Hafke Jens B, Hafke Yuliya, Smith J Andrew C, Lüttge Ulrich, Thiel Gerhard
Institut für Botanik, Technische Universität Darmstadt, Schnittspahnstr. 3, 64287 Darmstadt, Germany.
Plant J. 2003 Jul;35(1):116-28. doi: 10.1046/j.1365-313x.2003.01781.x.
Electrophysiological studies using the patch-clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current-voltage relations of the tonoplast were dominated by a strongly inward-rectifying anion-selective channel that was active at cytoplasmic-side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage-dependent gating of a 3-pS malate-selective ion channel, which showed a half-maximal open probability at -43 mV. Also, the time-averaged unitary currents following a step to a negative voltage corresponded to the time-dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion-selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent Km of 2.5 mm malate2- activity), a selectivity sequence of fumarate2- > malate2- > Cl- > maleate2- approximately citrate3-, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 microM niflumate reversibly blocked the activity of the 3-pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 microm-2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3-pS, inward-rectifying, anion-selective channel represents the principal pathway for malate influx into the vacuole.
利用膜片钳技术对景天酸代谢（CAM）植物落地生根叶片叶肉细胞分离出的液泡进行了电生理研究，以表征负责夜间苹果酸积累的苹果酸转运系统。在膜两侧均存在苹果酸的情况下，液泡膜的电流-电压关系主要由一个强内向整流阴离子选择性通道主导，该通道在细胞质侧负电压时活跃。宏观电导的整流反映在一个3皮安的苹果酸选择性离子通道的电压依赖性门控上，该通道在-43毫伏时显示出半数最大开放概率。此外，向负电压阶跃后的时间平均单位电流与宏观电流的时间依赖性动力学相对应，表明该通道的活性是阴离子选择性内向整流器的基础。内向整流器对苹果酸表现出饱和动力学（苹果酸2-活性的表观Km为2.5毫米），选择性顺序为富马酸2-＞苹果酸2-＞Cl-＞马来酸2-≈柠檬酸3-，并且在较高pH值下活性更高（表观pK为7.1，在pH约8.0时活性最大）。所有这些特性与在分离的液泡膜囊泡中观察到的苹果酸转运特性密切一致。此外，100微摩尔的尼氟酸可逆地阻断了3皮安通道的活性，并同等程度地抑制了宏观电流和苹果酸向液泡膜囊泡的转运。在生理电压下记录的宏观电流密度和估计的通道密度0.2微米-2足以解释该CAM植物中观察到夜间苹果酸积累速率，这表明3皮安的内向整流阴离子选择性通道代表了苹果酸流入液泡的主要途径。
