Berger H A, Travis S M, Welsh M J
Department of Internal Medicine, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242, USA.
Am J Physiol. 1998 Mar;274(3):L305-12. doi: 10.1152/ajplung.1998.274.3.L305.
While studying the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), we found that addition of F- to the cytosolic surface of excised, inside-out membrane patches reversibly increased Cl- current in a dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP. F- increased current even in the presence of deferoxamine, which chelates Al3+, suggesting that stimulation was not due to AlF4-. F- also stimulated current in a CFTR variant that lacked a large part of the R domain, suggesting that the effect was not mediated via this domain. Studies of single channels showed that F- increased the open-state probability by slowing channel closure from bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F- influenced regulation by the cytosolic domains, most likely the nucleotide-binding domains (NBDs). Consistent with this, we found that mutation of a conserved Walker lysine in NBD2 changed the relative stimulatory effect of F- compared with wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no effect. Based on these and previous data, we speculate that F- interacts with CFTR, possibly via NBD2, and slows the rate of channel closure.
在研究囊性纤维化跨膜传导调节因子（CFTR）的调节机制时，我们发现，向切除的内向外膜片的胞质面添加氟离子（F⁻），会以剂量依赖的方式可逆地增加氯离子电流。这种刺激需要预先磷酸化以及ATP的存在。即使在存在去铁胺（一种螯合Al³⁺的物质）的情况下，F⁻也能增加电流，这表明刺激并非由AlF₄⁻引起。F⁻还能刺激一种缺少大部分R结构域的CFTR变体中的电流，这表明该效应不是通过这个结构域介导产生的。单通道研究表明，F⁻通过减缓通道从活动爆发中关闭的速度来增加开放状态概率；爆发之间的平均关闭时间和单通道电导没有改变。这些结果表明，F⁻影响胞质结构域（最有可能是核苷酸结合结构域（NBDs））的调节。与此一致的是，我们发现NBD2中一个保守的沃克赖氨酸突变改变了F⁻与野生型CFTR相比的相对刺激作用，而NBD1中沃克赖氨酸的突变则没有影响。基于这些以及之前的数据，我们推测F⁻可能通过NBD2与CFTR相互作用，并减缓通道关闭的速度。
