Schmid A, Burckhardt G, Gögelein H
Max-Planck-Institut für Biophysik, Frankfurt/Main, Federal Republic of Germany.
J Membr Biol. 1989 Nov;111(3):265-75. doi: 10.1007/BF01871011.
Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl- channels with a single-channel conductance of 73 +/- 2 pS in symmetrical 140 mM KCl solution (n = 25). The current-voltage relationship was linear in the range of -60 to +80 mV, but channel kinetic properties depended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10-15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2-6 msec. Clamp voltages more negative than -50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl- = Br- = I- greater than SO4(2-) approximately F-. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1.0 mM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1 mM) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl(-)-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes.
通过差速离心分离并在Percoll梯度上富集的大鼠肾皮质内吞小泡，既含有一个电生H⁺转运系统，又含有一条传导性氯离子途径。我们采用脱水/复水方法，将富集的内体小泡制剂的小泡融合，从而使其能用于膜片钳技术。在融合的小泡中，我们在对称的140 mM KCl溶液中观察到单通道电导为73±2 pS的Cl⁻通道（n = 25）。电流-电压关系在-60至+80 mV范围内呈线性，但通道动力学特性取决于钳制电位。在正电位时，可分辨出两个电导亚水平，通道的平均开放时间为10 - 15毫秒。在负电压时，只能分辨出一个亚状态，平均开放时间降至2 - 6毫秒。钳制电压比-50 mV更负会导致通道可逆失活。该通道对阴离子的选择性高于阳离子。离子替代实验揭示了阴离子通透性顺序为Cl⁻ = Br⁻ = I⁻＞SO₄²⁻≈F⁻。葡萄糖酸盐、甲磺酸盐和环己基氨基磺酸盐不可通透。阴离子通道阻滞剂4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS，1.0 mM）和5-硝基-2-(3-苯丙基氨基)-苯甲酸（NPPB，0.1 mM）完全抑制通道活性。与从放射性标记的Cl⁻通量测量以及内吞小泡悬浮液中H⁺泵活性研究获得的数据进行比较表明，这里描述的通道参与了在ATP驱动的H⁺摄入内体过程中维持电中性。
