Functional reconstitution of purified human Hv1 H+ channels

Abstract

Voltage-dependent H(+) (Hv) channels mediate proton conduction into and out of cells under the control of membrane voltage. Hv channels are unusual compared to voltage-dependent K(+), Na(+), and Ca(2+) channels in that Hv channel genes encode a voltage sensor domain (VSD) without a pore domain. The H(+) currents observed when Hv channels are expressed heterologously suggest that the VSD itself provides the pathway for proton conduction. In order to exclude the possibility that the Hv channel VSD assembles with an as yet unknown protein in the cell membrane as a requirement for H(+) conduction, we have purified Hv channels to homogeneity and reconstituted them into synthetic lipid liposomes. The Hv channel VSD by itself supports H(+) flux.

Fig. 1

Proton flux by vesicles containing recombinant Hv channels. (A) SDS-PAGE gel showing purified Hv channels. Lane 1: molecular weight marker, 2: final wash, and 3: Human Hv-1D4 eluted with 0.4 mg/ml 1D4 peptide. The gene for the full length human Hv channels (GenBank accession no: 91992153) with a C-terminal 1D4 tag (ARAAGGTETSQVAPA) was ligated into the PICZ-c vector (Invitrogen Life Technologies). This vector was transformed into a His+ strain of SMD1163 Pichia pastoris and selected as described . Transformed cells were grown in 1 L cultures of BMG media (Yeast Nirtogen Base, 100 mM sodium phosphate pH 6.3 and 1 % glycerol) at 30 ° C until an optical density of ~20 was reached. BMG media was exchanged for BMM media (BMG with 1 % MeOH instead of glycerol) and grown at 24° C for 24 hours. Frozen pellets were lysed with a mixer mill (Retsch, Inc. Model MM301) and resuspended in buffer (500 mM NaCl, 50 mM TRIS-HCl, pH 8.5, 2 mM β-mercaptoethanol, 0.1 µg/ml deoxyribonuclease I, 0.1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1.0 mM phenylmethysulfonyl fluoride and 2.0 mM Ethylenediaminetetraacetic acid (EDTA). The pH was adjusted to 8.5 with NaOH, and 0.15 g DDM (n-dodecyl-β-D-maltopyranoside, Anatrace) per g of cells was added prior to a 2–3 hour extraction at room temperature followed by centrifugation at 31000 × g for 25 min. Supernatant was added to 1D4 antibody-linked sepharose affinity resin previously equilibrated with buffer A (500 mM NaCl, 50 mM TRIS-HCl, pH 7.5, 1 mM EDTA and 1 mM DDM) and rotated at room temperature for 2 hours. The resin was collected on a column, washed with buffer A (4 × 5 column volumes) and eluted with buffer A containing 0.4 mg/ml 1D4 peptide. Protein was loaded on a Superdex-200 gel filtration column in 20 mM TRIS-HCl, pH 7.5, 150 mM KCl, 50 mM NaCl and 4 mM DM (n-decyl-β-D-maltopyranoside, Anatrace, anagrade) (Buffer B). The fractions corresponding to Hv channels were pooled and concentrated to 1.0 mg/ml for reconstitution into lipid vesicles. (B) Fluorescence-based H⁺ flux assay. Vesicles (cyan) loaded with high concentration of K⁺ are diluted into low concentration K⁺ buffer containing the fluorescence dye ACMA (9-amino-6-chloro-methoxyacridine). Addition of valinomycin (red), a K⁺ selective ionophore, results in K⁺ efflux, which generates a driving force for H⁺ influx. If there is a H⁺ channel (blue) in the vesicle membrane, pH inside the vesicle will decrease. This pH decrease is monitored by ACMA because the protonated form, which becomes trapped inside vesicles, loses fluorescence whereas unprotonated ACMA diffuses freely across the membrane . (C) Fluorescence-based H⁺ flux assay for vesicles with and without Hv1 colored blue and red, respectively (n = 5). Error bars indicate standard error of the mean. Valinomycin and CCCP are added at the indicated time points. The fluorescence data for vesicles containing Hv channels was obtained using a published procedure with the following modification . A mixture of 6:6:3:3:1 of POPC:POPE:POPS:SM:PI (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine, 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine, 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phospho-L-Serine, Sphingomyelin, and L-α-Phosphatidylinositol, obtained from Avanti) was prepared based on the composition of human neutrophil plasma membrane . The lipid mixture was dried under an Argon stream and then resuspended to 10 mg/ml in dialysis buffer (20 mM HEPES, pH 7.0, 150 mM KCl, 10% glycerol, 0.2 mM EGTA and 2 mM 2-mercaptoethanol). The lipid mixture was then sonicated in a bath sonicator three times for 2 minutes. Decylmaltoside (DM) was added to the lipid mixture to 10 mM and rotated at room temperature for 1hr. Protein was added to the lipid mixture in a 1:100 (wt:wt) protein to lipid ratio and an additional 10 mM DM was added. As a control empty vesicles were made in which only buffer B was added to the lipids. The protein-lipid mixture was rotated at room temperature for ~3 hours then placed into dialysis membrane (molecular weight cut off of 50 KDa) and dialyzed in 4 L of dialysis buffer for 5 days at RT exchanging buffer daily. Vesicles were then harvested and flash frozen in liquid nitrogen and stored at −80° C. Vesicles were thawed in room temperature water and then sonicated once in a bath sonicator for 5 seconds and then diluted 20 fold into flux buffer (20 mM HEPES, pH 7.0, 150 mM NaCl, 7.5 mM KCl, 10% glycerol, 0.2 mM EGTA, 0.5 mg/ml BSA, 2 mM 2-mercaptoethanol and 2 µM ACMA) in a quartz cuvette. Data were collected on a Spex Fluorolog 3–11 spectrofluorometer in time acquisition mode at 30-second intervals with excitation at 410 nm, emission 490 nm, with bandwidth 5 nm and an integration time 2 s. A baseline was collected for 150 s before the addition of 20 nM valinomycin. After the fluorescence stabilized carbonyl cyanide m-chloro phenyl hydrazone (CCCP) was added to 2 µM rendering all vesicles H⁺ permeable and a minimum baseline was collected for 150 seconds. Data are scaled by (F_i − F_min)/(F_max−F_min), where F_max is the average value of the starting baseline and F_min is the average value of the minimum baseline. F_max−F_min (the total reduction in fluorescence after CCCP addition) was ~ 25 % for all vesicles.

Fig. 2

Proton flux into vesicles containing Hv channels at various protein to lipid ratios. (A) Fluorescence-based H⁺ flux assay for vesicles containing a decreasing number of Hv channels. Protein to lipid ratios of 1:100 (dark blue, n = 5), 1:500 (pink, n = 2), 1:1000 (orange, n = 3), 1:5000 (yellow, n = 3), 1:10,000 (cyan, n = 4), 1:20,000 (light green, n = 3), 1:40,000 (green, n =4), 1:60,000 (violet, n = 3) and empty vesicles (red squares, n = 4) are plotted (error bars represent the standard error of the mean, range of mean for 1:500). Protein and vesicles were prepared as described in the Fig. 1 legend. (B) Sucrose cushion of vesicles containing Hv channels. Numbers denote the fractions collected from top to bottom. Lipid vesicles containing Hv1, with protein to lipid ratio 1: 100 (wt:wt), were layered on a sucrose gradient (From top to bottom, 140 µl sample plus 60 µl dialysis buffer, 600 µl 7 % sucrose, and 1 ml 27 % sucrose in dialysis buffer). The gradients were then centrifuged at 135,000 × g in a Sorvall RP55-S swinging bucket rotor for 2 hours and then fractionated into 8 × 225 µl fractions. A 15 µl sample of each fraction was then mixed with 15 µl 2x running buffer and run on a 12% gel (SDS-PAGE) and stained with Coomassie blue. (C) Determination of the fraction of functional Hv channels. Plot of μ versus the ratio of fluorescence decay contributed by Hv containing vesicles over the total fluorescence decay by addition of CCCP where μ is the ratio of the number of channels over number of vesicles calculated with $$\mu =\frac{8g_{\mathit{\text{Hv}}}\pi r^2{M}_L}{g_L\sigma M_{\mathit{\text{Hv}}}},$$ where g_Hv and g_L are the grams of Hv channel and lipid added, r is the estimated average radius of a vesicle (100 nm), M_L is the molecular weight of the average lipid molecule (754 Da), σ is the estimated area per lipid molecule (63 Ų 13) and M_Hv is the molecular mass of the Hv channel dimer (70,000 Da). Protein to lipid ratios are as in Fig. 2A, 1:100 (μ = 43.0, n = 5), 1:500 (μ = 8.59, n = 2), 1:1000 (μ = 4.30, n = 3), 1:5000 (μ = 0.86, n = 3), 1:10,000 (μ = 0.43, n = 4), 1:20,000 (μ = 0.21, n = 3), 1:40,000 (μ = 0.11, n =4), 1:60,000 (μ = 0.07, n = 3) error bars represent the standard error of the mean (range of mean for 1:500). The two curves are derived from equation (1) with φ (fraction of functional Hv) = 1.0, θ (fraction of reconstitution deficient vesicles) = 0.15 (red) and ϕ = 0.5, θ = 0.15 (green). The inset is a close-up view along the x-axis indicating that the fit is superior with a curve corresponding to ϕ = 1.0, θ = 0.15.

Fig. 3

Comparison of dilution series data with theory. (A) Theoretical curves for the decrease of internal pH over time at the equivalent protein to lipid ratios as in Fig 2A, scaled with the theoretical fraction of empty vesicles. Curves are colored to match the equivalent experimental traces in Fig 3B; a theoretical curve corresponding to empty vesicles is not shown. The change in internal H⁺ concentration was calculated using the algorithm described in Moffat et al. with slight modifications. To account for the voltage-dependent gating property of the Hv channels the proton flux was calculated as: $$J_H=\frac{G_H(V_m-V_H)}{F\left(1+\text{exp}\left(\frac{-zF(V_m-V_{½})}{RT}\right)\right)}$$Where G_H is proton conductance, V_m is membrane voltage, V_H is the equilibrium potential for H⁺, F is Faraday’s constant, z is the effective charge (a value of 3.0 e was used 15), V_mid is the midpoint voltage of activation for Hv (a value of 40 mV was used 5), R is the ideal gas constant and T is the absolute temperature in Kelvin (298 K). The algorithm was run successively for a unit volume of one vesicle of radius 100 nm with n channels (where n = 1, 2, 3,…,30) either facing outside-in or outside-out (expressed as a multiplier of either 1 or −1 on the V_m in the two-state Boltzmann). This basis set of 60 vectors representing the internal pH change of the vesicle were combined to generate the expected flux of a population of vesicles each containing n channels according to: $$f(n,m)=\frac{n!}{m!(n-m)!}{\left(\frac{1}{2}\right)}^n$$ where n signifies the total number of channels and m the total number of channels facing outside-in. Since the flux due to channels facing outside-in is much greater than the flux due to channels facing outside-out (by more than 3 orders of magnitude) we applied the simplifying assumption that flux into any vesicle containing channels in both orientations was equal to the flux generated by only the channels facing outside-in. This operation results in a new basis set of 30 vectors that correspond to the H⁺ flux into populations of vesicles with total of n channels in either orientation (where n = 1, 2, 3,…,30). This new basis set was then applied to the distribution of vesicles with n channels at the various protein to lipid ratios used according to: $$f(n)={\left(\frac{\varphi }{(1-\theta )}\right)}^n\frac{{\mu }^n}{n!}\text{exp}\left(\frac{-\varphi \mu }{(1-\theta )}\right)$$ where f(n) is the fraction of vesicles with n channels, φ is the fraction of functional Hv (a value of 1.0 was used), θ is the fraction of reconstitution deficient vesicles (a value of 0.15 was used) and μ is the ratio of number of channels to number of vesicles (see Fig. 2C legend). Simulations were all preformed using MATLAB. (B) Experimental fluorescence traces from Fig. 2A highlighting the first 200 seconds after the addition of valinomycin.

Fig. 4

Specific H⁺ permeation through Hv1. Fluorescence-based H⁺ flux assay for vesicles containing Hv1 (dark blue, n=5), KvAP VSD (green, n=4), KvAP (dark green, n=4), paddle chimera (cyan). Empty vesicles are shown in red. Error bars indicate standard error of the mean. Valinomycin and CCCP are added at the indicated time points. KvAP, KvAP VSD, and paddle chimera channels were expressed and purified according to published procedures ; . Reconstitutions were carried out as described in Fig. 1C legend with the following protein to lipid ratios (wt:wt) 1:200 (KvAP VSD), 1:100 (KvAP), and 1: 50 (paddle chimera).