title: Detergent screening of the human voltage-gated proton channel using fluorescence-detection size-exclusion chromatography.
abstract: The human voltage-gated proton channel (Hv1) is a membrane protein consisting of four transmembrane domains and intracellular amino- and carboxy-termini. The protein is activated by membrane depolarization, similar to other voltage-sensitive proteins. However, the Hv1 proton channel lacks a traditional ion pore. The human Hv1 proton channel has been implicated in mediating sperm capacitance, stroke, and most recently as a biomarker/mediator of cancer metastasis. Recently, the three-dimensional structures for homologues of this voltage-gated proton channel were reported. However, it is not clear what artificial environment is needed to facilitate the isolation and purification of the human Hv1 proton channel for structural study. In the present study, we generated a chimeric protein that placed an enhanced green fluorescent protein (EGFP) to the amino-terminus of the human Hv1 proton channel (termed EGFP-Hv1). The chimeric protein was expressed in a baculovirus expression system using Sf9 cells and subjected to detergent screening using fluorescence-detection size-exclusion chromatography. The EGFP-Hv1 proton channel can be solubilized in the zwitterionic detergent Anzergent 3-12 and the nonionic n-dodecyl-β-d-maltoside (DDM) with little protein aggregation and a prominent monomeric protein peak at 48 h postinfection. Furthermore, we demonstrate that the chimeric protein exhibits a monomeric protein peak, which is distinguishable from protein aggregates, at the final size-exclusion chromatography purification step. Taken together, we can conclude that solubilization in DDM will provide a useable final product for further structural characterization of the full-length human Hv1 proton channel.
authors.affiliation: Department of Pharmacology & Neuroscience, UNT Health Science Center, Fort Worth, Texas.
authors.forename: Amruta
authors.initials: A
authors.lastname: Agharkar
authors.forename: Jennifer
authors.initials: J
authors.lastname: Rzadkowolski
authors.forename: Mandy
authors.initials: M
authors.lastname: McBroom
authors.forename: Eric B
authors.initials: EB
authors.lastname: Gonzales
mesh_terms: D000818:Animals; D016466:CHO Cells; D002850:Chromatography, Gel; D003412:Cricetulus; D003902:Detergents / Q000737:chemistry* / Q000494:pharmacology; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D012995:Solubility / Q000187:drug effects; D018411:Spodoptera
chemical_list: D003902:Detergents; C510235:HVCN1 protein, human; D007473:Ion Channels; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: detergent screen; electrophysiology; fluorescent-detection size-exclusion chromatography; voltage-gated proton channel