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Biophys J. 2016 Oct 4;111(7):1429-1443. doi: 10.1016/j.bpj.2016.08.030.

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes.

Author information

1
Unitat de Fisiología, Departament de Ciències Fisiològiques II, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Spain; U-750, CIBERER, ISCIII, Spain.
2
Istituto di Biofisica, CNR, Genoa, Italy.
3
ICFO-Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology, Castelldefels, Spain.
4
Unitat de Neurobiologia, Departament Patologia i Terapèutica Experimental IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat.
5
Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat.
6
Istituto di Biofisica, CNR, Genoa, Italy. Electronic address: pusch@ge.ibf.cnr.it.
7
Unitat de Fisiología, Departament de Ciències Fisiològiques II, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Spain; U-750, CIBERER, ISCIII, Spain. Electronic address: restevez@ub.edu.

Abstract

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

PMID:
27705766
PMCID:
PMC5052465
DOI:
10.1016/j.bpj.2016.08.030
[Indexed for MEDLINE]
Free PMC Article

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