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Figure 1. Patch-clamp fluorometry of Venus-Hv1. (A) Fluorescence images of an inside-out patch held at the indicated membrane potential. The image in the bottom right corner is a superposition of the fluorescence image at 100 mV (green) and the transmitted light image of the pipette to show the location of the membrane patch within the pipette. Bar, 10 µm. (B) Proton currents from the patch in A elicited by depolarizing voltage-clamp pulses. (C) Conductance versus voltage curve derived from the currents in B. The continuous line is a fit to Eq. 2 with parameters qa = 1.63 eo and V0.5 = −33.8 mV. (D) Fluorescence measured from the whole patch area at each voltage was normalized to the fluorescence at −100 mV (circles). A ratio greater than one indicates an increase (dequenching) of the fluorescence. The fluorescence of a region of membrane attached to the pipette but outside of the patch is also plotted (squares). The red continuous line is the I-V curve from the currents in A, whereas the red dashed curve is the fit to the G-V curve in C. The bath solution contained 100 mM MES buffer, and the pH of solutions is indicated.
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Figure 2. Patch-clamp fluorometry of Venus-Hv1 and Hv1-Venus constructs. (A) Fluorescence images of an inside-out patch from an oocyte expressing Hv1 channels labeled with Venus in the N terminus. The patch was held at the indicated membrane potential. (B) G-V curve from proton currents from the patch in A elicited by depolarizing voltage-clamp pulses. The continuous line is a fit to Eq. 2 with parameters qa = 1.31 eo and V0.5 = −31.2 mV. (C) Fluorescence measured from the whole area of the patch in A at each voltage, plotted as normalized intensity with respect to the fluorescence intensity at −100 mV. (D) Fluorescence images from an inside-out patch from oocytes expressing the Hv1 construct labeled at the C terminus. (A and D) Bars, 10 µm. (E) G-V curve from proton currents from the same patch in D elicited by depolarizing voltage-clamp pulses. The continuous line is a fit to Eq. 2 with parameters qa = 1.56 eo and V0.5 = −39.6 mV. (F) Fluorescence measured from the whole patch area and analyzed as in C. For the analysis in C and F, a ratio greater than one indicates an increase (dequenching) of the fluorescence as the patch is held at increased positive potentials. The red continuous line is the I-V curve from the currents in the corresponding patch, whereas the dashed curve is the fit to the G-V curve. The buffer concentration for these experiments was 10 mM MES, and the pH gradient is as in Fig. 1.
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Figure 3. Patch-clamp fluorometry of Venus-Hv1 at pHi 7. (A) Fluorescence images of an inside-out patch held at the indicated membrane potential. Bars, 10 µm. (B) Proton currents from the patch in A elicited by depolarizing voltage-clamp pulses, as indicated by the schematic protocol. (C) Conductance versus voltage curve derived from the currents in B. The continuous line is a fit to Eq. 2 with parameters qa = 1.62 eo and V0.5 = 33.6 mV. (D) Fluorescence measured from the whole patch area at each voltage was normalized to the fluorescence at −100 mV. A ratio equal to one at all voltages indicates the absence of dequenching of the fluorescence of Venus. The red continuous line is the I-V curve from the currents in B. The bath solution contained 10 mM HEPES buffer.
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Figure 4. Fluorescence dequenching and proton depletion. (A) Fluorescence image of a typical patch expressing Hv1-Venus and approximation of its geometry by a truncated cone with the indicated dimensions used in the model in Eq. 4. (B) The value of the observed dequenching at 80 mV is plotted as a function of the current for that particular patch also at 80 mV. All of the experiments in this figure were obtained with an intracellular solution of pH 5.5, with the exception of those patches represented by the open circles. The continuous red curve is the dequenching as a function of current magnitude predicted by Eq. 4. The parameters that went into Eq. 4 were rp = 10 µm, ro = 5 µm, lp = 15 µm, Ka = 7.08 × 10−7, αo = 0.1812, fpKa = 5.6, DBH = 5 × 10−10 m2/s, and Btotal = 10 mM. The dotted line indicates lack of dequenching and describes the recordings from N-labeled channels at pHi 7.
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Figure 5. Measurement of the pKa of fluorescent constructs. (A) Representative membrane sheet from oocytes injected with the Venus-Hv1 construct. The membrane was imaged under different pH conditions, indicated at the top of each frame. Fluorescence was measured as the mean of an ROI encompassing a large area of the membrane sheet or from individual clusters of channels with the same results. Bar, 20 µm (same for all images). (B) The fluorescence was normalized to the value at the highest pH for each construct to build the full titration curve. The data were fitted to the titration function (Eq. 3). The pKa for Venus-Hv1 (open circles) is 5.63 and 7.62 for VenusH148G-Hv1 (closed circles). The data are the mean of five experiments for both constructs, and error bars are the standard error of the mean.
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Figure 6. Recovery of fluorescence dequenching in VenusH148G-Hv1 at pHi 7. (A) Fluorescence images of an inside-out patch from an oocyte expressing the construct VenusH148G-Hv1 held at the indicated membrane potential. Bar, 10 µm. (B) Proton currents from the patch in A elicited by depolarizing voltage-clamp pulses. (C) Conductance versus voltage curve derived from the currents in B. The continuous line is a fit to Eq. 2 with parameters qa = 1.47 eo and V0.5 = 35.4 mV. (D) Fluorescence dequenching measured from the whole area of the patch in A. The red curve is the I-V curve for the same patch. (E) The fluorescence from 28 patches similar to A was measured at 80 mV, normalized to the fluorescence at −100 mV, and plotted as a function of the current magnitude at 80 mV, as in Fig. 4. A ratio greater than one at all voltages indicates dequenching of the fluorescence of VenusH148G. The bath solution contained 10 mM HEPES buffer. The continuous red line is the predicted dequenching by Eq. 4 with parameters rp = 10 µm, ro = 5 µm, lp = 15 µm, Ka = 2.81 × 10−8, αo = 0.26, fpKa = 7.6, DBH = 5 × 10−10 m2/s, and Btotal = 10 mM.
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Figure 7. Diffusion model calculations. (A) Schematic representation of the proton depletion zone generated by an outward proton current through an Hv1 channel. (B) Calculations using Eq. 5 of the extent of the depletion of protons as a function of the radial coordinate, r, for different values of the bulk proton concentration, c∞, as indicated; the value of q = 14 fA is the same for all curves as well as DH = 7,000 µm2/s. (C) Calculation of the effect of the presence of buffer. The black curve was calculated with DH = 7,000 µm2/s and Eq. 5. The effect of the presence of buffer is illustrated by the dotted line, which was calculated with Eq. 6. The values of the constants are c∞ = 3.16 µM, [B] = 100 mM, a = 1 nm, kb = 1010 M−1s−1, and DH = 7,000 µm2/s. Data are presented as molar proton concentration (left axis) and corresponding pH (right axis). (D) Experimental (red) and simulated (black) currents in response to a 100-mV depolarization. Simulated current trace was generated taking into account a pH change during current development of 1 pH unit, which changes the single-channel current from 5 fA to 1.4 fA. The current is given by Eq. 7 in the form I(H,t)=N·(1−e−t/τ)[5−(5−1.4)(1−e−t/τ)], with τ = 200 ms and N as an arbitrary whole number chosen to match the size of the experimental current.
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