title: Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protons.
abstract: The ClC transport protein family comprises both Cl(-) ion channel and H(+)/Cl(-) and H(+)/NO(3)(-) exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl(-) passage and in addition serves as a staging post for H(+) exchange. This same conserved glutamate acts as a gate to regulate Cl(-) flow in ClC channels. The activity of ClC-2, a genuine Cl(-) channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than approximately 7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly-facing histidine 532 at the N-terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification-dependent activation of ClC-2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl(-) acts as a voltage-independent modulator, as though regulating the pK(a) of the protonatable residue. Our results suggest that voltage dependence of ClC-2 is given by hyperpolarisation-dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl(-) efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts.
authors.affiliation: Centro de Estudios Científicos, Valdivia, Chile.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Niemeyer
authors.forename: L Pablo
authors.initials: LP
authors.lastname: Cid
authors.forename: Yamil R
authors.initials: YR
authors.lastname: Yusef
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Briones
authors.forename: Francisco V
authors.initials: FV
authors.lastname: Sepúlveda
mesh_terms: D000818:Animals; D017920:Antiporters / Q000378:metabolism; D000090007:CLC-2 Chloride Channels; D002460:Cell Line; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D003198:Computer Simulation; D019143:Evolution, Molecular; D018698:Glutamic Acid; D006168:Guinea Pigs; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D014162:Transfection
chemical_list: D017920:Antiporters; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; C114260:hydrogen-chloride symporter; D018698:Glutamic Acid; D006639:Histidine