Ferrera Loretta, Pincin Chiara, Moran Oscar
Istituto di Biofisica, CNR, Via De Marini 6, 16149, Genoa, Italy.
J Membr Biol. 2007 Dec;220(1-3):1-9. doi: 10.1007/s00232-007-9066-4. Epub 2007 Sep 18.
The human cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) transporter ATPases. This protein forms a Cl(-) channel with a complex regulation; gene mutations cause cystic fibrosis disease. We investigated the interaction between the protein and the flavone UCCF-029 using the patch-clamp technique in the excised inside-out configuration in order to study the molecular mechanism of action for this potentiator on completely phosphorylated channel (25 U/ml protein kinase A) and a relatively low level of ATP (0.3 mM: ). Low concentrations of UCCF-029 (<50 nM: ) increase the open probability (p (o)), favoring the channel transition to an activated state, while high UCCF-029 (>50 nM: ) levels determine inhibition of the CFTR by a reduction of the total open time. Our data suggest that this drug can potentiate CFTR by binding to a specific site on the nucleotide binding domain, promoting dimer formation. The response of CFTR to variable concentrations of ATP is not modified by application of the potentiator UCCF-029 at either low, activatory, concentration or high, inhibitory, levels. Hence, we conclude that the potentiator may not interfere with binding of ATP but probably acts at an independent site in the protein, interacting directly with CFTR to modulate channel activity.
人类囊性纤维化跨膜传导调节因子（CFTR）是三磷酸腺苷（ATP）结合盒（ABC）转运ATP酶超家族的成员。该蛋白形成一个具有复杂调节机制的氯离子通道；基因突变会导致囊性纤维化疾病。我们使用膜片钳技术，在切除的内向外构型中研究了该蛋白与黄酮UCCF - 029之间的相互作用，以探讨这种增强剂对完全磷酸化通道（25 U/ml蛋白激酶A）和相对低水平ATP（0.3 mM）作用的分子机制。低浓度的UCCF - 029（<50 nM）增加开放概率（p(o)），促使通道转变为激活状态，而高浓度的UCCF - 029（>50 nM）则通过减少总开放时间来抑制CFTR。我们的数据表明，这种药物可通过结合核苷酸结合域上的特定位点来增强CFTR，促进二聚体形成。在低浓度（激活浓度）或高浓度（抑制浓度）下应用增强剂UCCF - 029，均不会改变CFTR对不同浓度ATP的反应。因此，我们得出结论，该增强剂可能不会干扰ATP的结合，但可能作用于蛋白中的一个独立位点，直接与CFTR相互作用以调节通道活性。
