Separate ion pathways in a Cl-/H+ exchanger

J Gen Physiol. 2005 Dec;126(6):563-70. doi: 10.1085/jgp.200509417.


CLC-ec1 is a prokaryotic CLC-type Cl(-)/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl-. A critical glutamate residue, E148, was previously shown to be required for Cl(-)/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl- transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl- transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl- transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl- and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chloride Channels / chemistry*
  • Chloride Channels / metabolism
  • Chlorides / metabolism
  • Crystallography
  • Escherichia coli / genetics
  • Hydrogen-Ion Concentration
  • Ion Transport
  • Molecular Sequence Data
  • Mutation
  • Protein Conformation
  • Proton Pumps / chemistry*
  • Proton Pumps / metabolism


  • Chloride Channels
  • Chlorides
  • Proton Pumps