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.

Keywords: detergent screen; electrophysiology; fluorescent-detection size-exclusion chromatography; voltage-gated proton channel.

Figure 1

Expression and functional analysis of chimeric EGFP-Hv1 Hv1 proton channel transiently expressed in Chinese hamster ovary (CHO) cells. (A) Cells expressing the EGFP-Hv1 proton channel 24-h post-transfection as seen through a fluorescent microsope (FITC filter cube). (B) Typical whole-cell patch clamp recordings of expressed chimeric EGFP-Hv1 proton channel. Whole cell patch clamp recording were performed 24-h post-transfection. The EGFP-Hv1 proton channels were clamped at an initial Vh of −60 mV, and subjected to +10 mV voltage steps (500 ms duration) to +130 mV. The voltage step to +80 mV and corresponding response is highlighted in red. Scale bars are in ms and pA, respectively.

Figure 2

Fluorescence-detection size-exclusion chromatography time course analysis of chimeric EGFP-Hv1 proton channel solubilized in dodecyl maltoside. Crude supernatant of EGFP-Hv1 proton channel containing plasma membranes from Sf9 cells were analyzed at (A) 24 h, (B) 48 h, (C) 72 h, and (D) 96 h. Chromatogram peaks associated with protein void (Void), chimeric protein (EGP-Hv1), and free monomeric EGFP (EGFP) are shown. Chimeric protein (black) and free monomeric EGFP (green) are scaled for comparison. Elution volume (mL) and Fluorescence are shown. au, arbitrary units.

Figure 3

FSEC analysis of Hv1 channel protein solubilized in βOG detergent. (A) 24 h and (B) 48 h after infection in SF9 cells. Chromatogram peaks associated with protein void (Void), chimeric protein (EGP-Hv1), and free monomeric EGFP (EGFP) are shown. Chimeric protein (blue) and free monomeric EGFP (green) are scaled for comparison. Chromatogram profiles are scaled for comparison. Elution volume (mL) and fluorescence are shown. au, arbitrary units.

Figure 4

FSEC analysis of Hv1 channel protein solubilized in LDAO detergent. (A) 24 h and (B) 48 h after infection in SF9 cells. Chimeric protein (orange) and free monomeric EGFP (green) are scaled for comparison. Chromatogram profiles are scaled for comparison. Elution volume (mL) and fluorescence are shown. au, arbitrary units.

Figure 5

FSEC analysis of Hv1 channel protein solubilized in Anzergent detergent. (A) 24 h and (B) 48 h after infection in SF9 cells. Chromatogram peaks associated with protein void (Void), chimeric protein (EGP-Hv1), and free monomeric EGFP (EGFP) are shown. Chimeric protein (purple) and free monomeric EGFP (green) are scaled for comparison. Chromatogram profiles are scaled for comparison. Elution volume (mL) and fluorescence are shown. au, arbitrary units.

Figure 6

Isolation and purification of the chimeric EGFP-Hv1 proton channel. (A) Cobalt metal affinity chromatography elution profile of isolated EGFP-Hv1 proton channel is shown. Axes of the chromatography profile are labeled Elution volume (mL) and Absorbance at 280 nm (mau), respectively. Equilibration, wash, and elution with increasing concentrations of imidazole are depicted (dashed line). Small gray vertical marks along the x-axis represent protein 3 mL protein fractions. (B) Size-exclusion chromatography profile of chimeric EGFP-Hv2 protein is shown. Chromatography axes are the same as panel A. Chimeric monomeric EGFP-Hv1 proton channels is labeled. Small gray vertical marks along x-axis indicate the 0.3 mL protein fractions obtained using a Superose 6/10–300 GL (GE) column.