A novel method for direct application of phospholipids to giant excised membrane patches in the study of sodium-calcium exchange and sodium channel currents.
A novel method for direct application of phospholipids to giant excised membrane patches in the study of sodium-calcium exchange and sodium channel currents.
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一种在钠钙交换和钠通道电流研究中直接将磷脂应用于巨大切除膜片的新方法。
DOI：
10.1007/bf00374927
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Hilgemann,DW
中科院分区：
文献类型：
--
作者：
Collins,A;Hilgemann,DW
在离体的巨大心肌细胞膜上研究了膜磷脂对Na ~+-Ca ~（2+）交换和Na ~+通道电流的影响。将磷脂悬浮在α-生育酚乙酸酯和己烷的惰性载体中，然后在电流记录期间直接施加到贴片电极的侧面，距离尖端很短。磷脂酰丝氨酸强烈刺激外向Na+-Ca ~（2+）交换电流，并改变细胞质Na+和Ca ~（2+）依赖的次级调节动力学。磷脂酰胆碱可部分逆转这种作用。长期治疗与磷脂酰丝氨酸消除失活瞬态后，通常观察到的快速应用细胞质Na+，但离开细胞质Ca 2+的依赖性很大程度上完好无损。在这种情况下，随后的胰凝乳蛋白酶治疗细胞质钙依赖性，但没有进一步的刺激作用，表明最大限度地减轻磷脂酰丝氨酸的失活。虽然这些结果表明，磷脂酰丝氨酸作用于细胞质的，蛋白酶敏感的调节结构域的交换器，磷脂酰丝氨酸也刺激了交换电流后，由胰凝乳蛋白酶失调，表明对交换机制本身的影响。在其他心肌细胞的准备，心脏Na+电流在巨大的补丁进行了时间依赖性的负移的电压依赖性的稳态失活。磷脂酰丝氨酸从胞质小叶的损失（即磷脂酰丝氨酸分布的跨双层不对称性的损失）似乎不是根本原因，因为磷脂酰丝氨酸没有逆转这种转变，尽管在相同的补丁中的Na+-Ca 2+交换电流的刺激。
Effects of membrane phospholipids on Na+-Ca2+exchange and Na+channel currents were studied in giant excised cardiac sarcolemmal patches. Phospholipids were suspended in an inert vehicle ofα-tocopherol acetate and hexane and were then directly applied to the side of patch electrodes at a short distance from the tip during current recording. Phosphatidylserine strongly stimulated outward Na+-Ca2+exchange current and altered the kinetics of cytoplasmic Na+- and Ca2+-dependent secondary modulation. This effect was partially reversed by phosphatidylcholine. Prolonged treatment with phosphatidylserine eliminated the inactivation transient normally observed upon rapid application of cytoplasmic Na+but left cytoplasmic Ca2+dependence largely intact. In such cases, subsequent chymotrypsin treatment removed cytoplasmic Ca2+dependence, but had no further stimulatory effect, indicating maximum alleviation of inactivation by phosphatidylserine. While these results indicate that phosphatidylserine acts on a cytoplasmic, protease-sensitive regulatory domain of the exchanger, phosphatidylserine also stimulated the exchange current after deregulation by chymotrypsin, indicating an effect on the exchange mechanism itself. As in other myocyte preparations, cardiac Na+currents in giant patches undergo a time-dependent negative shift in the voltage dependence of steady-state inactivation. Loss of phosphatidylserine from the cytoplasmic leaflet (i.e. loss of transbilayer asymmetry of phosphatidylserine distribution) does not appear to be the underlying cause, since phosphatidylserine did not reverse this shift, despite stimulation of Na+-Ca2+exchange current in the same patches.