Somasundaram B, Mahaut-Smith M P
Physiological Laboratory, Cambridge, United Kingdom.
J Biol Chem. 1995 Jul 14;270(28):16638-44. doi: 10.1074/jbc.270.28.16638.
The activation of a monovalent cation current was studied in rat megakaryocytes using patch clamp techniques combined with photometric measurements of intracellular concentrations of Ca2+ ([Ca2+]i) and Na+. ADP evoked a release of [Ca2+]i and transiently activated a monovalent cation-selective channel, which, at negative potentials and under physiological conditions, would be expected to carry an inward Na+ current. The single channel conductance, estimated by noise analysis from whole cell currents at -50 to -60 mV was 9 picosiemens. Thapsigargin-induced [Ca2+]i increases failed to stimulate the monovalent cation current, suggesting that neither [Ca2+]i nor the depletion of internal Ca2+ stores were activators of this conductance. However, buffering of [Ca2+]i changes with 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid showed that both activation and inactivation of the current were accelerated by a rise in [Ca2+]i. The monovalent cation conductance was activated by internal perfusion with inositol 1,4,5-trisphosphate, both in the presence and in the absence of a rise in [Ca2+]i. Internal perfusion with inositol 2,4,5-trisphosphate, the poorly metabolizable isomer of inositol trisphosphate, similarly activated the monovalent cation current, whereas 1,3,4,5-tetrakisphosphate neither activated a current nor modified the ADP-induced monovalent current. Heparin, added to the pipette, blocked activation of the channel by ADP. The intracellular concentration of Na+, monitored by sodium-binding benzofuran isopthalate, increased by 10-20 mM in response to ADP under pseudophysiological conditions. We conclude the existence of a novel nonselective cation channel in the plasma membrane of rat megakaryocytes, which is activated by IP3 and can lead to increases in cytosolic Na+ after stimulation by ADP.
采用膜片钳技术结合对细胞内钙离子（[Ca2+]i）和钠离子浓度的光度测量，研究了大鼠巨核细胞中单价阳离子电流的激活情况。ADP引起[Ca2+]i释放，并短暂激活单价阳离子选择性通道，在负电位和生理条件下，预计该通道会携带内向钠离子电流。通过对-50至-60 mV全细胞电流进行噪声分析估计，单通道电导为9皮西门子。毒胡萝卜素诱导的[Ca2+]i升高未能刺激单价阳离子电流，这表明[Ca2+]i和细胞内钙库的耗竭均不是该电导的激活剂。然而，用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸缓冲[Ca2+]i变化表明，[Ca2+]i升高会加速电流的激活和失活。在存在和不存在[Ca2+]i升高的情况下，用肌醇1,4,5-三磷酸进行内部灌注均可激活单价阳离子电导。用肌醇三磷酸代谢不良的异构体肌醇2,4,5-三磷酸进行内部灌注，同样可激活单价阳离子电流，而1,3,4,5-四磷酸既不激活电流，也不改变ADP诱导的单价电流。加入移液管中的肝素可阻断ADP对通道的激活。在假生理条件下，用钠结合苯并呋喃间苯二甲酸监测，ADP作用下细胞内钠离子浓度升高10 - 20 mM。我们得出结论，大鼠巨核细胞质膜中存在一种新型非选择性阳离子通道，该通道由IP3激活，ADP刺激后可导致胞质钠离子增加。
