Uncoupling and turnover in a Cl-/H+ exchange transporter

J Gen Physiol. 2007 Apr;129(4):317-29. doi: 10.1085/jgp.200709756.

Abstract

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl(-) for one H(+) via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl(-) ion located near the center of the membrane. Mutations at this position lead to "uncoupling," such that the H(+)/Cl(-) transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl(-) gradient, but the extent and rate of this H(+) pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl(-) transport that is not linked to counter-movement of H(+), i.e., a "leak." The unitary Cl(-) transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is approximately 4,000 s(-1) for wild-type protein, and the uncoupled mutants transport Cl(-) at similar rates.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Antiporters / chemistry*
  • Antiporters / genetics
  • Antiporters / metabolism*
  • Chloride Channels / chemistry*
  • Chloride Channels / genetics
  • Chloride Channels / metabolism*
  • Chlorides / metabolism
  • Dimerization
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Liposomes / metabolism
  • Models, Chemical
  • Molecular Sequence Data
  • Mutation
  • Poisson Distribution
  • Protein Structure, Tertiary
  • Protons

Substances

  • Antiporters
  • Chloride Channels
  • Chlorides
  • ClC protein, E coli
  • Escherichia coli Proteins
  • Liposomes
  • Protons
  • chloride-base exchanger

Associated data

  • GENBANK/P37019