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J. Biol. Chem., Vol. 283, Issue 9, 5364-5369, February 29, 2008

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Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog^*

Silvia G. Bompadre¹, Min Li, and Tzyh-Chang Hwang

From the Dalton Cardiovascular Research Center and the Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, Missouri 65211

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N⁶-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity.

Received for publication, November 16, 2007

^* This work was supported by National Institutes of Health Grants NIHR01DK55835 and NIHR01HL53455 (to T. C. H.) and NIHK01DK075408 (to S. G. B.) and by Research Grant D06G0 from the Cystic Fibrosis Foundation (to S. G. B.). 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 text and two supplemental figures.

¹ To whom correspondence should be addressed: Dalton Cardiovascular Research Center, University of Missouri-Columbia, 134 Research Park Dr., Columbia, MO 65211. Tel.: 573-882-2271; Fax: 573-884-4232; E-mail: BompadreS{at}missouri.edu.

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