Wiedmer T, Sims P J
J Membr Biol. 1985;84(3):249-58. doi: 10.1007/BF01871388.
The fluorescent potentiometric indicator diS-C3-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts--bearing membrane-assembled C5b67 complexes--were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (-7 mV) and ghosts (O mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential--and the consequent onset of hemolysis--is accelerated by raising external [K+], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K+] or [Na+], no change in membrane potential (from O mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from O mV by selectively increasing the K+ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was less than 5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmembrane equilibration of Na+ and K+ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (Em) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K+ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the near-equilibrium condition of potassium-containing red cells when suspended at high external K+. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.
荧光电位指示剂二磺酸-3-(5)（diS-C3-(5)）已被用于研究补体系统C5b-9膜攻击复合物组装引起的膜电位变化。通过在C8缺陷的人血清中进行免疫激活，产生了EAC1-7人红细胞和带有膜组装C5b67复合物的重封红细胞血影。对这些细胞中间体进行的研究表明，EAC1-7红细胞和血影的膜电位分别与对照红细胞（-7 mV）和血影（0 mV）没有变化。向EAC1-7红细胞中添加补体蛋白C8和C9会导致膜电位呈剂量依赖性去极化，这发生在溶血之前。这种溶血前膜电位的去极化以及随之而来的溶血开始，会因提高细胞外[K+]而加速，这表明跨膜阳离子梯度的扩散平衡是细胞溶解事件的限速因素。对于悬浮在高细胞外[K+]或[Na+]中的EAC1-7重封血影，添加C8/C9后未检测到膜电位（相对于0 mV）的变化。当用缬氨霉素选择性增加K+电导使EAC1-7血影的膜电位偏离0 mV时，添加C8和C9后观察到膜呈剂量依赖性去极化。在这些实验中，血影膜的溶解破坏小于5%。这项研究得出的结论包括：（i）测量到的红细胞唐南电位溶血前去极化直接证实了免疫溶血的胶体渗透理论。（ii）Na+和K+通过C5b-9孔的跨膜扩散平衡导致膜电位（Em）呈剂量依赖性去极化，这似乎是靶红细胞溶解破裂的限速因素。（iii）在高K+介质中观察到的增强免疫溶血不能归因于跨C5b-9孔的阳离子选择性电导，可能与高细胞外K+悬浮时含钾红细胞的接近平衡状态有关。这些实验表明，羰花青染料荧光指示剂可用于监测在细胞溶解和非细胞溶解条件下免疫损伤质膜引起的电化学变化。讨论了该方法在检测补体损伤细胞质膜亚溶解病理生理变化中的潜在应用。
