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J. Biol. Chem., Vol. 280, Issue 51, 41997-42003, December 23, 2005

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State-dependent Chemical Reactivity of an Engineered Cysteine Reveals Conformational Changes in the Outer Vestibule of the Cystic Fibrosis Transmembrane Conductance Regulator^*

From the School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are gated by binding and hydrolysis of ATP at the nucleotide-binding domains (NBDs). We used covalent modification of CFTR channels bearing a cysteine engineered at position 334 to investigate changes in pore conformation that might accompany channel gating. In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET⁺), which increases conductance, occurred only during channel closed states. This suggests that the rate of reaction of the cysteine was greater in closed channels than in open channels. R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET⁺. Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs.

Received for publication, September 19, 2005 , and in revised form, October 12, 2005.

^* This work was supported by National Institutes of Health Grant DK-056481 and American Heart Association Established Investigator Grant 0140174N (to N. A. M.). 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.

¹ To whom correspondence should be addressed: School of Biology, Georgia Institute of Technology, 310 Ferst Dr., Atlanta, GA 30332-0230. Tel.: 404-385-2955; Fax: 404-894-0519; E-mail: Nael.McCarty{at}biology.gatech.edu.

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