title: A conserved pore-lining glutamate as a voltage- and chloride-dependent gate in the ClC-2 chloride channel.
abstract: ClC-2 is a ubiquitously expressed, two-pore homodimeric Cl- channel opened by hyperpolarisation. Little is known about its gating mechanisms. Crystallographic and functional studies in other ClC channels suggest that a conserved glutamate residue carboxylate side-chain can close protopores by interacting with a Cl--binding site in the pore. Competition for this site is thought to provide the molecular basis for gating by extracellular Cl-. We now show that ClC-2 gating depends upon intra- but not extracellular Cl- and that neutralisation of E217, the homologous pore glutamate, leads to loss of sensitivity to intracellular Cl- and voltage. Experiments testing for transient activation by extracellular protons demonstrate that E217 is not available for protonation in the closed channel state but becomes so after opening by hyperpolarisation. The results suggest that E217 is a hyperpolarisation-dependent protopore gate in ClC-2 and that access of intracellular Cl- to a site normally occupied by its side-chain in the pore stabilises the open state. A remaining hyperpolarisation-dependent gate might correspond to that closing both pores simultaneously in other ClC channels.
authors: Centro de Estudios Científicos (CECS), Av. Arturo Prat 514, Casilla 1469, Valdivia, Chile.
authors: María Isabel
authors: MI
authors: Niemeyer
authors: L Pablo
authors: LP
authors: Cid
authors: Leandro
authors: L
authors: Zúñiga
authors: Marcelo
authors: M
authors: Catalán
authors: Francisco V
authors: FV
authors: Sepúlveda
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000494:pharmacology*; D017124:Conserved Sequence; D018698:Glutamic Acid; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D014162:Transfection
chemical_list: D018118:Chloride Channels; D002712:Chlorides; D018698:Glutamic Acid