Identification of a new malate channel serving nocturnal vacuolar malate influx in the cam-plant Kalanchoe daigremontiana

Abstract

We examined the electrical properties of the tonoplast from isolated vacuoles of Kalanchoe diagremontiana and detected in whole-vacuole patch clamp measurements a specific conductance with a high selectivity for anions. Changing the anion composition of the cytoplasmic medium we found a shift of the equilibrium voltage, which was consistent with the activity of an anion selective conductance. The following selectivity sequence was obtained for this transporter: fumurate > malate » chloride > maleate » citrate. One prominent feature of this anion conductance was its voltage dependency showing a strong rectification with the result that significant conductance was only present at voltages more negative than about 0 mV. This means that the conductance preferentially catalyses passive malate influx from the cytoplasm into the vacuole. High resolution recordings allowed identification of a single channel with a unitary conductance of 3 pS which has the same selectivity and voltage dependency as the macroscopic current, i.e. the aforementioned anion conductance is indeed due to the activity of a low conductance anion channel. Considering the measured malate current density at physiological tonoplast voltages and the geometry of the vacuoles we can estimate that the malate fluxes through this channel are sufficient to account for the malate accumulation in CAM. The selectivity sequence determined in the patch clamp assay is very similar with that reported from experiments using radio-tracer fluxes. An estimation of the affinity of the channel for malate in the electrical assay (3 mM) resembles that determined in biochemical transport assays (3-5 mM). The channel was blocked by the anion channel inhibitor niflumic acid. The same pharmacological sensitivity was obtained in biochemical transport assays for malate accumulation into isolated vacuoles.