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J. Biol. Chem., Vol. 282, Issue 12, 9098-9104, March 23, 2007

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Functional Analysis of Mutations in the Putative Binding Site for Cystic Fibrosis Transmembrane Conductance Regulator Potentiators

INTERACTION BETWEEN ACTIVATION AND INHIBITION^*

Olga Zegarra-Moran¹, Martino Monteverde, Luis J. V. Galietta, and Oscar Moran

From the Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Largo Gerolamo Gaslini 5, I-16148 Genova, Italy and the Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, I-16149 Genova, Italy

An increasing number of compounds able to potentiate the activity of mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been identified by high throughput screening or by individual search of derivatives of known active compounds. Several lines of evidence suggest that most CFTR potentiators act through the same mechanism, probably by binding to the nucleotide binding domains to promote the activity of the protein and then, with lower affinity, to an inhibitory site. With the aim of identifying the activating binding site, we recently modeled the nucleotide binding domain dimer and predicted a common binding site for potentiators in its interface. To validate this model experimentally, we mutated some of the residues involved in the putative binding site, i.e. Arg⁵⁵³, Ala⁵⁵⁴, and Val¹²⁹³. The activity of CFTR potentiators was measured as apical membrane currents on polarized cells stably expressing wild type or mutated proteins. CFTR activity was elicited by application of a membrane-permeable cAMP analogue followed by increasing concentrations of potentiators. We found that all three mutants responded to cAMP, although the affinity of R553Q was higher than that of wild type CFTR. In R553Q and V1293G mutants, the dissociation constant of potentiators for the activating site was increased, whereas the dissociation constant for the inhibitory site was reduced. Our results show that the mutated residues are part of the activating binding site for potentiators, as suggested by the molecular model. In addition, these results suggest that the activating and inhibitory sites are not independent of each other.

Received for publication, December 13, 2006 , and in revised form, January 22, 2007.

^* This work was supported by grants from the Italian Cystic Fibrosis Foundation (FFC 6/2003 and FFC 4/2006). 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. Tel.: 390105636403; Fax: 390103779797; E-mail: ozegarra{at}unige.it.

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