Caci Emanuela, Caputo Antonella, Hinzpeter Alexandre, Arous Nicole, Fanen Pascale, Sonawane Nitin, Verkman A S, Ravazzolo Roberto, Zegarra-Moran Olga, Galietta Luis J V
Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, L.go Gerolamo Gaslini 5, 16147 Genova, Italy.
Biochem J. 2008 Jul 1;413(1):135-42. doi: 10.1042/BJ20080029.
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.
囊性纤维化跨膜传导调节因子（CFTR）是一种上皮细胞氯离子通道，噻唑烷酮化合物CFTR(inh)-172能以高亲和力和选择性对其进行抑制。在本研究中，我们提供证据表明CFTR(inh)-172直接作用于CFTR。我们在CFTR蛋白第六个跨膜螺旋的氨基酸残基中引入突变，该结构域在通道孔形成中起重要作用。将334 - 352位的碱性和亲水性氨基酸替换为丙氨酸残基，并使用功能测定法评估对CFTR(inh)-172的敏感性。我们发现347位精氨酸突变为丙氨酸会使CFTR(inh)-172的抑制效力降低20 - 30倍。将Arg347突变为其他氨基酸也会降低抑制效力，天冬氨酸会导致CFTR(inh)-172活性几乎完全丧失。本研究结果提供证据表明CFTR(inh)-172直接与CFTR相互作用，且Arg347对这种相互作用很重要。
