Uncoupling and turnover in a Cl-/H+ exchange transporter.
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.
Walden
Michael
M
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA.
Accardi
Alessio
A
Wu
Fang
F
Xu
Chen
C
Williams
Carole
C
Miller
Christopher
C
D019943:Amino Acid Substitution; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D016012:Poisson Distribution; D017434:Protein Structure, Tertiary; D011522:Protons
D017920:Antiporters; D018118:Chloride Channels; D002712:Chlorides; C508370:ClC protein, E coli; D029968:Escherichia coli Proteins; D008081:Liposomes; D011522:Protons; C093920:chloride-base exchanger