Allen G J, Sanders D
Department of Biology, University of York, UK.
Plant J. 1996 Dec;10(6):1055-69. doi: 10.1046/j.1365-313x.1996.10061055.x.
Activity of vacuolar ion channels can be regulated by the cytosolic free Ca2+ concentration ([Ca2+]cyt). Using the whole-vacuole mode of patch-clamp with Vicia faba guard cell vacuoles, three distinct cation currents were apparent that were differentially regulated by [Ca2+]cyt. At 'zero' to 100 nM [Ca2+]cyt, instantaneous currents typical of Fast Vacuolar (FV) channels were activated. A 10 fold KCl gradient directed out of the vacuole increased FV currents (up to fivefold) at negative potentials compared with the currents in symmetrical KCl. At [Ca2+]cyt higher than 100 nM, instantaneous currents became smaller and voltage-independent (non-rectifying) and were typical of Vacuolar K(+)-selective (VK) channels. These currents were less sensitive to a KCl gradient than were the FV currents, being stimulated less than twofold at negative potentials. Reversal potentials measured in the presence of a KCl gradient indicated a high K+ permeability of both FV and VK currents. At [Ca2+]cyt higher than 600 nM time-dependent currents elicited by positive potentials were typical of Slow Vacuolar (SV) channel activation. When the Ca2+ mole fraction in the cytosolic or luminal solution was varied the reversal potential of SV currents (determined by tail current analysis) passed through maximum or minimum values. The resultant calculated apparent permeability ratios varied with ionic conditions but indicated high Ca2+ and K+ permeabilities. If a Cl- permeability was assumed then the apparent PCa was lower. However, substitution of Cl- by the larger (impermeant) anion gluconate had no effect on the reversal potential of SV tail currents in the presence of Ca2+ and a K+ gradient, demonstrating that the assumption of Cl- permeability of the SV channel is invalid. Single-channel SV currents also decreased with increasing cytosolic Ca2+ mole fraction. These data indicate that the SV channel is highly cation selective, shows characteristics typical of a multi-ion pore and derives ion selectivity by Ca2+ binding. The SV channel currents could also be Mg(2+)-activated and were demonstrated to be Mg(2+)-permeable in the absence of Ca2+. The apparent permeability ratio (PMg:PK) also varied under different ionic conditions. The results indicate not only that FV, VK and SV channels are all present in a single cell type, but also that each is differentially regulated by [Ca2+]cyt. The respective roles of these channels in vacuolar ion release are discussed, and possible conditions are presented in which these channels could be activated by disparate signalling pathways during stomatal closure.
液泡离子通道的活性可受胞质游离钙离子浓度（[Ca2+]cyt）的调节。利用蚕豆保卫细胞液泡的膜片钳全液泡模式，可明显观察到三种不同的阳离子电流，它们受[Ca2+]cyt的调节方式存在差异。在“零”至100 nM的[Ca2+]cyt浓度下，快速液泡（FV）通道典型的瞬时电流被激活。与对称KCl溶液中的电流相比，从液泡向外的10倍KCl梯度在负电位时可增加FV电流（高达五倍）。当[Ca2+]cyt高于100 nM时，瞬时电流变小且与电压无关（非整流），这是液泡K+选择性（VK）通道的典型特征。这些电流对KCl梯度的敏感性低于FV电流，在负电位时其刺激增加不到两倍。在存在KCl梯度的情况下测得的反转电位表明FV和VK电流都具有较高的K+通透性。当[Ca2+]cyt高于600 nM时，正电位引发的时间依赖性电流是慢液泡（SV）通道激活的典型特征。当胞质或液泡内溶液中的Ca2+摩尔分数发生变化时，SV电流的反转电位（通过尾电流分析确定）会经过最大值或最小值。由此计算出的表观渗透率比值随离子条件而变化，但表明Ca2+和K+具有较高的通透性。如果假设存在Cl-通透性，则表观PCa较低。然而，在存在Ca2+和K+梯度的情况下，用较大的（不可渗透的）阴离子葡萄糖酸盐替代Cl-对SV尾电流的反转电位没有影响，这表明假设SV通道具有Cl-通透性是无效的。单通道SV电流也随着胞质Ca2+摩尔分数的增加而减小。这些数据表明，SV通道具有高度的阳离子选择性，显示出多离子孔的典型特征，并通过Ca2+结合获得离子选择性。SV通道电流也可被Mg(2+)激活，并且在没有Ca2+的情况下被证明是Mg(2+)可渗透的。表观渗透率比值（PMg:PK）在不同的离子条件下也有所变化。结果表明，不仅FV、VK和SV通道都存在于单一细胞类型中，而且每种通道都受[Ca2+]cyt的差异调节。讨论了这些通道在液泡离子释放中的各自作用，并提出了在气孔关闭期间这些通道可能被不同信号通路激活的可能条件。
