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J. Biol. Chem., Vol. 283, Issue 8, 4957-4966, February 22, 2008

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Conformational Changes in a Pore-lining Helix Coupled to Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating^*

From the Laboratory of Kidney and Electrolyte Metabolism, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10–1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.

Received for publication, March 14, 2007 , and in revised form, November 28, 2007.

^* This work was supported by the NHLBI, National Institutes of Health intramural budget Grant ZO1-HL001286. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ To whom correspondence should be addressed: Laboratory of Kidney and Electrolyte Metabolism, NHLBI, National Institutes of Health, 9000 Rockville Pike, Bldg. 10, Rm. 7D13, Bethesda, MD 20892. Tel.: 301-402-1311; E-mail: raghuramv{at}mail.nih.gov.

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