Gunderson K L, Kopito R R
Department of Biological Sciences, Stanford University, California 94305-5020.
J Biol Chem. 1994 Jul 29;269(30):19349-53.
Single channel analysis of artificial lipid planar bilayers reconstituted with wild-type human cystic fibrosis transmembrane regulator (CFTR) revealed a 10.3 pS Cl- selective channel that was activated upon phosphorylation with protein kinase A. Gating of this channel was described by a simple kinetic model consisting of a single open burst state and two closed states. The open probability of CFTR channels in bilayers increased as a function of increasing Mg-ATP concentration and exhibited negative cooperativity, suggesting the interaction of two or more ATP binding sites in channel gating. Mg-ATP increased channel open probability by decreasing the duration of the long-lived closed state but had no effect on either the mean open time or the fast closed state. ADP inhibited channel opening by precisely antagonizing the effect of ATP, suggesting that ADP inhibits the CFTR channel by competing with ATP for binding. Poorly hydrolyzable ATP analogs such as AMP-PNP and ATP gamma S, polyphosphates such as pyrophosphate (PPi) and tripolyphosphate (PPPi), and orthovanadate failed to support channel activity alone. When applied in the presence of ATP, these compounds all caused the CFTR channel to "lock" into a prolonged open burst state. These data support a model in which hydrolysis of ATP leads to closure of channels that have been opened by ATP.
对用野生型人类囊性纤维化跨膜调节因子（CFTR）重构的人工脂质平面双层膜进行单通道分析，发现了一个10.3 pS的Cl-选择性通道，该通道在用蛋白激酶A磷酸化后被激活。这个通道的门控由一个简单的动力学模型描述，该模型由一个单一的开放爆发状态和两个关闭状态组成。双层膜中CFTR通道的开放概率随Mg-ATP浓度的增加而增加，并表现出负协同性，这表明在通道门控中有两个或更多的ATP结合位点相互作用。Mg-ATP通过缩短长寿命关闭状态的持续时间来增加通道开放概率，但对平均开放时间或快速关闭状态均无影响。ADP通过精确拮抗ATP的作用来抑制通道开放，这表明ADP通过与ATP竞争结合来抑制CFTR通道。水解性差的ATP类似物如AMP-PNP和ATPγS、多磷酸盐如焦磷酸（PPi）和三聚磷酸（PPPi）以及原钒酸盐单独均不能支持通道活性。当在ATP存在的情况下应用时，这些化合物都会使CFTR通道“锁定”在一个延长的开放爆发状态。这些数据支持一个模型，即ATP的水解导致已被ATP打开的通道关闭。
