title: Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes.
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.
authors.affiliation: Unitat de Fisiología, Departament de Ciències Fisiològiques II, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Spain; U-750, CIBERER, ISCIII, Spain.
authors.forename: Héctor
authors.initials: H
authors.lastname: Gaitán-Peñas
authors.affiliation: Istituto di Biofisica, CNR, Genoa, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Gradogna
authors.affiliation: ICFO-Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology, Castelldefels, Spain.
authors.forename: Lara
authors.initials: L
authors.lastname: Laparra-Cuervo
authors.affiliation: Unitat de Neurobiologia, Departament Patologia i Terapèutica Experimental IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat.
authors.forename: Carles
authors.initials: C
authors.lastname: Solsona
authors.affiliation: Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat.
authors.forename: Victor
authors.initials: V
authors.lastname: Fernández-Dueñas
authors.affiliation: Unitat de Fisiología, Departament de Ciències Fisiològiques II, IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat, Spain; U-750, CIBERER, ISCIII, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Barrallo-Gimeno
authors.affiliation: Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental IDIBELL-Universitat de Barcelona, L'Hospitalet de Llobregat.
authors.forename: Francisco
authors.initials: F
authors.lastname: Ciruela
authors.affiliation: ICFO-Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology, Castelldefels, Spain.
authors.forename: Melike
authors.initials: M
authors.lastname: Lakadamyali
authors.affiliation: Istituto di Biofisica, CNR, Genoa, Italy. Electronic address: pusch@ge.ibf.cnr.it.
authors.forename: Michael
authors.initials: M
authors.lastname: Pusch
authors.affiliation: 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.
authors.forename: Raúl
authors.initials: R
authors.lastname: Estévez
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000838:Anions / Q000737:chemistry / Q000378:metabolism*; D002229:Carbenoxolone / Q000737:chemistry / Q000494:pharmacology; D005110:Extracellular Space / Q000737:chemistry / Q000187:drug effects; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000737:chemistry / Q000494:pharmacology; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration; D010539:Permeability; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D013654:Taurine / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000737:chemistry; D014981:Xenopus
chemical_list: D000838:Anions; C485976:LRRC8A protein, human; D008164:Luminescent Proteins; D008565:Membrane Proteins; D018377:Neurotransmitter Agents; D014867:Water; D013654:Taurine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D002229:Carbenoxolone