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The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7.

Authors
  • Costa, Alex
  • Gutla, Paul Vijay Kanth
  • Boccaccio, Anna
  • Scholz-Starke, Joachim
  • Festa, Margherita
  • Basso, Barbara
  • Zanardi, Ilaria
  • Pusch, Michael
  • Schiavo, Fiorella Lo
  • Gambale, Franco
  • Carpaneto, Armando
Type
Published Article
Journal
The Journal of Physiology
Publisher
Wiley (Blackwell Publishing)
Publication Date
Aug 01, 2012
Volume
590
Issue
Pt 15
Pages
3421–3430
Identifiers
DOI: 10.1113/jphysiol.2012.230227
PMID: 22641774
Source
Medline
License
Unknown

Abstract

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.

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