Obermeyer G, Armstrong F, Blatt M R
Department of Biological Sciences, University of London, Wye College, Kent, United Kingdom.
J Membr Biol. 1994 Feb;137(3):249-59. doi: 10.1007/BF00232593.
The efficacy and mechanism of alpha-dendrotoxin (DTX) block of K+ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K+ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K+ (K+o) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K+ current (IK,in), but was ineffective in blocking current through the outward-rectifying K+ channels (IK,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for IK,in, neither altering its activation at voltages negative of -120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of IK,in followed a simple titration function with an apparent K1/2 for block of 2.2 nM in 3 mM K+o. However, DTX block was dependent on the external K+ concentration. Raising K+o from 3 to 30 mM slowed block and resulted in a 60-70% reduction in its efficacy (apparent Ki = 10 mM in 10 nM DTX). The effect of K+ in protecting IK,in was competitive with DTX and specific for permeant cations. A joint analysis of IK,in block with DTX and K+ concentration was consistent with a single class of binding sites with a Kd for DTX of 240 pM. A Kd of 410 microM for extracellular K+ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K+ (K1/2 approximately 1 mM) for IK,in activation; they parallel features of K+ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K+ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K+ inward rectifier.
研究了α-树眼镜蛇毒素（DTX）对蚕豆气孔保卫细胞中钾离子通道电流的阻断作用及其机制。在电压钳制条件下，测定完整保卫细胞中内向整流钾离子通道和外向整流钾离子通道所携带的电流，并将阻断作用表征为DTX和细胞外钾离子（K⁺ₒ）浓度的函数。加入浴液中的0.1 - 30 nM DTX可阻断内向整流钾离子电流（Iꜭ,꜀ꜛ），但即使在30 nM的浓度下，对通过外向整流钾离子通道的电流（Iꜭ,ꜝꜗ）也无阻断作用。DTX阻断作用与钳制电压无关，对Iꜭ,꜀ꜛ的电压依赖性动力学也无显著影响，既不改变其在-120 mV负电压下的激活，也不改变其在更正电压下的失活。未发现DTX作用存在使用依赖性的证据。Iꜭ,꜀ꜛ的阻断遵循简单的滴定函数，在3 mM K⁺ₒ中，阻断的表观K₁/₂为2.2 nM。然而，DTX阻断作用依赖于细胞外钾离子浓度。将K⁺ₒ从3 mM提高到30 mM会减缓阻断作用，并导致其效能降低60 - 70%（在10 nM DTX中，表观Kᵢ = 10 mM）。钾离子对Iꜭ,꜀ꜛ的保护作用与DTX具有竞争性，且对渗透性阳离子具有特异性。对Iꜭ,꜀ꜛ被DTX阻断与钾离子浓度的联合分析与一类单一的结合位点一致，该位点对DTX的Kd为240 pM。同时也表明细胞外钾离子的Kd为410 μM。这些结果补充了先前的研究，这些研究表明存在一个需要细胞外钾离子（K₁/₂约为1 mM）来激活Iꜭ,꜀ꜛ的结合位点；它们与许多动物细胞中DTX和相关肽毒素对钾离子通道的阻断特征相似，证明了植物质膜钾离子通道在生理条件下对纳摩尔毒素浓度的敏感性；这些数据还突出了一个主要差异：在保卫细胞中，DTX的作用似乎对钾离子内向整流器具有特异性。
