Shorofsky S R, Izu L, Wier W G, Balke C W
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201, USA.
Circ Res. 1998 Mar 9;82(4):424-9. doi: 10.1161/01.res.82.4.424.
The goal of this study was to examine the relationship between Ca2+ entry through L-type Ca2+ channels and local [Ca2+]i transients (Ca2+ sparks) in single rat cardiac ventricular cells. L-type Ca2+ channels were activated by depolarization of cell-attached membrane patches, and [Ca2+]i was measured simultaneously as fluo 3 fluorescence using laser scanning confocal microscopy. Patch depolarization with Ca2+ as the charge carrier (10 or 110 mmol.L(-1)) significantly increased the probability of the occurrence of Ca2+ sparks (Ca2+ spark rate) only in the volume of cytoplasm located immediately beneath the membrane patch (basal Ca2+ spark rate, 119 Ca2+ sparks.cell(-1).s(-1); patch depolarization Ca2+ spark rate, 610 Ca2+ sparks.cell(-1).s(-1); P<.005). With Ba2+ in the pipette solution (10 mmol.L(-1)), patch depolarization was not associated with an increased Ca2+ spark rate at the position of the pipette or at any other sites distant from the pipette. Therefore, Ca2+ entry and not voltage per se was a necessary event for the occurrence of Ca2+ sparks. Under identical experimental conditions, patch depolarization experiments opened single L-type Ca2+ channels with a single-channel conductance of 19 pS with Ba2+ as the charge carrier. Although single-channel openings could not be resolved when Ca2+ was the charge carrier, ensemble averages yielded an inward current of up to 0.75 pA. The results suggest that voltage-activated Ca2+ entry through one or a small number of L type Ca2+ channels triggers the release of Ca2+ only from the sarcoplasmic reticulum in direct proximity to those L-type Ca2+ channels. The relatively low probability of triggering Ca2+ sparks may have resulted from some alteration of excitation-contraction coupling associated with the technique of the cell-attached patch clamp.
本研究的目的是检测在单个大鼠心室肌细胞中，通过L型钙通道的Ca2+内流与局部[Ca2+]i瞬变（Ca2+火花）之间的关系。通过对细胞贴附膜片进行去极化来激活L型钙通道，同时使用激光扫描共聚焦显微镜以fluo 3荧光测量[Ca2+]i。以Ca2+作为电荷载体（10或110 mmol·L(-1)）进行膜片去极化，仅在紧邻膜片下方的细胞质区域显著增加了Ca2+火花出现的概率（基础Ca2+火花率，119次Ca2+火花·细胞(-1)·秒(-1)；膜片去极化Ca2+火花率，610次Ca2+火花·细胞(-1)·秒(-1)；P<0.005）。当移液管溶液中含有Ba2+（10 mmol·L(-1)）时，膜片去极化与移液管位置或移液管远处任何其他位点的Ca2+火花率增加无关。因此，Ca2+内流而非电压本身是Ca2+火花发生的必要事件。在相同实验条件下，以Ba2+作为电荷载体进行膜片去极化实验，可打开单通道电导为19 pS的单个L型钙通道。虽然以Ca2+作为电荷载体时无法分辨单通道开放，但总体平均值产生高达0.75 pA的内向电流。结果表明，通过一个或少数L型钙通道的电压激活Ca2+内流仅触发紧邻这些L型钙通道的肌浆网释放Ca2+。触发Ca2+火花的概率相对较低可能是由于与细胞贴附膜片钳技术相关的兴奋 - 收缩偶联的某些改变所致。
