Electrodiffusion, barrier, and gating analysis of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.
Electrodiffusion, barrier, and gating analysis of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.
复制标题
用vallyomycin或gramicidin处理的人红细胞中DIDS不敏感的氯化物电导的电辐射，屏障和门控分析。
DOI：
10.1085/jgp.109.2.201
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发表时间：
1997-02
影响因子：
3.8
通讯作者：
Novak, T S
中科院分区：
文献类型：
--
作者：
Freedman, J C;Novak, T S
DIDS不敏感的Cl−电导的电流-电压曲线已在来自5名供体的人红细胞中测定。使用流式细胞术和差分激光散射从细胞收缩率估计电流。使用质子离子载体FCCP从未缓冲的悬浮液的细胞外pH估计膜电位。细胞体积的高斯分布的宽度在细胞收缩期间保持不变，表明细胞之间具有均匀的Cl−电导。用DIDS预处理30 min后，缬氨霉素诱导K+和Cl−的净流出，并在持续存在DIDS的情况下进行测量;在25°C和37°C下，抑制最大值均为高于1 μM DIDS的约65%。将缬氨霉素或短杆菌肽在不同[K+]o下诱导的DIDS不敏感净Cl−流出的非线性电流-电压曲线与基于（1）电扩散理论、（2）单势垒模型、（3）单占据、多势垒模型和（4）电压门控机制的预测进行比较。电扩散精确地描述了测量的跨膜电压和[K+]o之间的关系。在我们的实验条件下（pH 7.5，23°C，1-3 μM缬氨霉素或60 ng/ml短杆菌肽，1.2%红细胞比容），用10 μM DIDS，恒定场渗透率比PK/PCl为74 ± 9，对应于PCl的73%抑制。将恒定场电流-电压方程拟合到测量的Cl−电流，得出PCl = 0.13 h−1（使用DIDS），而没有DIDS时为0.49 h−1，与大多数先前的研究结果一致。然而，内向整流DIDS不敏感的Cl−电流与电扩散和某些单占据多势垒模型不一致。这些数据可以通过位于跨膜电场中心附近的单个屏障或电压门控通道机制得到很好的描述，根据该机制，最大电导为0.055 ± 0.005 S/g Hb，一半通道在−27 ± 2 mV时打开，等效门控电荷为−1.2 ± 0.3。
Current-voltage curves for DIDS-insensitive Cl− conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl− conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K+ and Cl− were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl− effluxes, induced by valinomycin or gramicidin at varied [K+]o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K+]o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio PK/PCl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of PCl. Fitting the constant field current-voltage equation to the measured Cl− currents yields P Cl = 0.13 h−1 with DIDS, compared to 0.49 h−1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl− current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.