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Removal of the Fe(III) site promotes activation of the human cystic fibrosis transmembrane conductance regulator by high-affinity Zn(II) binding

Abstract

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-ethylmalemide promotes channel opening, glutathionylation of C1344 in NBD2 inhibits channel opening. Our recent studies demonstrated that the N-ethylmalemide-induced potentiation involves a high-affinity inhibitory Fe3+ site at the interface between the R domain and intracellular loop 3 (ICL3). However, it is unkown 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+ potentiates the activity of the human CFTR channel by targeting C1344 once the interfacial Fe3+ bridge has been disrupted. The results showed that internal nanomolar Zn2+ increased their activity by about two to three fold 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-ethylmalemide modification or a C1344A mutation. It is interesting that this Zn2+-trigered potentiation was not found in the wild type human CFTR to which endogenous Fe3+ has been bound. Thus, high-affinity binding of Zn2+ to C1344 in NBD2 may stimulate human CFTR activity in a phosphorylation-dependent manner but primary binding of Fe3+ to the ICL3-R interface may prohibit this stimulation.

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Publication details

The article was received on 15 Nov 2017, accepted on 08 Jan 2018 and first published on 08 Jan 2018


Article type: Communication
DOI: 10.1039/C7MT00315C
Citation: Metallomics, 2018, Accepted Manuscript
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    Removal of the Fe(III) site promotes activation of the human cystic fibrosis transmembrane conductance regulator by high-affinity Zn(II) binding

    G. Wang, Metallomics, 2018, Accepted Manuscript , DOI: 10.1039/C7MT00315C

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