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FEBS J. 2016 Jul;283(13):2458-75. doi: 10.1111/febs.13752. Epub 2016 Jun 15.

External Zn(2+) binding to cysteine-substituted cystic fibrosis transmembrane conductance regulator constructs regulates channel gating and curcumin potentiation.

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Department of Physiology and Pharmacology, Oregon Health & Sciences University, Portland, OR, USA.
Department of Drug Research and Development, Institute of Biophysical Medico-chemistry, Reno, NV, USA.
Department of Physiology and Membrane Biology, University of California School of Medicine, Davis, CA, USA.
Truman State University, Kirksville, MO, USA.
Department of Physiology, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, USA.


The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by ATP binding-induced dimerization of nucleotide-binding domains, the interaction between the phosphorylated regulatory (R) domain and the curcumin-sensitive interface between intracellular loop (ICL) 1 and ICL4, and the resultant inward-to-'outward' reorientation of transmembrane domains. Although transmembrane helices (TM) 2 and TM11 link the ICL1-ICL4 interface with the interface between extracellular loop (ECL) 1 and ECL6, it is unknown whether both interfaces are gating-coupled during the reorientation. Herein, R334C and T1122C mutations were used to engineer two Zn(2+) bridges near and at the ECL1-ECL6 interface, respectively, and the gating effects of a Zn(2+) disturbance at the ECL1-ECL6 interface on the stimulatory ICL1/ICL4-R interaction were determined. The results showed that both Zn(2+) bridges inhibited channel activity in a dose- and Cl(-) -dependent manner, and the inhibition was reversed by a washout or suppressed by thiol-specific modification. Interestingly, their Cl(-) -dependent Zn(2+) inhibition was weakened at higher Zn(2+) concentrations, their Zn(2+) affinity was stronger in the resting state than in the activated state, and their activation current noises were decreased by external Zn(2+) binding. More importantly, the external Zn(2+) inhibition was reversed by internal curcumin in the R334C construct but not in the T1122C mutant. Therefore, although both Zn(2+) bridges may promote channel closure, external Zn(2+) may disturb the ECL1-ECL6 interface and thus prevent the stimulatory ICL1/ICL4-R interaction and curcumin potentiation via a gating coupling between these two interfaces.


ABC transporter; extracellular loop; intracellular loop; long-range gating coupling; metal disturbance

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