining analyses by total internal reflection fluorescent microscopy revealed the direct interaction between JP2 and Cav1. Mouse mesenteric arterial tissues were organ-cultured and concomitantly treated with siRNA using reversible permeabilization procedures. Knockdown of JP2 in mMASMCs significantly decreased the colocalization of Cav1 and RyRs and the amplitude of STOCs. The contraction induced by the inhibition of BKCa channels/STOC activity and subsequent membrane depolarization in siJP2-treated mMASMCs was significantly smaller than that in siControl. These results suggested that the novel interaction of JP2 with Cav1 seems to provide a structural/functional basis for Ca2+-mediated crosstalk between RyRs and BKCa channels, and likely positions the PM-SR junctional sites to convert Ca2+ spark signals into hyperpolarizing signals in SMCs lacking transverse tubular system. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HAEM Modulation of Arterial Smooth Muscle Cell Large-Conductance Ca2+-Activated K+ (BK) Channel Activity

Several hemolytic diseases are associated with vasospasm. Haem release during hemolysis can build up to toxic levels. Under normal conditions haem is degraded by haem oxygenase to carbon monoxide (CO), bilverdin and Fe2+. The mechanisms causing vasospasm are poorly understood. Arterial smooth muscle cells (SMCs) express BK channels which produce spontaneous transient outward currents (STOCs) when activated by Ca2+ sparks. STOCs hyperpolarize SMCs resulting in cell relaxation and vasodilation. Previous studies show that intracellular haem inhibits whereas CO enhances BK channel activity in inside-out patches. We hypothesize that haem interaction with BK channels might contribute to the vasospasm. Therefore, the current study aims to investigate the mechanism of action of haem by examining the effects of extracellular haem and CO on whole-cell BK currents. SMCs were isolated from the mesenteric artery of male Wistar rats. Depolarizing pulse-induced BK currents and STOCs were recorded using whole-cell patch techniques. CO was applied using CO-releasing molecule 3 (CORM-3) which was prepared before each use. Extracellular haem (5 µM) increased pulse-induced BK currents and STOC amplitudes by 2.3 ± 0.5 (p≤0.01, n=9) and 1.28 ± 0.09 (p≤0.05, n=5) times respectively. Extracellular CORM-3 (30 µM) had no significant effect on pulse induced BK currents (n=8) but doubled STOC frequency (2.06 ± 0.61, p≤0.05, n=6) without affecting STOC amplitude (n=6). In the presence of the HO-inhibitor, zinc protoporphyrin-IX, haem increased STOC amplitude (1.5 ± 0.04, p≤0.001, n=4) and frequency (2.4 ± 0.5, p≤0.05, n=4). In summary, our results suggest that the stimulatory effect of haem on STOCs has a component that is independent of its degradation product, CO. Furthermore, other intracellular factors could be contributing to the positive haem effects. Information gained from further studies could improve our understanding of the changes in vascular tone that occur during haemolytic diseases.

Abstract 043: Influence of Sex, Hormones and Age on Cerebrovascular Function: Role of Large Conductance Potassium Channel Subunits

Although adult females are protected, the cerebrovascular disease is greater in aged post-menopause females than in age matched males, but the role of vascular function and large conductance potassium (BK) channel is not clear. We hypothesize that “compared to adult males, cerebral vasculature of intact adult female rats displays an attenuated myogenic response and greater intrinsic tone due to differential expression and function of BK channel subunits. Aging or menopause exaggerates myogenic response, diminishes intrinsic tone and thus, vasodilatory capacity in females more than males due to a reduction in the BK subunit function.” Our results suggest that middle cerebral arteries (MCA) of intact adult females had attenuated myogenic response compared to adult males and ovariectomy (OVX) females (% change in diameter: 16±8, -25±4 and -49±7). Development of intrinsic tone was greater in intact adult females than adult males and OVX females (% intrinsic tone: 47±5, 27±4 and 15±5). The ratio of BK β1 to α subunits was less in intact adult females than adult males and OVX females (0.4±0.07, 2±0.3 and 0.6±0.1). Spontaneous transient outward currents (STOC) were higher in smooth muscle cells of intact adult females compared to adult males (pA: 204±27 and 128±7). While aging exaggerated the myogenic response in females, it had no effect in males (fold change 4±1 Vs 0.8±0.5). However, intrinsic tone was decreased in aged females but increased in aged males (% change -43±9 Vs 37± 6). Aging decreased the amplitude of STOCs but with a greater magnitude of effect in females (fold change females: -4.9±1; males: -0.8±0.2). While aging increased BKα subunit, it decreased β1 subunit protein (β1 change: females -52±8; males -37±13). Acute activation of BKα subunit by NS1619, while had no effect in intact adult females (-5±3%), it dilated the MCA of adult males (32±9%). However, acute activation of BKβ1 by estradiol while relaxed MCA of adult females (35±7%) and males (22±2%), it had less effect in OVX females (-7±8%) and aged females (4±9%). In conclusion, a higher incidence of cerebrovascular disease in aged and menopause female may be due to sex-specific changes in vascular properties and BK channels that could result in diminished estradiol-mediated vasodilation.

190 Haem and carbon monoxide (co) modulation of large-conductance ca2+-activated k+ (bk) channel activity

Haemolysis is a distinctive feature of certain diseases such as haemorrhagic stroke and sickle cell anaemia. The haem released from red blood cells during haemolysis can build up to toxic...

T-type voltage-gated Ca2+ channels do not contribute to the negative feedback regulation of myogenic tone in murine superior epigastric arteries.

T‐type voltage‐gated Ca2+ channels (CaV3.2 VGCC) have been hypothesized to control spontaneous transient outward currents (STOCs) through large‐conductance Ca2+‐activated K+ channels (BKCa), and contribute to the negative‐feedback regulation of myogenic tone. We tested this hypothesis in superior epigastric arteries (SEAs) isolated from male C57BL/6 mice. SEAs were isolated and enzymatically dissociated to obtain single smooth muscle cells (SMCs) for whole‐cell recording of paxilline‐sensitive (PAX, 1 μmol/L) STOCs at −30 mV, or cannulated and studied by pressure myography (80 cm H2O, 37°C). The CaV3.2 blocker Ni2+ (30 μmol/L) had no effect on STOC amplitude (20.1 ± 1.7 pA vs. 20.6 ± 1.7 pA; n = 12, P = 0.6), but increased STOC frequency (0.79 ± 0.15 Hz vs. 1.21 ± 0.22 Hz; n = 12, P = 0.02). Although Ni2+ produced concentration‐dependent constriction of isolated, pressurized SEAs (logEC50 = −5.8 ± 0.09; E max = 72 ± 5% constriction), block of BKCa with PAX had no effect on vasoconstriction induced by 30 μmol/L Ni2+ (in the absence of PAX = 66 ± 4% constriction vs. in the presence of 1 μmol/L PAX = 65 ± 4% constriction; n = 7, P = 0.06). In contrast to Ni2+, the nonselective T‐type blocker, mibefradil, produced only vasodilation (logEC50 = −6.9 ± 0.2; E max = 74 ± 8% dilation), whereas the putative T‐type blocker, ML218, had no significant effect on myogenic tone between 10 nmol/L and 10 μmol/L (n = 6–7, P = 0.59). Our data do not support a role for CaV3.2 VGCC in the negative‐feedback regulation of myogenic tone in murine SEAs and suggest that Ni2+ may constrict SEAs by means other than block of CaV3.2 VGCC.

Aging increases capacitance and spontaneous transient outward current amplitude of smooth muscle cells from murine superior epigastric arteries

Large conductance Ca(2+)-activated K(+) channels (BKCa) contribute to negative feedback regulation of smooth muscle cell (SMC) tone. However, the effects of aging on BKCa function are unclear. We tested the hypothesis that aging alters SMC BKCa function in superior epigastric arteries (SEAs) by using perforated patch recording of enzymatically isolated SMCs from 3- to 4-mo-old male C57BL/6 mice (Young) and 24- to 26-mo-old male C57BL/6 mice (Old). SMC capacitance from Young (15.7 ± 0.4 pF; n = 110) was less than Old (17.9 ± 0.5 pF; n = 104) (P < 0.05). SMCs displayed spontaneous transient outward currents (STOCs) at membrane potentials more positive than -30 mV; depolarization increased STOC amplitude and frequency (P < 0.05; n = 19-24). STOC frequency in Young (2.2 ± 0.6 Hz) was less than Old (4.2 ± 0.7 Hz) at -10 mV (P < 0.05, n = 27-30), with no difference in amplitude (1.0 ± 0.1 vs. 0.9 ± 0.1 pA/pF, respectively). At +30 mV, STOC amplitude in Young (3.2 ± 0.3 pA/pF) was less than Old (5.0 ± 0.5 pA/pF; P < 0.05, n = 61-67) with no difference in frequency (3.9 ± 0.4 vs. 3.2 ± 0.3 Hz, respectively). BKCa blockers (1 μM paxilline, 100 nM iberiotoxin, 1 mM tetraethylammonium) or a ryanodine receptor antagonist (100 μM tetracaine) inhibited STOCs (n ≥ 6; P < 0.05 each). Western blots revealed increased expression of BKCa α-subunit protein in Old. Pressure myography revealed no effect of age on SEA maximal diameter, myogenic tone, or paxilline-induced constriction (n = 10-12; P > 0.05). Enhanced functional expression of SMC BKCa-dependent STOCs in Old may represent an adaptation of resistance arteries to maintain functional integrity.

Aging increases the amplitude of spontaneous transient outward currents in murine resistance artery smooth muscle cells

Large conductance Ca2+‐activated K+ channels (BKCa) contribute to negative feedback regulation of microvascular tone. However, the effects of aging on BKCa function are unclear. The purpose of this study was to determine: 1) if enzymatically‐isolated smooth muscle cells (SMCs) from skeletal muscle resistance arteries display BKCa‐ and ryanodine receptor (RyR)‐dependent spontaneous transient outward currents (STOCs); and 2) whether aging affects STOC properties. Using perforated patch clamp recording, individual SMCs from Young (≥ 3 months) and Old (≥ 24 months) male C57BL/6 mice displayed STOCs at membrane potentials ≥ −30 mV, with the amplitude and frequency of STOCs increasing with membrane depolarization (n = 19–24; p &lt; 0.05). STOCs were inhibited by BKCa channel blockers (1 μM paxilline, 100 nM iberiotoxin) and by a RyR antagonist (100 μM tetracaine) (n ≥ 5; p &lt; 0.05 each). At +30 mV holding potential, STOC amplitude = 3.2 ± 0.32 pA/pF and frequency = 3.88 ± 0.41 Hz (n = 67) in SMCs from Young. STOC amplitude increased to 4.97 ± 0.5 pA/pF (n = 61; p &lt; 0.05) in SMCs from Old, with no significant difference in frequency (3.15 ± 0.31 Hz; p &gt;; 0.05). SMCs from murine resistance arteries display BKCa‐ and RyR‐dependent STOCs and aging increases STOC amplitude. Age‐related change in BKCa activity may impact the regulation of SMC function and contribute to age‐induced alterations in muscle blood flow regulation. (NIH R01HL086483).

Abnormal Ca2+ Spark/STOC Coupling in Cerebral Artery Smooth Muscle Cells of Obese Type 2 Diabetic Mice

Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca2+ spark and STOC activities, measured by confocal microscopy and patch-clamp technique, respectively, occur in isolated CASMCs of an experimental model of type-2 diabetes (db/db mouse). We found that mean Ca2+ spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca2+ released through Ca2+ sparks in db/db CASMCs, though Ca2+ spark frequency remained. Interestingly, the frequency of large-amplitude Ca2+ sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from −50 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel β1/α subunit ratio found in db/db vascular tissues. Taken together, Ca2+ spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK β1 subunit/α subunit ratio which underlies the significant reduction of Ca2+ spark/STOC coupling in CASMCs of diabetic animals.

Functional and electrophysiological identification of large conductance voltage‐ and Ca2+‐activated K+ (BK) channels in human urinary bladder smooth muscle

The large conductance voltage‐ and Ca2+‐activated K+ (BK or KCa1.1) channel is a key player in controlling excitability and contractility of animal urinary bladder smooth muscle (UBSM), however its role in human UBSM is unknown. Here, using the BK channel specific inhibitor‐iberiotoxin, functional studies and the perforated patch‐clamp technique, we identified the BK channel in human UBSM. In isolated UBSM strips iberiotoxin increased spontaneous phasic contraction amplitude and force (n=10). In isolated UBSM cells, voltage‐step depolarizations caused large K+ currents (∼1 nA; n=15). About 80% of these K+ currents were inhibited by iberiotoxin. In the voltage range of −40 to +40 mV, these cells also revealed spontaneous transient outward currents (STOCs). STOCs amplitude and frequency increased with increasing the holding potential toward positive values (n=20). STOCs were inhibited by iberiotoxin (n=4). In the presence of ryanodine, when the STOCs were blocked, single channels with large single channel conductance (120.6 pS) were observed (n=10). Their amplitude and open probability were voltage‐dependent. In current‐clamp mode, iberiotoxin caused membrane potential depolarization (n=4). The data reveal that the BK channel is the major voltage‐dependent K+ channel expressed in human UBSM cells that plays a key role in determining cell excitability and contractility. Supported by DK070909 to G.V.P.

Calcium Mobilization and Spontaneous Transient Outward Current Characteristics upon Agonist Activation of P2Y2 Receptors in Smooth Muscle Cells

A quantitative model is provided that links the process of metabotropic receptor activation and sequestration to the generation of inositol 1,4,5-trisphosphate, the subsequent release of calcium from the central sarcoplasmic reticulum, and the consequent release of calcium from subsarcolemma sarcoplasmic reticulum that acts on large-conductance potassium channels to generate spontaneous transient outward currents (STOCs). This model is applied to the case of STOC generation in vascular A7r5 smooth muscle cells that have been transfected with a chimera of the P2Y2 metabotropic receptor and green fluorescent protein (P2Y2-GFP) and exposed to the P2Y2 receptor agonist uridine 5′-triphosphate. The extent of P2Y2-GFP sequestration from the membrane on exposure to uridine 5′-triphosphate, the ensuing changes in cytosolic calcium concentration, as well as the interval between STOCs that are subsequently generated, are used to determine parameter values in the model. With these values, the model gives a good quantitative prediction of the dynamic changes in STOC amplitude observed upon activation of metabotropic P2Y2 receptors in the vascular smooth muscle cell line.

Ca(2+) spark sites in smooth muscle cells are numerous and differ in number of ryanodine receptors, large-conductance K(+) channels, and coupling ratio between them.

Ca(2+) sparks are highly localized Ca(2+) transients caused by Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors (RyR). In smooth muscle, Ca(2+) sparks activate nearby large-conductance, Ca(2+)-sensitive K(+) (BK) channels to generate spontaneous transient outward currents (STOC). The properties of individual sites that give rise to Ca(2+) sparks have not been examined systematically. We have characterized individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca(2+) sparks using wide-field digital microscopy and patch-clamp recording of STOC in whole cell mode. We used a signal mass approach to measure the total Ca(2+) released at a site and to estimate the Ca(2+) current flowing through RyR [I(Ca(spark))]. The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca(2+) signal mass, I(Ca(spark)), STOC amplitude, and 5-ms isochronic STOC amplitude. Sites that failed to generate STOC did so consistently, while those at the remaining sites generated STOC without failure, allowing the sites to be divided into STOC-generating and STOC-less sites. We also determined the average number of spark sites, which was 42/cell at a minimum and more likely on the order of at least 400/cell. We conclude that 1) spark sites differ in the number of RyR, BK channels, and coupling ratio of RyR-BK channels, and 2) there are numerous Ca(2+) spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored.

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C.

Localized Ca(2+) transients resulting from inositol trisphosphate (IP(3))-dependent Ca(2+) release couple to spontaneous transient outward currents (STOCs) in murine colonic myocytes. Confocal microscopy and whole cell patch-clamp techniques were used to investigate coupling between localized Ca(2+) transients and STOCs. Colonic myocytes were loaded with fluo 3. Reduction in external Ca(2+) ([Ca(2+)](o)) reduced localized Ca(2+) transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca(2+) transients and STOCs showed increased coupling strength between Ca(2+) transients and STOCs when [Ca(2+)](o) was reduced. Gd(3+) (10 microM) did not affect Ca(2+) transients but increased STOC amplitude and frequency. Similarly, an inhibitor of Ca(2+) influx, 1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C (PKC) inhibitor, GF-109203X, also increased the amplitude and frequency of STOCs but had no effect on Ca(2+) transients. Phorbol 12-myristate 13-acetate (1 microM) reduced STOC amplitude and frequency but did not affect Ca(2+) transients. 4alpha-Phorbol (1 microM) had no effect on STOCs or Ca(2+) transients. Single channel studies indicated that large-conductance Ca(2+)-activated K(+) (BK) channels were inhibited by a Ca(2+)-dependent PKC. In summary 1) Ca(2+) release from IP(3) receptor-operated stores activates Ca(2+)-activated K(+) channels; 2) Ca(2+) influx through nonselective cation channels facilitates activation of PKC; and 3) PKC reduces the Ca(2+) sensitivity of BK channels, reducing the coupling strength between localized Ca(2+) transients and BK channels.

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, 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 approximately 100 nM above a resting concentration of approximately 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 microM 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.