Anno2012
AutoriCosta A; Gutla PVK; Boccaccio A; Scholz-Starke J; Festa M; Basso B; Zanardi I; Pusch M; Lo Schiavo F; Gambale F; Carpaneto A
AbstractFunctional 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.
RivistaJournal Of Physiology
ISSN0022-3751
Impact factor
Volume590
Pagina inizio3430
Pagina fine3421
Autori IBFFranco GAMBALE, Armando CARPANETO, Michael PUSCH, Barbara BASSO POZZI, Paul Vijay Kanth GUTLA, Ilaria ZANARDI, Joachim Johannes SCHOLZ STARKE, Margherita FESTA
Linee di Ricerca IBFMD.P01.001.001, MD.P01.005.001, MD.P01.009.001
Sedi IBFIBF.GE, IBF.MI