Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
J Biol Chem. 2012 Mar 23;287(13):10156-10165. doi: 10.1074/jbc.M112.342972. Epub 2012 Feb 1.
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.
囊性纤维化跨膜电导调节因子 (CFTR) 氯离子通道是 ATP 结合盒 (ABC) 蛋白家族的成员，该家族的大多数成员都作为主动转运体发挥作用。人们认为，主动转运的 ABC 蛋白在跨膜底物途径的“外向”和“内向”构象之间交替。在 CFTR 中，基于与其他 ABC 蛋白的同源性，假定外向构象对应于通道开放状态。我们使用膜片钳记录技术，从细胞内和细胞外溶液中定量测定进入 CFTR 两个不同跨膜区域的半胱氨酸反应探针的进入速度。两种探针，大的[2-磺基乙基]甲硫基磺酸盐 (MTSES) 分子和可渗透的 Au(CN)(2)(-) 离子，被应用于膜的两侧，以修饰取代亮氨酸 102（第一跨膜区）和苏氨酸 338（第六跨膜区）的半胱氨酸。通道的核苷酸结合域突变会改变通道的开启和关闭。我们发现，对于 MTSES 和 Au(CN)(2)(-)，从细胞质侧进入这两个半胱氨酸的速度在开放通道中更快，而从细胞外侧进入相同位点的速度在关闭通道中更快。这些结果与跨膜区域的交替进入一致，但开放状态面向内部，关闭状态面向外部。因此，我们的发现促使对当前 CFTR 结构和机制模型进行修订，并对所有 ABC 蛋白的转运机制产生更广泛的影响。我们的研究结果还表明，在 CFTR 渗透途径中，功能性和非功能性（“残余”）门的可能位置。
