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Vacuolar malate uptake is mediated by an anion‐selective inward rectifier
Author(s) -
Hafke Jens B.,
Hafke Yuliya,
Smith J. Andrew C.,
Lüttge Ulrich,
Thiel Gerhard
Publication year - 2003
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2003.01781.x
Subject(s) - inward rectifier potassium ion channel , biophysics , ion transporter , conductance , crassulacean acid metabolism , ion channel , malic acid , vesicle , chemistry , patch clamp , gating , membrane potential , biochemistry , membrane , biology , physics , photosynthesis , receptor , citric acid , condensed matter physics
Summary 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 K m of 2.5 m m malate 2− activity), a selectivity sequence of fumarate 2− > malate 2− > Cl − > maleate 2– ≈ citrate 3– , and greater activity at higher pH values (with an apparent p K 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 µ m 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 µm −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.