The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by ATP binding at the interface of two cytoplasmic nucleotide binding domains (NBDs) and phosphorylation of the regulatory (R) domain by protein kinase A (PKA). The human CFTR has two functionally active thiol groups for gating regulation by chemical modification. Although modification of C832 in the R domain with N-ethylmaleimide promotes channel opening, glutathionylation of C1344 in NBD2 inhibits channel opening. Our recent studies demonstrated that the N-ethylmaleimide-induced potentiation involves a high-affinity inhibitory Fe³⁺ site at the interface between the R domain and intracellular loop 3 (ICL3). However, it is unknown whether the glutathionylation-evoked inhibition implies another stimulatory metal site. Here, Fe³⁺-insensitive mutations at the R–ICL3 interface were employed to further examine whether Zn²⁺ potentiated the activity of the human CFTR channel by targeting C1344 once the interfacial Fe³⁺ bridge was disrupted. The results showed that internal nanomolar Zn²⁺ increased its activity by about two- to threefold at a low level of protein kinase A, and the increase was reversed by EDTA or DTT or reduced glutathione but suppressed by a high level of protein kinase A, N-ethylmaleimide modification or a C1344A mutation. It is interesting that this Zn²⁺-triggered potentiation is not found in the wild type human CFTR to which endogenous Fe³⁺ is bound. Thus, the high-affinity binding of Zn²⁺ to C1344 in NBD2 may stimulate human CFTR activity in a phosphorylation-dependent manner, but the primary binding of Fe³⁺ to the ICL3–R interface may prohibit this stimulation.

中文翻译：

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Fe（iii）位点的去除通过高亲和性Zn（ii）结合促进人类囊性纤维化跨膜电导调节剂的激活†

囊性纤维化跨膜电导调节剂（CFTR）氯化物通道通过两个胞质核苷酸结合域（NBDs）界面上的ATP结合和蛋白激酶A（PKA）调节（R）域的磷酸化而被激活。人CFTR具有两个功能活跃的巯基，可通过化学修饰进行门控调节。尽管用N-乙基马来酰亚胺修饰R结构域中的C832可促进通道开放，但NBD2中C1344的谷胱甘肽酰化可抑制通道开放。我们最近的研究表明，N-乙基马来酰亚胺诱导的增强涉及高亲和力抑制Fe ³⁺位点位于R结构域和细胞内环3（ICL3）之间的界面。然而，尚不清楚谷胱甘肽化引起的抑制是否暗示另一个刺激性金属位点。在这里，一旦界面Fe ³⁺桥被破坏，R–ICL3界面上的Fe ^3+不敏感突变被用来进一步检查Zn ²⁺是否通过靶向C1344来增强人CFTR通道的活性。结果表明，内部纳米摩尔Zn ²⁺在低水平的蛋白激酶A时，其活性增加了约2到3倍，而EDTA或DTT或还原型谷胱甘肽却使这种增加被逆转，但高水平的蛋白激酶A则抑制了这种增长，ñ-乙基马来酰亚胺修饰或C1344A突变。有趣的是，在与内源性Fe ³⁺结合的野生型人CFTR中未发现这种Zn ²⁺触发的增强作用。因此，Zn ²⁺与NBD2中C1344的高亲和力结合可能以磷酸化依赖性的方式刺激人的CFTR活性，但Fe ³⁺与ICL3-R界面的初步结合可能会阻止这种刺激。