Werth J L, Usachev Y M, Thayer S A
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA.
J Neurosci. 1996 Feb 1;16(3):1008-15. doi: 10.1523/JNEUROSCI.16-03-01008.1996.
The free intracellular Ca2+ concentration ([Ca2+]i) is governed by the balance between the activation of Ca2+ channels and buffering and efflux processes. We tested the hypothesis that Ca2+ efflux pathways are susceptible to modulation. The whole-cell patch-clamp technique was used in combination with Indo-1-based microfluorometry to record Ca2+ current and [Ca2+]i simultaneously from single rat dorsal root ganglion (DRG) neurons grown in culture. Depolarizing test pulses (-80 to 0 mV, 100-300 msec) elicited [Ca2+]i transients that recovered to basal levels by a process best-fit with a single exponential (tau = 5.1 +/- 0.4 sec; n = 14) and were independent of Ca2+ load (40-500 pC) over this range of test pulses. [Ca2+]i transients recorded in whole-cell configuration were similar to those elicited by a brief train of action potentials in unclamped neurons. Inhibition of Ca2+ sequestration into intracellular stores with thapsigargin had no effect on the kinetics of recovery. Inhibition of plasma membrane Ca2+ ATPase (PMCA) function by including a peptide inhibitor (C28R2) in the patch pipette significantly slowed recovery to basal [Ca2+]i (tau = 9.9 +/- 0.8 sec; n = 4). Preincubation with calmidazolium, a calmodulin antagonist, produced modest slowing of Ca2+ efflux. Phorbol dibutyrate, an activator of protein kinase C (PKC), accelerated Ca2+ efflux only when the PMCA had been inhibited by C28R2. We conclude that in DRG neurons PMCAs are responsible for lowering [Ca2+]i after small Ca2+ loads and that PMCA-mediated Ca2+ efflux is modulated by calmodulin- and PKC-signaling pathways.
细胞内游离钙离子浓度（[Ca2+]i）受钙离子通道激活与缓冲及外流过程之间平衡的调控。我们检验了钙离子外流途径易于被调节这一假说。采用全细胞膜片钳技术结合基于 Indo-1 的显微荧光测定法，同时记录培养的单个大鼠背根神经节（DRG）神经元的钙离子电流和[Ca2+]i。去极化测试脉冲（-80 至 0 mV，100 - 300 毫秒）引发的[Ca2+]i 瞬变通过与单指数函数最佳拟合的过程恢复到基础水平（时间常数 tau = 5.1 ± 0.4 秒；n = 14），并且在该测试脉冲范围内与钙离子负荷（40 - 500 pC）无关。在全细胞配置中记录的[Ca2+]i 瞬变与未钳制神经元中一串短暂动作电位引发的瞬变相似。用毒胡萝卜素抑制钙离子向细胞内储存库的螯合对恢复动力学没有影响。通过在膜片吸管中加入肽抑制剂（C28R2）抑制质膜钙离子 ATP 酶（PMCA）功能，显著减慢了恢复到基础[Ca2+]i 的速度（tau = 9.9 ± 0.8 秒；n = 4）。用钙调蛋白拮抗剂 calmidazolium 预孵育使钙离子外流适度减慢。佛波酯，一种蛋白激酶 C（PKC）激活剂，仅在 PMCA 被 C28R2 抑制时加速钙离子外流。我们得出结论，在 DRG 神经元中，小钙离子负荷后 PMCAs 负责降低[Ca2+]i，并且 PMCA 介导的钙离子外流受钙调蛋白和 PKC 信号通路调节。
