title: Glutamate 268 regulates transport probability of the anion/proton exchanger ClC-5.
abstract: The Cl(-)/H(+) exchange mediated by ClC transporters can be uncoupled by external SCN(-) and mutations of the proton glutamate, a conserved residue at the internal side of the protein. We show here for the mammalian ClC transporter ClC-5 that acidic internal pH led to a greater increase in currents upon exchanging extracellular Cl(-) for SCN(-). However, transport uncoupling, unitary current amplitudes, and the voltage dependence of the depolarization-induced activation were not altered by low pH values. Therefore, it is likely that an additional gating process regulates ClC-5 transport. Higher internal [H(+)] and the proton glutamate mutant E268H altered the ratio between ClC-5 transport and nonlinear capacitance, indicating that the gating charge movements in ClC-5 arise from incomplete transport cycles and that internal protons increase the transport probability of ClC-5. This was substantiated by site-directed sulfhydryl modification of the proton glutamate mutant E268C. The mutation exhibited small transport currents together with prominent gating charge movements. The charge restoration using a negatively charged sulfhydryl reagent reinstated also the WT phenotype. Neutralization of the charge of the gating glutamate 211 by the E211C mutation abolished the effect of internal protons, showing that the increased transport probability of ClC-5 results from protonation of this residue. S168P (a mutation that decreases the anion affinity of the central binding site) reduced also the internal pH dependence of ClC-5. These results support the idea that protonation of the gating glutamate 211 at the central anion-binding site of ClC-5 is mediated by the proton glutamate 268.
authors.affiliation: Institut für Neurophysiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Grieschat
authors.forename: Alexi K
authors.initials: AK
authors.lastname: Alekov
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D020125:Mutation, Missense; D011522:Protons; D013438:Sulfhydryl Compounds / Q000494:pharmacology
chemical_list: C097503:CLC-5 chloride channel; D018118:Chloride Channels; D011522:Protons; D013438:Sulfhydryl Compounds; D018698:Glutamic Acid