Astill D S, Rychkov G, Clarke J D, Hughes B P, Roberts M L, Bretag A H
Centre for Advanced Biomedical Studies, University of South Australia, North Terrace, Adelaide, Australia.
Biochim Biophys Acta. 1996 Apr 26;1280(2):178-86. doi: 10.1016/0005-2736(95)00281-2.
Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.
利用杆状病毒系统，骨骼肌氯离子通道CIC-1（大鼠）以及将精氨酸304替换为谷氨酸的点突变体在培养的Sf-9昆虫细胞中高水平表达。全细胞膜片钳记录显示出大的内向整流电流，内向电流最大值可达15 nA，外向电流最大值可达2.5 nA。在电压阶跃正向至+40 mV时出现饱和现象，而在电压阶跃负向至-60 mV时产生失活电流，该电流由一个稳态成分和两个指数衰减成分组成，对于-120 mV的电压阶跃，τ1 = 6.14±0.92 ms，τ2 = 36.5±3.29 ms（标准差），n = 7。在外翻膜片配置中记录的电流常常出乎意料地大，可达同一细胞全细胞电流的5%，这表明Sf-9细胞的质膜中通道分布不均匀。研究了包括蒽-9-羧酸盐（A9C）、尼氟酸和高铼酸盐在内的多种氯离子通道阻滞剂的药理学特性，首次表明高铼酸盐是C1C-1的有效阻滞剂，且其作用机制复杂。此外，发现A9C的效力取决于外部氯离子浓度。与在肌肉细胞本身的研究一样，除了应用茚满氧基乙酸衍生物IAA94/95外，阻断作用迅速有效且易于逆转，IAA94/95起效长达10分钟，并且与细胞内作用位点一致，通过冲洗很难逆转。将位置304处高度保守的精氨酸突变为谷氨酸并没有显著改变通道的行为。
