Quantification of Hv1-induced proton translocation by a lipid-coupled Oregon Green 488-based assay

Abstract

Passive proton translocation across membranes through proton channels is generally measured with assays that allow a qualitative detection of the H-transfer. However, if a quantitative and time-resolved analysis is required, new methods have to be developed. Here, we report on the quantification of pH changes induced by the voltage-dependent proton channel Hv1 using the commercially available pH-sensitive fluorophore Oregon Green 488-DHPE (OG488-DHPE). We successfully expressed and isolated Hv1 from Escherichia coli and reconstituted the protein in large unilamellar vesicles. Reconstitution was verified by surface enhanced infrared absorption (SEIRA) spectroscopy and proton activity was measured by a standard 9-amino-6-chloro-2-methoxyacridine assay. The quantitative OG488-DHPE assay demonstrated that the proton translocation rate of reconstituted Hv1 is much smaller than those reported in cellular systems. The OG488-DHPE assay further enabled us to quantify the K-value of the Hv1-inhibitor 2-guanidinobenzimidazole, which matches well with that found in cellular experiments. Our results clearly demonstrate the applicability of the developed in vitro assay to measure proton translocation in a quantitative fashion; the assay allows to screen for new inhibitors and to determine their characteristic parameters. Graphical abstract ᅟ.

Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers

“…Using Oregon Green 488 DHPE, the proton-pumping activity of the reconstituted F-type ATPase was observed by monitoring the interior pH changes of the proteoliposome as a function of time (Figure 4E) [142]. Oregon Green 488 DHPE was also used to quantify the Hv1-induced proton transport [143].…”

HIFs: New arginine mimic inhibitors of the Hv1 channel with improved VSD–ligand interactions

“…Among the residues involved in 2GBI binding, phenylalanine (Phe) 150, located in the S2 helix, was found to play a particularly important role ( Hong et al, 2014 ). The WT channel is inhibited by 2GBI in the micromolar concentration range ( Gerdes et al, 2018 ; Hong et al, 2013 ). However, when F150 is mutated to alanine, inhibition occurs in the nanomolar range ( Hong et al, 2013 ; Fig.…”

Synthesis and Purification of Lipid-conjugated Fluorescent pH Sensors

“…Rhodamine-based dyes provide an alternative due to their improved brightness and photostability; however, for reconstituted systems, problems with insufficient encapsulation during protein reconstitution and leakage out of vesicles were reported ( Schubert, 2003 ; Leiding et al, 2009 ). More recently, membrane-bound pH sensors based on lipid-conjugated fluorophores have been utilized ( Kemmer et al, 2015; Veshaguri et al, 2016; Schwamborn et al 2017; Gerdes et al 2018; Kühnel et al, 2019 ). These lipid-conjugated pH sensors efficiently co-reconstitute with membrane proteins into large liposomes, thereby avoiding loss during reconstitution and allowing studies in reconstituted systems down to the single vesicle level ( Kemmer et al, 2015 ; Veshaguri et al, 2016 ; Kühnel et al, 2019 ).…”