He Z, Tong Q, Quednau B D, Philipson K D, Hilgemann D W
Department of Physiology, Cardiovascular Research Laboratories, UCLA School of Medicine, Los Angeles, California 90095-1760, USA.
J Gen Physiol. 1998 Jun;111(6):857-73. doi: 10.1085/jgp.111.6.857.
We have cloned the squid neuronal Na+-Ca2+ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na+-Ca2+ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca2+ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na+-dependent 45Ca2+ uptake; the uptake was inhibited by injection of Ca2+ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na+-dependent inactivation, secondary activation by cytoplasmic Ca2+, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATP gamma S, in the presence of F- (0.2 mM) and vanadate (50 microM), and both effects reversed on application of a phosphatidylinositol-4',5'-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATP gamma S. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4',5'-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5-10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na+ and Ca2+ transport reactions. Outward current transients associated with Na+ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca2+ extrusion were much larger. For NCX-SQ1, charge movements of Ca2+ transport could be defined in voltage jump experiments with a low cytoplasmic Ca2+ (2 microM) in the presence of high extracellular Ca2+ (4 mM). The rates of charge movements showed "U"-shaped dependence on voltage, and the slopes of both charge-voltage and rate-voltage relations (1,600 s-1 at 0 mV) indicated an apparent valency of -0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.
我们克隆了鱿鱼神经元钠钙交换体NCX-SQ1，将其在非洲爪蟾卵母细胞中表达，并在巨大的膜片上对其调节特性和离子转运特性进行了表征。鱿鱼交换体与犬钠钙交换体（NCX1.1）有58%的同源性。在NCX1中确定具有功能重要性的区域高度保守。与迄今为止的交换体序列不同，NCX-SQ1在跨膜片段3和4之间有一个潜在的蛋白激酶C磷酸化位点（苏氨酸184），在钙结合区域有一个酪氨酸激酶位点（酪氨酸462）。与NCX1相比，NCX-SQ1的大细胞内环中有47个氨基酸的缺失。与NCX1类似，NCX-SQ1在非洲爪蟾卵母细胞中的表达诱导了胞质钠依赖性45钙摄取；摄取被注射钙螯合剂所抑制。在巨大的膜片中，NCX-SQ1外向交换电流表现出钠依赖性失活、胞质钙的二次激活以及胰凝乳蛋白酶的激活。在F-（0.2 mM）和钒酸盐（50 microM）存在的情况下，ATP和ATP硫酯ATPγS都能强烈刺激NCX-SQ1交换电流，且两种效应在应用磷脂酰肌醇-4',5'-二磷酸抗体后都能逆转。NCX1电流受ATP刺激，但不受ATPγS刺激。与NCX1电流一样，NCX-SQ1电流也受到磷脂酰肌醇-4',5'-二磷酸脂质体的强烈刺激。与鱿鱼轴突中的结果相反，NCX-SQ1不受磷酸精氨酸（5-10 mM）的刺激。胰凝乳蛋白酶处理后，NCX-SQ1的外向和内向交换电流比NCX1电流更强烈地依赖电压。进行了离子浓度跃变实验以估计钠和钙转运反应的相对电中性。与钠外流相关的外向电流瞬变对于NCX-SQ1比NCX1小得多，与钙外流相关的内向电流瞬变则大得多。对于NCX-SQ1，在高细胞外钙（4 mM）存在下，低胞质钙（2 microM）的电压跃变实验中可以定义钙转运的电荷移动。电荷移动速率表现出对电压的“U”形依赖性，电荷-电压和速率-电压关系的斜率（0 mV时为1600 s-1）表明基础反应的表观价为-0.6个电荷。显然，当离子被封闭到结合位点时，NCX-SQ1中比NCX1有更多的负电荷移入膜电场。
