Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis |
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Authors: | Caci Emanuela Caputo Antonella Hinzpeter Alexandre Arous Nicole Fanen Pascale Sonawane Nitin Verkman A S Ravazzolo Roberto Zegarra-Moran Olga Galietta Luis J V |
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Institution: | Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, L.go Gerolamo Gaslini 5, 16147 Genova, Italy. |
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Abstract: | CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction. |
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