Scott-Ward T S, Li H, Schmidt A, Cai Z, Sheppard D N
Department of Physiology, School of Medical Sciences University of Bristol University Walk, BS8 1TD Bristol, UK.
Mol Membr Biol. 2004 Jan-Feb;21(1):27-38. doi: 10.1080/09687680310001597758.
Niflumic acid is widely used to inhibit Ca(2+) -activated Cl(-) channels. However, the chemical structure of niflumic acid resembles that of diphenylamine-2-carboxylate, a drug that inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. To investigate how niflumic acid inhibits CFTR Cl(-) channel, we studied recombinant wild-type human CFTR in excised inside-out membrane patches. When added to the intracellular solution, niflumic acid caused a concentration- and voltage-dependent decrease of CFTR Cl(-) current with half-maximal inhibitory concentration (K(i)) of 253 microM and Hill co-efficient of approximately 1, at -50 mV. Niflumic acid inhibition of single CFTR Cl(-) channels was characterized by a very fast, flickery block that decreased dramatically current amplitude without altering open-probability. Consistent with these data, spectral analysis of CFTR Cl(-) currents suggested that channel block by niflumic acid was described by the closed <--> open <--> blocked kinetic scheme with blocker on rate (k(on)) = 13.9 x 10(6) M(-1)s(-1), off rate (k(off))=3348 s(-1) and dissociation constant (K(d)) = 241 microM, at -50 mV. Based on these data, we tested the effects of niflumic acid on transepithelial Cl(-) secretion and cyst growth using type I MDCK epithelial cells. Niflumic acid (200 microM) inhibited cAMP-stimulated, bumetanide-sensitive short-circuit current by 55%. Moreover, the drug potently retarded cyst growth. We conclude that niflumic acid is an open-channel blocker of CFTR that inhibits Cl(-) permeation by plugging the channel pore. It or related agents might be of value in the development of new therapies for autosomal dominant polycystic kidney disease.
尼氟酸被广泛用于抑制钙激活氯离子通道。然而，尼氟酸的化学结构与二苯胺-2-羧酸盐相似，二苯胺-2-羧酸盐是一种抑制囊性纤维化跨膜传导调节因子（CFTR）氯离子通道的药物。为了研究尼氟酸如何抑制CFTR氯离子通道，我们在切除的内向外膜片中研究了重组野生型人CFTR。当添加到细胞内溶液中时，尼氟酸导致CFTR氯离子电流呈浓度和电压依赖性下降，在-50 mV时，半数最大抑制浓度（K(i)）为253 μM，希尔系数约为1。尼氟酸对单个CFTR氯离子通道的抑制表现为非常快速的闪烁阻断，这种阻断会显著降低电流幅度而不改变开放概率。与这些数据一致，CFTR氯离子电流的光谱分析表明，尼氟酸对通道的阻断可以用关闭<-->开放<-->阻断动力学方案来描述，在-50 mV时，阻断剂结合速率（k(on)）= 13.9×10(6) M(-1)s(-1)），解离速率（k(off)）=3348 s(-1)，解离常数（K(d)）= 241 μM。基于这些数据，我们使用I型MDCK上皮细胞测试了尼氟酸对跨上皮氯离子分泌和囊肿生长的影响。尼氟酸（200 μM）抑制了cAMP刺激的、布美他尼敏感的短路电流达55%。此外，该药物有力地阻碍了囊肿生长。我们得出结论，尼氟酸是CFTR的开放通道阻断剂，通过堵塞通道孔来抑制氯离子通透。它或相关药物可能在常染色体显性多囊肾病新疗法的开发中具有价值。
