Site-directed fluorescence studies of a prokaryotic ClC antiporter.
Channels and transporters of the ClC family serve a variety of physiological functions. Understanding of their gating and transport mechanisms remains incomplete, with disagreement over the extent of protein conformational change involved. Using site-directed fluorescence labeling, we probe ClC-ec1, a prokaryotic ClC, for transport-related structural rearrangements. We specifically label cysteines introduced at several positions in the R helix of ClC-ec1 with AlexaFluor 488, an environment-sensitive fluorophore, and demonstrate that the labeled mutants show H+/Cl- transport activity indistinguishable from that of the wild-type protein. At each position that we examined we observe fluorescence changes upon acidification over the same pH range that is known to activate transport. The fluorescence change is also sensitive to Cl- concentration; furthermore, the Cl- and H+ dependencies are coupled as would be expected if the fluorescence change reflected a conformational change required for transport. Together, the results suggest that the changes in fluorescence report protein conformational changes underlying the transport process. Labeled transporters mutated to remove a glutamate critical to proton-coupled chloride transport retain pH-dependent fluorescence changes, suggesting that multiple residues confer pH dependence on the transport mechanism. These results have implications for models of transport and gating in ClC channels and transporters.
Bell
Susan P
SP
Membrane Transport Biophysics Unit, Porter Neuroscience Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Curran
Patricia K
PK
Choi
Sean
S
Mindell
Joseph A
JA
D001692:Biological Transport; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008301:Maleimides / Q000737:chemistry; D008954:Models, Biological; D009154:Mutation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
C504424:Alexa Fluor 488 C5-maleimide; D018118:Chloride Channels; C508370:ClC protein, E coli; D029968:Escherichia coli Proteins; D008301:Maleimides; D003545:Cysteine