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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.

Author information

1
Department of Physiology and Pharmacology, Oregon Health & Sciences University, Portland, OR, USA.
2
Department of Drug Research and Development, Institute of Biophysical Medico-chemistry, Reno, NV, USA.
3
Department of Physiology and Membrane Biology, University of California School of Medicine, Davis, CA, USA.
4
Truman State University, Kirksville, MO, USA.
5
Department of Physiology, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, USA.

Abstract

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.

KEYWORDS:

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

PMID:
27175795
DOI:
10.1111/febs.13752
[Indexed for MEDLINE]
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