External Zn2+ binding to cysteine-substituted cystic fibrosis transmembrane conductance regulator constructs regulates channel gating and curcumin potentiation

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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 Zn2+ bridges near and at the ECL1–ECL6 interface, respectively, and the gating effects of a Zn2+ disturbance at the ECL1–ECL6 interface on the stimulatory ICL1/ICL4-R interaction were determined. The results showed that both Zn2+ 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 Zn2+ inhibition was weakened at higher Zn2+ concentrations, their Zn2+ affinity was stronger in the resting state than in the activated state, and their activation current noises were decreased by external Zn2+ binding. More importantly, the external Zn2+ inhibition was reversed by internal curcumin in the R334C construct but not in the T1122C mutant. Therefore, although both Zn2+ bridges may promote channel closure, external Zn2+ 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.

An enzyme specifically reacts with a single substrate like a key and a lock. Here we demonstrate a novel intramolecular key and lock model in which the exact fit of the phosphorylated R domain (key) into an active site (key hole), the interface of intracellular loops 1 and 4 of the CFTR chloride channel (enzyme), is critical for channel opening.

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