Schultz B D, DeRoos A D, Venglarik C J, Singh A K, Frizzell R A, Bridges R J
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Am J Physiol. 1996 Aug;271(2 Pt 1):L192-200. doi: 10.1152/ajplung.1996.271.2.L192.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A- and ATP-regulated Cl- channel located in the apical membranes of epithelial cells. Previously Sheppard and Welsh (J. Gen. Physiol. 100: 573-591, 1992) showed that glibenclamide, a compound which binds to the sulfonylurea receptor and thus blocks nucleotide-dependent K+ channels, reduced CFTR whole cell current. The aim of this study was to identify the mechanism underlying this inhibition in cell-free membrane patches containing CFTR Cl- channels. Exposure to gliben-clamide caused a reversible reduction in current carried by CFTR which was paralleled by a decrease in channel open probability (Po). The decrease in Po was concentration dependent, and half-maximum inhibition (ki) occurred at 30 microM. Fluctuation analysis indicated a flickery-type block of open CFTR channels. Event duration analysis supported this notion by showing that the glibenclamide-induced decrease in Po was accompanied by interruptions of open bursts [i.e., an apparent reduction in the burst duration (Tburst)] with only a slight reduction in closed time (Tc). The plot of the corresponding open-to-closed (Tburst-1) and closed-to-open (Tc-1) rates as a function of glibenclamide concentration were consistent with a pseudo-first-order open-blocked mechanism and provided estimates of the on rate (kon = 1.17 microM-1S-1), the off rate (koff = 16 s-1), and the dissociation constant (Kd = 14 microM). The difference between the Ki (30 microM) and the Kd (14 microM) is the result expected for a closed-open-blocked model with an initial Po of 0.47. Since the initial Po was 0.50 +/- 0.02 (n = 12), we can conclude that glibenclamide blocks CFTR by a closed-open-blocked mechanism.
囊性纤维化跨膜传导调节因子（CFTR）是一种蛋白激酶A和ATP调节的氯离子通道，位于上皮细胞的顶端膜中。此前，谢泼德和威尔士（《普通生理学杂志》100:573 - 591，1992年）表明，格列本脲是一种与磺酰脲受体结合从而阻断核苷酸依赖性钾通道的化合物，它能降低CFTR全细胞电流。本研究的目的是确定在含有CFTR氯离子通道的无细胞膜片中这种抑制作用的潜在机制。暴露于格列本脲会导致CFTR携带的电流可逆性降低，同时通道开放概率（Po）也会降低。Po的降低呈浓度依赖性，半数最大抑制浓度（ki）为30微摩尔。波动分析表明，开放的CFTR通道存在闪烁型阻断。事件持续时间分析支持了这一观点，即格列本脲引起的Po降低伴随着开放爆发的中断[即爆发持续时间（Tburst）明显缩短]，而关闭时间（Tc）仅略有缩短。相应的开放到关闭（Tburst - 1）和关闭到开放（Tc - 1）速率与格列本脲浓度的关系图符合伪一级开放阻断机制，并提供了结合速率（kon = 1.17微摩尔-1秒-¹）、解离速率（koff = 16秒-¹）和解离常数（Kd = 14微摩尔）的估计值。Ki（30微摩尔）和Kd（14微摩尔）之间的差异是初始Po为0.47的关闭-开放-阻断模型所预期的结果。由于初始Po为0.50±0.02（n = 12），我们可以得出结论，格列本脲通过关闭-开放-阻断机制阻断CFTR通道。
