Zhang Wen-Hao, Ryan Peter R, Sasaki Takayuki, Yamamoto Yoko, Sullivan Wendy, Tyerman Steve D
Key laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, PR China.
Plant Cell Physiol. 2008 Sep;49(9):1316-30. doi: 10.1093/pcp/pcn107. Epub 2008 Aug 2.
TaALMT1 encodes a putative transport protein associated with Al(3+)-activated efflux of malate from wheat root apices. We expressed TaALMT1 in Nicotiana tabacum L. suspension cells and conducted a detailed functional analysis. Protoplasts were isolated for patch-clamping from cells expressing TaALMT1 and from control cells (empty vector transformed). With malate(2-) as the permeant anion in the protoplast, an inward current (anion efflux) that reversed at positive potentials was observed in protoplasts expressing TaALMT1 in the absence of Al(3+). This current was sensitive to the anion channel antagonist niflumate, but insensitive to Gd(3+). External AlCl(3) (50 microM), but not La(3+) and Gd(3+), increased the inward current in TaALMT1-transformed protoplasts. The inward current was highly selective to malate over nitrate and chloride (P(mal) >> P(NO3) >or= P(Cl), P(mal)/P(Cl) >or=18, +/-Al(3+)), under conditions with higher anion concentration internally than externally. The anion currents displayed a voltage and time dependent deactivation at negative voltages. Voltage ramps revealed that inward rectification was caused by the imposed anion gradients. Single channels with conductances between 10 and 17 pS were associated with the deactivation of the current at negative voltages, agreeing with estimates from voltage ramps. This study of the electrophysiological function of the TaALMT1 protein in a plant heterologous expression system provides the first direct evidence that TaALMT1 functions as an Al(3+)-activated malate(2-) channel. We show that the Al(3+)-activated currents measured in TaALMT1-transformed tobacco cells are identical to the Al(3+)-activated currents observed in the root cells of wheat, indicating that TaALMT1 alone is likely to be responsible for those endogenous currents.
TaALMT1编码一种假定的转运蛋白，该蛋白与铝（3+）激活的苹果酸从小麦根尖流出有关。我们在烟草悬浮细胞中表达了TaALMT1，并进行了详细的功能分析。从表达TaALMT1的细胞和对照细胞（空载体转化细胞）中分离原生质体用于膜片钳实验。以苹果酸（2-）作为原生质体中的通透阴离子，在无铝（3+）的情况下，在表达TaALMT1的原生质体中观察到一种内向电流（阴离子外流），该电流在正电位时反转。该电流对阴离子通道拮抗剂尼氟灭酸敏感，但对钆（3+）不敏感。外部氯化铝（50微摩尔）可增加TaALMT1转化原生质体中的内向电流，而镧（3+）和钆（3+）则无此作用。在内含阴离子浓度高于外部的条件下，内向电流对苹果酸的选择性远高于硝酸盐和氯离子（P（苹果酸）>>P（硝酸根）≥P（氯离子），P（苹果酸）/P（氯离子）≥18，±铝（3+））。阴离子电流在负电压下表现出电压和时间依赖性失活。电压斜坡显示内向整流是由施加的阴离子梯度引起的。电导在10至17皮安之间的单通道与负电压下电流的失活有关，这与电压斜坡的估计结果一致。在植物异源表达系统中对TaALMT1蛋白电生理功能的这项研究提供了首个直接证据，证明TaALMT1作为铝（3+）激活的苹果酸（2-）通道发挥作用。我们表明，在TaALMT1转化的烟草细胞中测得的铝（3+）激活电流与在小麦根细胞中观察到的铝（3+）激活电流相同，这表明TaALMT1可能单独负责那些内源电流。
