Side-dependent inhibition of a prokaryotic ClC by DIDS.
The x-ray structure of the Escherichia coli chloride/proton antiporter ClC-ec1 provides a structural paradigm for the widespread and diverse ClC family of chloride channels and transporters. To maximize the usefulness of this paradigm, it is important to directly relate structure to function via studies of ClC-ec1 itself; however, few functional studies of this protein have been performed. In an endeavor to develop new tools for functional analysis of ClC-ec1, we have discovered that this transporter is inhibited by the stilbenedisulfonate 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In planar lipid bilayers, DIDS inhibits ClC-ec1 activity reversibly, with an apparent affinity in the micromolar range. Since ClC-ec1 is randomly oriented in the bilayers, ascertaining whether DIDS inhibits from the intracellular or extracellular side required an indirect approach. Using the ClC-ec1 structure as a guide, we designed a strategy in which modification of Y445C was monitored in conjunction with inhibition by DIDS. We found that DIDS inhibits transporters specifically from the intracellular side. Transporters with their extracellular side exposed to DIDS function normally, maintaining stoichiometric proton/chloride antiport over a wide range of proton and chloride concentrations. The side-dependent nature of DIDS inhibition will be useful for generating "functionally oriented" preparations of ClC-ec1, in which DIDS is used to silence transporters in one orientation but not the other.
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.
Kimberly
K
Matulef
Merritt
M
Maduke
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D001665:Binding Sites; D001692:Biological Transport; D018118:Chloride Channels / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology*; D002712:Chlorides / Q000737:chemistry; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D011522:Protons; D013997:Time Factors
C086321:CLC-1 channel; D018118:Chloride Channels; D002712:Chlorides; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D011522:Protons; D003545:Cysteine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid