title: External Zn(2+) binding to cysteine-substituted cystic fibrosis transmembrane conductance regulator constructs regulates channel gating and curcumin potentiation.
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.
authors.affiliation: 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.
authors.forename: Guangyu
authors.initials: G
authors.lastname: Wang
authors.affiliation: Truman State University, Kirksville, MO, USA.
authors.forename: Rheeann
authors.initials: R
authors.lastname: Linsley
authors.affiliation: Department of Physiology, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, USA.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Norimatsu
mesh_terms: D000818:Animals; D003474:Curcumin / Q000378:metabolism* / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D005905:Glyburide / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000187:drug effects; D014981:Xenopus; D015032:Zinc / Q000494:pharmacology
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003474:Curcumin; D015032:Zinc; D005905:Glyburide
keywords: ABC transporter; extracellular loop; intracellular loop; long-range gating coupling; metal disturbance