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J. Biol. Chem., Vol. 280, Issue 10, 8945-8950, March 11, 2005

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Location of a Common Inhibitor Binding Site in the Cytoplasmic Vestibule of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore^*

Paul Linsdell

From the Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada

Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel is inhibited by a broad range of organic anions that enter the channel pore from its cytoplasmic end, physically occluding the Cl^– permeation pathway. These open channel blocker molecules are presumed to bind within a relatively wide pore inner vestibule that shows little discrimination between different large anions. The present study uses patch clamp recording to identify a pore-lining lysine residue, Lys-95, that acts to attract large blocker molecules into this inner vestibule. Mutations that remove the fixed positive charge associated with this amino acid residue dramatically weaken the blocking effects of five structurally unrelated open channel blockers (glibenclamide, 4,4'-dinitrostilbene-2,2'-disulfonic acid, lonidamine, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and taurolithocholate-3-sulfate) when applied to the cytoplasmic face of the membrane. Mutagenesis of Lys-95 also induced amino acid side chain charge-dependent rectification of the macroscopic current-voltage relationship, consistent with the fixed positive charge on this residue normally acting to attract Cl^– ions from the intracellular solution into the pore. These results identify Lys-95 as playing an important role in attracting permeant anions into the channel pore inner vestibule, probably by an electrostatic mechanism. This same electrostatic attraction mechanism also acts to attract larger anionic molecules into the relatively wide inner vestibule, where these substances bind to block Cl^– permeation. Thus, structurally diverse open channel blockers of CFTR appear to share a common molecular mechanism of action that involves interaction with a positively charged amino acid side chain located in the inner vestibule of the pore.

Received for publication, December 21, 2004 , and in revised form, January 3, 2005.

^* This work was supported by the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 902-494-2265; Fax: 902-494-1685; E-mail: paul.linsdell{at}dal.ca.

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