http://www.cnr.it/ontology/cnr/individuo/prodotto/ID188521

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

Type

Articolo in rivista (Classe)
Prodotto della ricerca (Classe)

Label

The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7 (Articolo in rivista) (literal)

Anno

2012-01-01T00:00:00+01:00 (literal)

Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#doi

10.1113/jphysiol.2012.230227 (literal)

Alternative label

Costa A; Gutla PVK; Boccaccio A; Scholz-Starke J; Festa M; Basso B; Zanardi I; Pusch M; Lo Schiavo F; Gambale F; Carpaneto A (2012)
The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7
in Journal of physiology (Lond., Print); WILEY-BLACKWELL PUBLISHING, INC, MALDEN (Stati Uniti d'America)
(literal)

Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#autori

Costa A; Gutla PVK; Boccaccio A; Scholz-Starke J; Festa M; Basso B; Zanardi I; Pusch M; Lo Schiavo F; Gambale F; Carpaneto A (literal)

Pagina inizio

3421 (literal)

Pagina fine

3430 (literal)

Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#url

http://jp.physoc.org/content/590/15/3421#target-3 (literal)

Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#numeroVolume

590 (literal)

Rivista

Journal of physiology (Rivista)

Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#numeroFascicolo

15 (literal)

Note

ISI Web of Science (WOS) (literal)

Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#affiliazioni

CNR, Ist Biofis, I-16149 Genoa, Italy; Univ Padua, I-35131 Padua, Italy; UOS Milano, Inst Biophys, CNR, I-20133 Milan, Italy (literal)

Titolo

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

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-7E245A 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. (literal)

Editore