The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7.
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-7(E245A) is 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.
University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy.
Alex
A
Costa
Paul Vijay Kanth
PV
Gutla
Anna
A
Boccaccio
Joachim
J
Scholz-Starke
Margherita
M
Festa
Barbara
B
Basso
Ilaria
I
Zanardi
Michael
M
Pusch
Fiorella Lo
FL
Schiavo
Franco
F
Gambale
Armando
A
Carpaneto
D000818:Animals; D017360:Arabidopsis; D018118:Chloride Channels / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D018515:Plant Leaves; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014617:Vacuoles / Q000502:physiology*; D014981:Xenopus
D018118:Chloride Channels; C490802:Clcn7 protein, rat; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins