External Zn²⁺ 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 nucleotidebinding 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 À -dependent manner, and the inhibition was reversed by a washout or suppressed by thiol-specific modification. Interestingly, their Cl À -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.

Lipid-dependent sequential allosteric activation of heat-sensing TRPV1 channels by anchor-stereoselective “hot” vanilloid compounds and analogs

“…In contrast, if the ligand binding sites are not cooperative, the Hill coefficient should not change when the potency is altered. For example, a single engineered Zn 2+ site in cystic fibrosis transmembrane conductance regulator (CFTR) always keeps a constant Hill coefficient of a Zn 2+ dose response although the site-mutation at the metal site decreases the Zn 2+ potency [ 22 ].…”

Ligand‐stereoselective allosteric activation of cold‐sensing TRPM8 channels by an H‐bonded homochiral menthol dimer with head‐to‐head or head‐to‐tail

“…66 Furthermore, as menthol shares the same binding pocket with WS-12 but the Hill coefficient of a WS-12 dose response is 1, 39 there is no cooperativity between four menthol binding pockets in homotetrameric TRPM8 through inter-subunit communication, just like a single engineered Zn 2+ site in CFTR. 67…”

PIP2-Dependent Thermoring Basis for Cold-Sensing of the TRPM8 Biothermometer

“…Previous studies demonstrated that these transition metal ions can bind to ion channels such as cystic fibrosis transmembrane conductance regulator (CFTR) and Slo BKCa and Kv4 channels, regulating channel gating. [29,[33][34][35][36][37][38][39] When the sandwiched transition metal cation interacts with M854 on S5, the additional swaping dynamic interaction between this metal site from one subunit and N958 on S6 from another adjacent subunit may facilitate the relative movement of the pore domain against the VSLD and the TRP domain in favor of opening of both upper and lower gates in the channel pore. Thus, the putative transition metal bridge of M854 on S5 with H835 on S4 and W789 on S3, together with the H-bond between R998 in the TRP domain and D772 or Q776 on S2, can prepare two smaller hairpins with 0 to 3-residue loops to stabilize the cold efficacy and to avoid cold denaturation (Figure 3).…”