Biophys J 89(3): 1721-1730

Side-Dependent Inhibition of a Prokaryotic ClC by DIDS

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305

Address reprint requests to Merritt Maduke, Dept. of Molecular and Cellular Physiology, B155 Beckman Center, 279 Campus Drive, Stanford, CA 94305. Tel.: 650-723-9075; Fax: 650-725-8021; E-mail: ude.drofnats@ekudam.

Received 2005 May 12; Accepted 2005 Jun 20.

Abstract

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.

Abstract

Reversal potentials under a wide range of chloride and proton concentrations and gradients. Solutions were buffered with 5 mM citrate-5 mM phosphate. V_rev “unoriented” represents the mean reversal potential ± SD (No. of bilayers) in the absence of DIDS. V_rev “oriented” represents the mean reversal potential ± SD (No. of bilayers) when 2.5 mM DIDS was added to either the cis or the trans chamber. E_Cl⁻ and E_H⁺ represent the calculated reversal potentials for Cl^{and H}^{, respectively. Activity coefficients of 0.688, 0.818, and 0.940 were used for 300 mM, 40 mM, and 3 mM Cl}^{, respectively (}45).

DIDS inhibits ClC-ec1 in a side-dependent manner. Values represent mean ± SD. Numbers in parentheses represent the number of bilayers tested. Conditions were as in Fig. 2, except in some experiments solutions were buffered with 5 mM phosphate in place of 40 mM HEPES. In each case, 2.5 mM DIDS was added to the cis chamber (300 mM KCl pH 5.0), the trans chamber (40 mM KCl pH 5.7), or both. The order of addition did not affect the amount of inhibition. The fact that the reversal potential (V_rev) does not change significantly upon addition of DIDS suggests that much of the residual current after DIDS inhibition is due to currents through ClC-ec1.

Potentiation is defined as the current after addition of MTSET divided by the current before addition of MTSET (mean ± SD). Numbers in parentheses represent the number of bilayers tested. Conditions were as in Fig. 4.

Acknowledgments

We thank Mila Gadzinski for technical assistance and Alessio Accardi and Christopher Miller for graciously donating the Y445C-ClC-ec1 construct and their adventitiously generated variant of BL21-DE3 cells found to contain low levels of contaminating porin channels. We also thank Richard Aldrich, Robert Blaustein, Anita Engh, Anthony Fodor, Weiyan Li, Gilbert Martinez, Joseph Mindell, and Richard Reimer for comments on the manuscript.

This work was supported by the Mathers Foundation and by the Esther Ehrman Lazard Faculty Scholar Award (M.M.). K.M. was supported by the Katherine McCormick Fellowship, the Stanford University School of Medicine Dean's Postdoctoral Award, and the Ruth L. Kirchstein National Research Service Award.

Acknowledgments

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