Parekh A B, Terlau H
Max-Planck-Institut für biophysikalische Chemie, Am Fassberg, D-37077 Göttingen, Germany.
Pflugers Arch. 1996 May;432(1):14-25. doi: 10.1007/s004240050100.
The regulation of capacitative Ca2+ influx in Xenopus oocytes was investigated using both the two electrode voltage-clamp (where Ca2+ is monitored through the Ca2+-dependent Cl- current) and patch-clamp techniques. Following stimulation of expressed 5-hydroxytryptamine (5-HT) receptors, capacitative Ca2+ influx deactivated in around 15 min. Following injection of [adenosine 5'-O-(3-Thiotriphosphate)] (ATP [gamma-S]), an ATP analogue that is readily used by protein kinases, capacitative Ca2+ influx activated by 5-HT application either did not deactivate or was prolonged around twofold. However, injection of adenylyl 5'-(beta,gamma-methylene)-diphosphonate (AMP-PCP), another ATP analogue that is not utilised by kinases, did not affect the time-course of Ca2+ influx. When capacitative Ca2+ influx was activated by readmission of Ca2+ to oocytes incubated in thapsigargin/0 Ca2+ solution for several hours, Ca2+ influx occurred and a weakly saturating relationship between external Ca2+ and Ca2+ influx was found. Ca2+ influx in thapsigargin-treated cells was unaffected by ATP [gamma-S]. ATP [gamma-s] and several kinases had no effect on the Ca2+-dependent Cl- current when the latter was activated by elevation of Ca2+ independent of capacitative Ca2+ influx. Protein kinase C slowly and partially inhibited the Cl- current. Outside-out patches taken from thapsigargin-treated cells failed to demonstrated any Ca2+ current or Ca2+-dependent Cl- current on reapplying high Ca2+ to the patch, despite the oocyte showing a large capacitative Ca2+ influx. The results suggest that a kinase, activated on receptor stimulation, prolongs the activation time-course of capacitative Ca2+ influx.
运用双电极电压钳（通过钙依赖氯电流监测钙离子）和膜片钳技术，研究了非洲爪蟾卵母细胞中钙池调控性钙离子内流。刺激表达的5-羟色胺（5-HT）受体后，钙池调控性钙离子内流在约15分钟内失活。注射[腺苷5'-O-(3-硫代三磷酸)]（ATP[γ-S]），一种蛋白激酶易于利用的ATP类似物后，5-HT诱导的钙池调控性钙离子内流要么不失活，要么延长约两倍。然而，注射另一种激酶无法利用的ATP类似物腺苷5'-(β,γ-亚甲基)-二磷酸酯（AMP-PCP），并不影响钙离子内流的时间进程。当将钙离子重新引入在毒胡萝卜素/无钙溶液中孵育数小时的卵母细胞，从而激活钙池调控性钙离子内流时，会发生钙离子内流，并且发现细胞外钙离子与钙离子内流之间存在弱饱和关系。毒胡萝卜素处理的细胞中的钙离子内流不受ATP[γ-S]的影响。当通过独立于钙池调控性钙离子内流的钙离子升高激活钙依赖氯电流时，ATP[γ-S]和几种激酶对该电流没有影响。蛋白激酶C缓慢且部分抑制氯电流。尽管卵母细胞显示出大量的钙池调控性钙离子内流，但从毒胡萝卜素处理的细胞获取的外侧向外膜片在重新向膜片施加高钙离子时，未能显示出任何钙离子电流或钙依赖氯电流。结果表明，受体刺激时激活的一种激酶延长了钙池调控性钙离子内流的激活时间进程。
