• Ion transport proteins in intracellular membranes of eukaryotic cells play key roles in many physiological and pathological processes. • The function of many of these transporters is poorly understood, because their intracellular localization makes them difficult to study. • Here, we used the large organelle of plant cells, the central vacuole, as a novel system to study an intracellular transporter from animal cells. • Our data showed that the lysosomal chloride transporter CLC-7 from rat constitutes a functional transport protein in the central vacuole of the model plant Arabidopsis thaliana (thale cress). • This novel approach has the potential to elucidate the transport properties of further, poorly studied intracellular ion channels and transporters. Functional characterization of intracellular transporters is hampered by the inaccessibility of animal endomembranes to standard electrophysiological techniques. Here, we used Arabidopsis mesophyll protoplasts as a novel heterologous expression system for the lysosomal chloride-proton exchanger CLC-7 from rat. Following transient expression of a rCLC-7:EGFP construct in isolated protoplasts, the fusion protein efficiently targeted to the membrane of the large central vacuole, the lytic compartment of plant cells. Membrane currents recorded from EGFP-positive vacuoles were almost voltage independent and showed time-dependent activation at elevated positive membrane potentials as a hallmark. The shift in the reversal potential of the current induced by a decrease of cytosolic pH was compatible with a 2Cl-/1H+exchange stoichiometry. Mutating the so-called gating glutamate into alanine (E245A) uncoupled chloride fluxes from the movement of protons, transforming the transporter into a chloride channel-like protein. Importantly, CLC-7 transport activity in the vacuolar expression system was recorded in the absence of the auxiliary subunit Ostm1, differently to recent data obtained in Xenopus oocytes using a CLC-7 mutant with partial plasma membrane expression. We also show that plasma membrane-targeted CLC-7E245Ais non-functional in Xenopus oocytes when expressed without Ostm1. In summary, our data suggest the existence of an alternative CLC-7 operating mode, which is active when the protein is not in complex with Ostm1. The vacuolar expression system has the potential to become a valuable tool for functional studies on intracellular ion channels and transporters from animal cells. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.
The Arabidopsis central vacuole as an expression system for intracellular transporters: Functional characterization of the Cl-/H+exchanger CLC-7
Carpaneto, Armando
2012-01-01
Abstract
• Ion transport proteins in intracellular membranes of eukaryotic cells play key roles in many physiological and pathological processes. • The function of many of these transporters is poorly understood, because their intracellular localization makes them difficult to study. • Here, we used the large organelle of plant cells, the central vacuole, as a novel system to study an intracellular transporter from animal cells. • Our data showed that the lysosomal chloride transporter CLC-7 from rat constitutes a functional transport protein in the central vacuole of the model plant Arabidopsis thaliana (thale cress). • This novel approach has the potential to elucidate the transport properties of further, poorly studied intracellular ion channels and transporters. Functional characterization of intracellular transporters is hampered by the inaccessibility of animal endomembranes to standard electrophysiological techniques. Here, we used Arabidopsis mesophyll protoplasts as a novel heterologous expression system for the lysosomal chloride-proton exchanger CLC-7 from rat. Following transient expression of a rCLC-7:EGFP construct in isolated protoplasts, the fusion protein efficiently targeted to the membrane of the large central vacuole, the lytic compartment of plant cells. Membrane currents recorded from EGFP-positive vacuoles were almost voltage independent and showed time-dependent activation at elevated positive membrane potentials as a hallmark. The shift in the reversal potential of the current induced by a decrease of cytosolic pH was compatible with a 2Cl-/1H+exchange stoichiometry. Mutating the so-called gating glutamate into alanine (E245A) uncoupled chloride fluxes from the movement of protons, transforming the transporter into a chloride channel-like protein. Importantly, CLC-7 transport activity in the vacuolar expression system was recorded in the absence of the auxiliary subunit Ostm1, differently to recent data obtained in Xenopus oocytes using a CLC-7 mutant with partial plasma membrane expression. We also show that plasma membrane-targeted CLC-7E245Ais non-functional in Xenopus oocytes when expressed without Ostm1. In summary, our data suggest the existence of an alternative CLC-7 operating mode, which is active when the protein is not in complex with Ostm1. The vacuolar expression system has the potential to become a valuable tool for functional studies on intracellular ion channels and transporters from animal cells. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.
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