Freedman J C, Novak T S, Bisognano J D, Pratap P R
Department of Physiology, State University of New York Health Science Center, Syracuse 13210.
J Gen Physiol. 1994 Nov;104(5):961-83. doi: 10.1085/jgp.104.5.961.
Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS-insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.
在存在和不存在阴离子转运抑制剂DIDS（4,4'-二异硫氰基-2,2'-二磺酸芪）及其活性较弱的类似物SITS（4-乙酰氨基-4'-异硫氰基芪-2,2'-二磺酸）的情况下，已直接测定了缬氨霉素或短杆菌肽诱导的净钾离子和氯离子外流，外部钾离子浓度用[K]o表示，且浓度各不相同。结果证实，在23℃下用10微摩尔/升DIDS或100微摩尔/升SITS预处理30分钟，在1毫摩尔/升[K]o且抑制剂持续存在的情况下，对传导性氯离子外流的抑制率仅为59 - 67%。当DIDS浓度、或DIDS预处理期间的温度或pH值升高时，1毫摩尔/升[K]o下10微摩尔/升DIDS的这种部分抑制作用保持不变。此前对这种部分抑制作用的观察促使人们推测人类红细胞中存在两条氯离子传导途径：一条由碳酸酐酶（带3蛋白）介导的对DIDS敏感的途径，以及一条对DIDS不敏感的途径。本实验表明，在[K]o对应于EK值在-35至0毫伏之间时，净氯离子外流中对DIDS不敏感的成分可忽略不计，缬氨霉素（1微摩尔/升）和短杆菌肽（0.06微克/毫升）作用下均≤0.1微摩尔/克血红蛋白/分钟。在较低的[K]o下，即EK低于约-35毫伏时，缬氨霉素（1微摩尔/升）作用下净氯离子外流中对DIDS不敏感的部分增加到2.6微摩尔/克血红蛋白/分钟，短杆菌肽（0.06微克/毫升）作用下增加到4.8微摩尔/克血红蛋白/分钟。根据平均细胞体积变化确定净通量，并根据未缓冲红细胞悬液外部pH值变化测量膜电位，推导出了对DIDS不敏感的氯离子传导的电流-电压曲线。虽然不同的[K]o对净氯离子外流的具体影响不太可能，但也不能完全排除，结果与以下假设一致：对DIDS不敏感的氯离子传导在Em约为-40毫伏时开启。
