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Figure 1. Extracellular protons reduce single-channel conductance of TRPV1 channels. (A) Representative single-channel currents at the indicated pH recorded from an outside-out patch of oocyte membrane expressing TRPV1. The external and internal solutions were symmetrical 150 mM KCl. Recordings at two membrane potentials were shown (±60 mV) and were low-pass filtered at 2 kHz. (B) All-point amplitude histograms of single-channel currents. The histograms were fit to sums of two Gaussian functions to determine the closed and open amplitudes. The solid lines represent best fits. (C) Dose–response curves for the unitary current amplitudes obtained at ±60 mV versus the extracellular pH. The solid lines represent fits to the Hill equation with pKa ∼ 7, Hill coefficient of n ∼ 0.6 for Vh = −60 mV, and pKa ∼ 6 and n ∼ 0.3 for Vh = +60 mV, respectively. (D) Normalized dose–response relationships showing different dependence on pH at different membrane potentials (±60 mV).
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Figure 2. Voltage dependence of proton block of single-channel conductance. (A and B) Representative single-channel currents recorded at two extracellular pHs with different voltages ranging from −100 to +100 mV. (A) pH 7.4 and (B) pH 5.5, recorded with symmetrical 150-mM KCl solutions. (C) Single-channel i-V curves at different values of pHos as indicated. The smooth lines were polynomial fits. (D; top plot) Ratio of the unitary current amplitudes at each pHo (7.4, 6.5, 5.5, and 4.5) to the amplitudes at pH 8.5 plotted as a function of voltage. The dashed lines represent the best fit to a Woodhull model, which corresponds to an electrical distance d = 0.5 for the binding site from the bulk solution, an apparent valence z = 1, a dissociation constant pKd,0 = 5.3 at 0 mV, and a pseudo Hill coefficient n = 0.4. The model was globally fit to the unitary current amplitudes at different pH values. (Bottom plot) pKd of proton inhibition estimated from the Hill equation fit at each voltage.
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Figure 3. Effects of ionic strength on proton block of TRPV1 channels. (A) Representative single-channel currents from TRPV1 channels recorded in symmetrical 1,000-mM KCl solutions at different voltages and extracellular pHs as indicated above traces. (B and C) Dose-dependent relationships of the unitary current amplitudes versus [K+] at −60 mV (B) and +60 mV (C), respectively. Note that the dose–response curves were saturated at different maximal current amplitudes with different pHs. Dashed lines represent predictions of the Gouy-Chapman-Stern model based on simultaneous fitting of the data at multiple extracellular pHs. The model assumes that protons exert their effects by one-to-one binding to negative surface charges on the membrane to lower local H+ and K+ concentrations. At high [K+], it predicted a common maximal current amplitude independent of extracellular pH. The fits correspond to the following parameter values: σ = 0.2e/nm2, pKa = 7 for protonation of surface charges, KS = 372 mM for the dissociation constant of K+ ion binding, n = 1.5 for the apparent Hill coefficient, and imax = 10 pA for the maximum current amplitude (−60 mV); σ = 0.1e/nm2, pKa = 7.2, KS = 160 mM, n = 1.1, and imax = 17 (+60 mV). The surface potentials Ψ0 at 2 M K+ for pHs 8.5, 7.4, and 5.5 were, respectively (in mV): −18, −15, and −0.6 (−60 mV), and −17, −12, and −0.4 (+60 mV). (D and E) Fits to a competitive inhibition model in which protons bind to the same sites occupied by K+ to block ion flows. Data at different pHs were fit simultaneously. The fit yields the following model parameters: KS = 219 mM for the dissociation constant of K+ binding, pKa = 6.5 for proton binding, n = 0.5 for the pseudo Hill coefficient, and imax = 15pA (Vh = −60 mV); Ks = 70 mM, pKa = 5.8, n = 0.7, and imax = 19 pA (Vh = +60 mV). (F and G) Effects of [K+] on proton inhibition at the indicated membrane potentials. The ratios of the unitary current amplitudes at pHs 7.4 and 5.5 to the amplitudes at pH 8.5 were plotted.
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Figure 4. Neutralization of D646 partially relieves proton block. (A) Representative single-channel current traces from the D646N mutant at different extracellular pHs. (Top row) Outward currents at +60 mV. (Bottom row) Inward currents at −60 mV. Solutions were symmetrical and contained 1 M KCl. (B–E) Plots of unitary current amplitudes versus ionic concentrations for the mutant channel at the indicated pHo (B, −60 mV; C, +60 mV). For comparison, similar plots for wild type are displayed in D and E.
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Figure 5. Neutralization of E636 reduces proton inhibition. (A) Representative single-channel currents from the E636Q mutant channel at different external pHs in symmetrical 1-M KCl solutions. Holding potential was either +60 mV (top row) or −60 mV (bottom row). (B and C) Plots of unitary current amplitudes versus [K+] at the indicated pHo. (B) Vh = −60 mV. (C) Vh = +60 mV. (D and E) Similar plots for wild type were shown for comparison.
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Figure 6. Double mutations of D646 and E636 abrogate proton block. (A and B) Examples of single-channel currents from the double mutant D646N/E636Q, showing little changes in the unitary current amplitudes with changes in extracellular pH at several ionic concentrations ([K+] = 25, 100, 500, and 1,000 mM). (A) Holding potential of +60 mV. (B) Holding potential of −60 mV. Solutions were symmetrical on both sides of membranes. (C and D) Plots of unitary current amplitudes versus [K+] at the indicated pH. (C) −60 mV. (D) +60 mV. Solid lines indicate double mutant, and dashed lines are for wild type. The unitary current amplitudes of the double mutant were similar to those of wild type at low pH and were independent of extracellular pH changes over a broad range of ionic concentrations (25–2,000 mM). The mutant did not show a detectable expression in oocytes. All recordings were from HEK293 cells.
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Figure 7. Proton gating is not involved. (A) Representative single-channel currents from the mutant V538L channels at different extracellular pHs. Currents were evoked by 1 µM capsaicin at either −60 mV (bottom row) or +60 mV (top row) in symmetrical solutions (2 M KCl). The V538L mutation, which abrogates proton activation of TRPV1, did not eliminate proton inhibition of single-channel conductance. (B) Similar recordings from the E600Q mutant channels. Proton block of unitary current amplitudes remained after neutralization of E600, which abolishes the potentiation effect of low pH on channel activation and gating. (C and D) Comparison of unitary current amplitudes of the mutant channels with wild type at different pH values. (C) Vh = −60 mV. (D) Vh = +60 mV. Both mutants and the wild type had similar current amplitudes at each tested pHo.
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Figure 8. A homology model for the pore region of TRPV1 depicting the positions of the residues important for proton block (Glu636 and Asp646). Residue Glu636 is located on the pore helix, and Asp646 is immediately above the selectivity filter (TIGMG). Approximately one helical turn below Glu636 is another charged residue Lys639 (shown in blue). The model was constructed by homology modeling using the crystal structure of the KcsA channel as a template. See Ryu et al. (2007) for the alignment of sequences between the two channels.
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Figure 9. K639 modulates channel conductance and proton inhibition. (A) Unitary current amplitudes of the K639Q mutant versus the wild-type channel at different pHs. The removal of the positive charge caused a reduction of the current amplitudes at all pHs. (B) Ratio of the unitary current amplitudes at each pH to the amplitudes at pH 8.5, showing an increase in the inhibition by protons after charge removal. Single-channel currents were recorded from transiently transfected HEK293 cells in the outside-out configuration. [K+] = 1 M, Vh = −60 mV, and data points are means ± SE (n ≥ 5).
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