Plastic recognition and electrogenic uniport translocation of 1^st-, 2^nd-, and 3^rd-row transition and post-transition metals by primary-active transmembrane P_1B-2-type ATPase pumps

Abstract

Transmembrane P1B-type ATPase pumps catalyze the extrusion of transition metal ions across cellular lipid membranes to maintain essential cellular metal homeostasis and detoxify toxic metals. Zn(II)-pumps of the P1B-2-type subclass,...

Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori

“…33 Application of FZ-3 in monitoring other transition metals revealed a turn-on fluorescent response with other substrates such as Fe( ii ), Co( ii ), Ni( ii ) and Cd( ii ) in the presence and absence of lipids. 35,36 However, in the putative application of FZ-3 to detect Ni( ii ) transport activity by NixA in proteoliposomes potential problems were encountered related to: (i) residual Zn( ii ) contamination in lipids isolated from native sources, and (ii) the FZ-3 significantly higher sensitivity for Zn( ii ) compared to Ni( ii ), which would affect batch-to-batch reproducibility. To circumvent these potential problems, we decided to exploit the comparable affinities of FZ-3 for Zn( ii ) and Ni( ii ) (or Co( ii )), and the significantly lower fluorescent turn-on response (fluorescence yield) for FZ-3-Ni( ii )/Co( ii ) complexes (compared to FZ-3-Zn( ii )).…”

“…Through this platform, which mimics the biophysical properties of cellular membranes, we determined key NixA transport properties. Three liposome-compatible and impermeable fluorescent probes were encapsulated in the proteoliposomes' lumen to characterize NixA metal translocation mechanism and kinetics: (1) a Ni( ii )-sensitive fluorescent probe, Fluozin-3-Zn( ii ) (FZ-3-Zn), 33–36 (2) the pH sensitive probe pyranine, 37 (3) and the membrane potential sensor oxonol VI. 38 Based on transport assays, we determine that NixA is a highly-selective, high-affinity Ni( ii ) uniporter, and that metal transport is electrogenic.…”