Pérez-Samartín A L, Miledi R, Arellano R O
Departamento de Neurociencias, Universidad del País Vasco, 48940 Leioa (Vizcaya), España.
J Physiol. 2000 Jun 15;525 Pt 3(Pt 3):721-34. doi: 10.1111/j.1469-7793.2000.00721.x.
Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique. In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application. In follicles, the permeability ratios for different anions with respect to Cl- were similar for both ICl,swell and Sin, with a sequence of: SCN- > I- > Br- >= NO3- >= Cl- > gluconate >= cyclamate > acetate > SO42-. Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was approximately 100 % slower at pH 8.0 compared with that at pH 6.0. Lanthanides inhibited ICl, swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 +/- 1.9 microM, while Sin was blocked up to 55 % with an apparent IC50 of 36 +/- 2.6 microM. Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 +/- 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to -20 mV. This single-channel activity was increased by application of ACh or ATP. The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling. All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity, strongly suggest that ICl,swell and Sin currents depend on the opening of the same type or a closely related class of volume-regulated chloride channels.
采用电生理技术研究了卵泡包被的非洲爪蟾卵母细胞（卵泡）的渗透压依赖性离子电流。使用双电极电压钳监测整个卵泡电流，采用膜片钳技术测量单通道活性。在保持于 -60 mV 的卵泡中，在外部低渗溶液中激活了两种氯离子电流。一种是由外部低渗引起的习惯性容积调节电流（ICl,swell），另一种是通过施加乙酰胆碱或 ATP 产生的缓慢且平稳的电流（Sin）。在卵泡中，对于 ICl,swell 和 Sin，不同阴离子相对于 Cl- 的通透率相似，顺序为：SCN- > I- > Br- >= NO3- >= Cl- > 葡萄糖酸盐 >= 环己基氨基磺酸盐 > 乙酸盐 > SO42-。细胞外 ATP 阻断了 Sin 的外向成分。此外，细胞外 pH 调节了由乙酰胆碱引起的 Sin 的失活动力学；例如，在 +80 mV 时，pH 8.0 时的失活比 pH 6.0 时慢约 100%。镧系元素抑制 ICl,swell 和 Sin。La3+ 完全抑制 ICl,swell，半数最大抑制浓度（IC50）为 17 ± 1.9 μM，而 Sin 被阻断高达 55%，表观 IC50 为 36 ± 2.6 μM。卵泡细胞的膜片钳记录显示，低渗刺激打开内向单通道电流。单通道电导（4.7 ± 0.4 pS）具有线性电流 - 电压关系，反转膜电位接近 -20 mV。施加乙酰胆碱或 ATP 可增加这种单通道活性。ICl,swell 的产生不受哌仑西平或甲溴东莨菪碱的影响，也不影响名为 Fin 的氯离子电流的嘌呤能激活。因此，ICl,swell 不是通过细胞肿胀期间释放的神经递质产生的。总之，对不同阴离子的同等区分、细胞外 pH 的相似调节作用、ATP 和 La3+ 的阻断作用以及相同的单通道活性，强烈表明 ICl,swell 和 Sin 电流取决于同类型或密切相关类别的容积调节氯离子通道的开放。
