Cloning, expression, and characterization of the squid Na+-Ca2+ exchanger (NCX-SQ1)
Cloning, expression, and characterization of the squid Na+-Ca2+ exchanger (NCX-SQ1)
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DOI：
10.1085/jgp.111.6.857
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发表时间：
1998-06-01
影响因子：
3.8
通讯作者：
Hilgemann, DW
中科院分区：
文献类型：
--
作者：
He, ZP;Tong, QS;Hilgemann, DW
我们已经克隆了鱿鱼神经元Na+-Ca 2+交换，NCX-SQ 1，表达在非洲爪蟾卵母细胞，其特点是巨大的切除膜补丁的监管和离子转运特性。鱿鱼交换器与犬Na+-Ca 2+交换器（NCX1.1）显示58%的同一性。被确定为在NCX 1中具有功能重要性的区域是非常保守的。迄今为止，在交换器序列中独特的是，NCX-SQ 1在跨膜区段3和4之间具有潜在的蛋白激酶C磷酸化位点（苏氨酸184），并且在Ca 2+结合区中具有酪氨酸激酶位点（酪氨酸462）。与NCX 1相比，NCX-SQ 1的大胞内环中缺失了47个氨基酸。与NCX 1类似，NCX-SQ 1在爪蟾卵母细胞中的表达诱导细胞质Na+依赖性Ca-45（2+）摄取;注射Ca 2+螯合剂抑制该摄取。在巨大的切除膜补丁，NCX-SQ 1外向交换电流显示Na+依赖性失活，二次激活细胞质Ca 2+，和激活糜蛋白酶。在F-（0.2 mM）和钒酸盐（50 μ M）存在下，ATP和ATP-硫酯ATP γ S强烈刺激NCX-SQ 1交换电流，并且在应用磷脂酰肌醇-4，5 '-二磷酸抗体时，两种作用都逆转。ATP可刺激NCX 1电流，但ATP γ S不刺激NCX 1电流。与NCX 1电流类似，磷脂酰肌醇-4 '，5'-二磷酸脂质体强烈刺激NCX-SQ 1电流。与鱿鱼轴突的结果相反，NCX-SQ 1不受磷酸精氨酸（5-10 mM）的刺激。经糜蛋白酶处理后，外向和内向NCX-SQ 1交换电流都比NCX 1电流具有更强的电压依赖性。离子浓度跳跃实验进行估计Na+和Ca 2+的运输反应的相对产电性。与Na+挤出相关的外向电流瞬变比NCX 1小得多，与Ca 2+挤出相关的内向电流瞬变大得多。对于NCX-SQ 1，钙离子转运的电荷运动可以在高细胞外钙离子（4 mM）的存在下，在低细胞质钙离子（2 μ M）的电压跳变实验中定义。电荷移动速率对电压呈“U”形依赖关系，电荷-电压和速率-电压关系的斜率（0 mV时为1，600 s（-1））表明潜在反应的表观电荷价为-0.6。显然，当离子被吸留到结合位点时，NCX-SQ 1中比NCX 1中更多的负电荷移动到膜场中。
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 Ca-45(2+) 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 mu M), 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 mu M) 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.