Korn S J, Horn R
Neurosciences Department, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
J Gen Physiol. 1989 Nov;94(5):789-812. doi: 10.1085/jgp.94.5.789.
The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.
采用全细胞贴片钳技术（标准贴片和穿孔贴片配置），研究钠钙交换对电压门控钙电流衰减以及钙依赖性氯通道介导的尾电流持续时间的影响。在GH3垂体细胞中研究钙电流；在AtT - 20垂体细胞中研究钙依赖性氯电流。用四乙铵（TEA⁺）或四甲铵（TMA⁺）替代细胞外钠来抑制钠钙交换。对照实验表明，用TEA⁺替代钠不会通过与钙依赖性氯通道直接相互作用或通过阻断钠氢交换产生其效应。用标准全细胞方法研究时，钙电流和钙依赖性氯电流在5 - 20分钟内衰减。抑制钠钙交换会加速衰减。相反，当使用穿孔贴片方法时，钙电流和钙依赖性氯电流的幅度在30 - 150分钟内保持稳定。在穿孔贴片记录的前30分钟内抑制钠钙交换不会导致电流衰减。在穿孔贴片实验中，钙依赖性氯电流失活速率在长达70分钟内也保持稳定，这表明内源性钙缓冲机制保持稳定。钙依赖性氯电流的持续时间与通过电压门控钙通道的钙内流数量呈正相关，并因抑制钠钙交换而延长。对于由更大的钙内流产生的更大电流，钠钙交换对氯电流的影响更大。然而，无论钙内流情况如何，抑制钠钙交换导致的氯尾电流延长幅度都较小。从含钠浴液切换到含TEA⁺浴液后的数十到数百毫秒内，尾电流会延长。抑制钠钙交换后，尾电流衰减动力学仍然复杂。这些数据强烈表明，在完整细胞中，钠钙交换在限制钙依赖性膜电流持续时间方面起直接但非排他性的作用。此外，这些研究表明，穿孔贴片技术是研究质膜细胞质表面附近功能相关钙瞬变调节的有用方法。
