Venglarik C J, Schultz B D, DeRoos A D, Singh A K, Bridges R J
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Biophys J. 1996 Jun;70(6):2696-703. doi: 10.1016/S0006-3495(96)79839-9.
Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel that is regulated by protein kinase A and cytosolic nucleotides. Previously, Sheppard and Welsh reported that the sulfonylureas glibenclamide and tolbutamide reduced CFTR whole cell currents. The aim of this study was to quantify the effects of tolbutamide on CFTR gating in excised membrane patches containing multiple channels. We chose tolbutamide because weak (i.e., fast-type) open channel blockers introduce brief events into multichannel recordings that can be readily quantified by current fluctuation analysis. Inspection of current records revealed that the addition of tolbutamide reduced the apparent single-channel current amplitude and increased the open-channel noise, as expected for a fast-type open channel blocker. The apparent decrease in unitary current amplitude provides a measure of open probability within a burst (P0 Burst), and the resulting concentration-response relationship was described by a simple Michaelis-Menten inhibition function. The concentration of tolbutamide causing a 50% reduction of Po Burst (540 +/- 20 microM) was similar to the concentration producing a 50% inhibition of short-circuit current across T84 colonic epithelial cell monolayers (400 +/- 20 microM). Changes in CFTR gating were then quantified by analyzing current fluctuations. Tolbutamide caused a high-frequency Lorentzian (corner frequency, fc > 300 Hz) to appear in the power density spectrum. The fc of this Lorentzian component increased as a linear function of tolbutamide concentration, as expected for a pseudo-first-order open-blocked mechanism and yielded estimates of the on rate (koff = 2.8 +/- 0.3 microM-1 s-1), the off rate (kon = 1210 +/- 225 s-1), and the dissociation constant (KD = 430 +/- 80 microM). Based on these observations, we propose that there is a bimolecular interaction between tolbutamide and CFTR, causing open channel blockade.
囊性纤维化跨膜传导调节因子（CFTR）是一种上皮氯离子通道，受蛋白激酶A和胞质核苷酸调控。此前，谢泼德和威尔士报道，磺脲类药物格列本脲和甲苯磺丁脲可降低CFTR全细胞电流。本研究的目的是量化甲苯磺丁脲对含有多个通道的切除膜片上CFTR门控的影响。我们选择甲苯磺丁脲是因为弱（即快速型）开放通道阻滞剂会在多通道记录中引入短暂事件，这些事件可通过电流波动分析轻松量化。对电流记录的检查显示，如快速型开放通道阻滞剂所预期的那样，添加甲苯磺丁脲会降低表观单通道电流幅度并增加开放通道噪声。单通道电流幅度的明显降低提供了爆发内开放概率（P0 Burst）的一种度量，由此产生的浓度-反应关系可用简单的米氏抑制函数描述。导致P0 Burst降低50%的甲苯磺丁脲浓度（540±20微摩尔）与使T84结肠上皮细胞单层短路电流抑制50%的浓度（400±20微摩尔）相似。然后通过分析电流波动来量化CFTR门控的变化。甲苯磺丁脲导致功率密度谱中出现高频洛伦兹峰（转折频率，fc>300赫兹）。该洛伦兹成分的fc随甲苯磺丁脲浓度呈线性增加，这是伪一级开放阻断机制所预期的，并得出了开启速率（koff = 2.8±0.3微摩尔-1秒-1）、关闭速率（kon = 1210±225秒-1）和解离常数（KD = 430±80微摩尔）的估计值。基于这些观察结果，我们提出甲苯磺丁脲与CFTR之间存在双分子相互作用，导致开放通道阻断。
