Plant and Cell Physiology Advance Access originally published online on August 2, 2008
Plant and Cell Physiology 2008 49(9):1316-1330; doi:10.1093/pcp/pcn107
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Characterization of the TaALMT1 Protein as an Al3+-Activated Anion Channel in Transformed Tobacco (Nicotiana tabacum L.) Cells
1 Key laboratory of Vegetation and Environmental Change, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, P.R. China
2 CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia
3 Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki, Okayama, 710-0046 Japan
4 School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
*Corresponding author: E-mail, steve.tyerman{at}adelaide.edu.au; Fax, +618 83037116.
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Abstract |
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TaALMT1 encodes a putative transport protein associated with Al3+-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 malate2– 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 Al3+. This current was sensitive to the anion channel antagonist niflumate, but insensitive to Gd3+. External AlCl3 (50 µM), but not La3+ and Gd3+, increased the inward current in TaALMT1-transformed protoplasts. The inward current was highly selective to malate over nitrate and chloride (Pmal >> PNO3 
PCl, Pmal/PCl 18, +/–Al3+), 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 Al3+-activated malate2– channel. We show that the Al3+-activated currents measured in TaALMT1-transformed tobacco cells are identical to the Al3+-activated currents observed in the root cells of wheat, indicating that TaALMT1 alone is likely to be responsible for those endogenous currents.
Keywords: ALMT1 - Anion channel - Aluminium - Malate - Al3+-gated - Patch-clamp
Abbreviations: 2,4-D, 2,4-Dichlorophenoxyacetic acid; BSA, bovine serum albumin; EGTA, glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid; Erev, reversal potential; ET8, aluminium tolerant line of wheat; G, conductance; GHK, Goldman–Hodgkin–Katz; I/V curve, current voltage curve; If, final current; Ii, initial current; Im, membrane current; PCl, permeability to Cl–; Pmal, permeability to malate2–; PNO3, permeability to NO3–; PVP, polyvinylpyrrolidone; TEA, tetraethylammonium; Vm, membrane voltage
(Received July 1, 2008; Accepted July 26, 2008)
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