title: Gating the glutamate gate of CLC-2 chloride channel by pore occupancy.
abstract: CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage-sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H(+). Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl(-), Br(-), SCN(-), and I(-)) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl(-)]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H(+) plays a minor role in dislodging the glutamate gate.
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, 78290 San Luis Potosí, México.
authors.forename: José J
authors.initials: JJ
authors.lastname: De Jesús-Pérez
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, 78290 San Luis Potosí, México.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Castro-Chong
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, R.O.C.
authors.forename: Ru-Chi
authors.initials: RC
authors.lastname: Shieh
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, 78290 San Luis Potosí, México.
authors.forename: Carmen Y
authors.initials: CY
authors.lastname: Hernández-Carballo
authors.affiliation: ReliaXpert, 78438 San Luis Potosí, México.
authors.forename: José A
authors.initials: JA
authors.lastname: De Santiago-Castillo
authors.affiliation: Physics Institute, Universidad Autónoma de San Luis Potosí, 78290 San Luis Potosí, México Arreola@dec1.ifisica.uaslp.mx.
authors.forename: Jorge
authors.initials: J
authors.lastname: Arreola
mesh_terms: D000838:Anions / Q000378:metabolism; D000090007:CLC-2 Chloride Channels; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*
chemical_list: D000838:Anions; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; D018698:Glutamic Acid