MECHANISM OF CARDIAC NA+-CA2+ EXCHANGE CURRENT STIMULATION BY MGATP - POSSIBLE INVOLVEMENT OF AMINOPHOSPHOLIPID TRANSLOCASE
MECHANISM OF CARDIAC NA+-CA2+ EXCHANGE CURRENT STIMULATION BY MGATP - POSSIBLE INVOLVEMENT OF AMINOPHOSPHOLIPID TRANSLOCASE
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DOI：
10.1113/jphysiol.1992.sp019254
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发表时间：
1992-08-01
影响因子：
5.5
通讯作者：
COLLINS, A
中科院分区：
文献类型：
--
作者：
HILGEMANN, DW;COLLINS, A
1.在豚鼠和兔心肌细胞膜片上，观察外向Na~+-Ca~(2+)交换电流对带电双亲分子和磷脂的敏感性。二甲胺(10 MM)、精胺(200-mU-M)和亚精胺(100-mU-M)对膜表面电位的筛选没有影响，而带正电荷的离子去污剂十六烷基三甲基铵和十二烷基三甲基铵对稳态外向交换电流(0.1-10-mU-M)有较强的抑制作用。预计会增加表面负电荷的干预措施包括用磷脂酶D处理细胞质表面，应用十二烷基硫酸盐(1-10-MU-m)，应用短链磷脂酰丝氨酸衍生物，二丙基磷脂酰丝氨酸(C10PS)，以及在用于覆盖电极的碳氢化合物混合物中加入1-3%的磷脂酰丝氨酸。每种干预措施都以与镁三磷酸腺苷类似的方式强烈刺激Na+-Ca~(2+)交换电流，减少外向交换电流(失活)在施加高细胞质钠时的部分衰减。K(I)约为1mU-m时，该电流可被戊氨酸抑制。已知与磷脂酰丝氨酸头基有关。胰凝乳酶对该交换体进行“去调控”后，戊氨酸不起作用。在吸管中加入0-2 mM的吡啶二硫代乙胺(一种氨基磷脂转位酶的氧化抑制剂)，可在不抑制基础外向交换电流或钠泵电流的情况下阻断镁三磷酸腺苷对外向交换电流的刺激。在细胞质一侧应用1.5 mM-DIAME，据报道减少了膜磷脂的不对称性，明显地逆转了用DIAME处理后和随后用二硫苏糖醇处理后的效果。Na+-Ca~(2+)交换电流再次被镁-三磷酸腺苷激活。当次级交换调节先前被胰凝乳酶去除时，联胺不起作用。由表面电位敏感的离子载体携带的钾电流。当胞外表面电荷被中和后，非肌动蛋白被镁三磷酸腺苷激活。当使用低离子强度的细胞质溶液时，效果最大(40-90%)。与镁-三磷酸腺苷处理过程中细胞质侧膜负电荷增加一致。当细胞质表面电荷被中和并使用低离子强度的胞外液时，镁ATP抑制非肌动蛋白携带的钾电流，这与胞外膜负电荷的减少一致。这些结果表明，镁三磷酸腺苷对Na~+-Ca~(2+)交换电流的刺激作用可能与荷电膜脂的变化有关，这种作用可能与跨膜有关。氧化敏感蛋白，涉及戊氨酸敏感部位，磷脂酰丝氨酸模拟镁ATP的作用，该作用扩展到简单的表面电位敏感的离子载体。所有结果都是一致的，镁三磷酸腺苷激活氨基磷脂转位酶是潜在的机制。
1. The sensitivity of outward Na+-Ca2+ exchange current to charged amphiphiles and phospholipids was tested in giant excised inside-out membrane patches from guinea-pig and rabbit myocytes.2. Screening of membrane surface potentials with dimethonium (10 mm), spermine (200-mu-M) and spermidine (100-mu-M) was without effect, while the positively charged ionic detergents hexadecyltrimethylammonium and dodecyltrimethylammonium strongly inhibited steady-state outward exchange current (0.1-10-mu-M).3. Interventions expected to increase negative surface charge included treatment of the cytoplasmic surface with phospholipase D, application of dodecylsulphate (1-10-mu-m), application of the short-chain phosphatidylserine derivative, dicapryl phosphatidylserine (C10PS), and inclusion of 1-3 % phosphatidylserine in the hydrocarbon mixture used to coat electrodes. Each intervention strongly stimulated Na+-Ca2+ exchange current in a similar way to MgATP, reducing the fractional decay of outward exchange current (inactivation) during application of high cytoplasmic sodium.4. The MgATP-stimulated exchange current was inhibited with a K(i) of approximately 1-mu-m by pentalysine. which is known to associate with phosphatidylserine head groups. After 'deregulation ' of the exchanger by chymotrypsin, pentalysine was without effect.5. Inclusion in the pipette of 0-2 mm-pyridyldithioethylamine (an oxidizing inhibitor of aminophospholipid translocase) abolished stimulation of outward exchange current by MgATP without inhibiting basal outward exchange current or sodium pump current.6. Application to the cytoplasmic side of 1.5 mM-diamide, which reportedly decreases membrane phospholipid asymmetry, apparently reversed the effect of MgATP.- After treatment with diamide and subsequently with dithiothreitol. Na+-Ca2+ exchange current was again stimulated by MgATP. Diamide was without effect when secondary exchange regulation had been previously removed by chymotrypsin.7. Potassium current carried by the surface potential-sensitive ionophore. nonactin, was stimulated by MgATP when extracellular surface charge had been neutralized. The effect was largest (40-90 %) when low ionic strength cytoplasmic solutions were employed. consistent with an increase of negative membrane charge on the cytoplasmic side during MgATP application.8. Potassium current carried by nonactin was inhibited by MgATP when cytoplasmic surface charge had been neutralized and extracellular solutions of low ionic strength were employed, consistent with a decrease of negative membrane charge on the extracellular side.9. These results indicate that the stimulatory effect of MgATP on Na+-Ca2+ exchange current could involve changes of charged membrane lipids, that the effect probably involves a transmembrane. oxidation-sensitive protein, that pentalysine-sensitive sites are involved, that phosphatidylserine mimics the effect of MgATP, and that the effect extends to a simple surface potential-sensitive ionophore. All results are consistent, with the activation by MgATP of an aminophospholipid translocase as the underlying mechanism.