Acid potentiation of the capsaicin receptor determined by a key extracellular site

Abstract

The capsaicin (vanilloid) receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. The response of VR1 to capsaicin or noxious heat is dynamically potentiated by extracellular protons within a pH range encountered during tissue acidosis, such as that associated with arthritis, infarction, tumor growth, and other forms of injury. A molecular determinant for this important physiological activity was localized to an extracellular Glu residue (E600) in the region linking the fifth transmembrane domain with the putative pore-forming region of the channel. We suggest that this residue serves as a key regulatory site of the receptor by setting sensitivity to other noxious stimuli in response to changes in extracellular proton concentration. We also demonstrate that protons, vanilloids, and heat promote channel opening through distinct pathways, because mutations at a second site (E648) selectively abrogate proton-evoked channel activation without diminishing responses to other noxious stimuli. Our findings provide molecular evidence for stimulus-specific steps in VR1 activation and offer strategies for the development of novel analgesic agents.

Figure 1

Initial screen for VR1 mutants with altered proton sensitivity. (A) Positions of substituted acidic residues in the VR1 channel are shown in the context of a putative transmembrane topology model based on studies of the related hTRP3 channel (45). (B) Analysis of proton- and capsaicin-activated currents in transfected HEK293 cells expressing VR1 mutants. Upper bars show the mean ratios of proton (pH 4.4): capsaicin (1 μM)-activated currents. Lower bars show average absolute amplitudes of capsaicin-activated current in transfected HEK293 cells expressing wild-type or mutant VR1 receptors. Data represent means ± SEM from 4–16 experiments. (C) Higher frequency of death among HEK293 cells expressing E600 mutant channels. Percentage of green fluorescent protein-positive (transfected) HEK293 cells that stained brightly with ethidium homodimer-1 (dead cells) is shown. Data represent means ± SEM of triplicate determinations from two independent experiments. Expression of E600Q and E600K mutants increases cell death. (D) Tenfold increase in capsaicin sensitivity in E600Q mutant channels. Capsaicin dose-response curves for VR1 wild-type and E600Q mutant channels. Oocytes were perfused for 1 min with solutions of increasing capsaicin concentrations, with an intermittent wash in capsaicin-free solution for 3 min. Baseline-subtracted steady-state currents were normalized to currents maximally activated by 200 nM capsaicin for E600Q and by 5 μM capsaicin for VR1 wild type. Averaged data were fitted with the Hill equation with average parameters obtained from fits to individual cells. For VR1, EC₅₀ = 520 ± 60 nM (n = 5), n_H = 2.5 ± 0.2. For E600Q, EC₅₀ = 40 ± 3 nM (n = 4), n_H = 2.7 ± 0.2. Maximal amplitudes were 3.0 ± 0.2 μA for wild type VR1 (5 μM capsaicin) or 3.2 ± 0.2 μA for E600Q (200 nM capsaicin).

Figure 2

Prepotentiated phenotype of E600Q and E600K mutant channels. (A) Modulation of heat-activated currents by extracellular protons in cells expressing VR1 wild-type (Upper) or E600Q mutant (Lower) channels. To compare current sizes, channels were first activated for 20 s by acidic solution (pH 4.0) at room temperature. [In oocytes, E600Q channels showed robust activation at low pH (see below)]. To record heat-activated currents, bath temperature was then elevated from room temperature to 47°C within 10 s. This procedure was repeated seven times in 2-min intervals. During the fifth heat application, bath pH was decreased from 7.6 to 6.3. (B) Sensitization of heat-activated currents and potentiation by protons in VR1 wild-type (n = 6) and E600Q mutant (n = 4) channels. Heat-evoked peak currents, recorded as in A, were normalized to pH 4-activated steady-state currents at room temperature. Averaged values for peak currents at the first (pH 7.6), fourth (pH 7.6), and fifth heat stimulus (pH 6.3) are shown. Error bars represent SEM. (C) Lower threshold for heat activation of E600K mutant channels. Solid lines show representative heat-activated currents at the first heat stimulus recorded from three independent oocytes (left axis). Open (E600K) and closed (VR1) circles display mean heat-activated currents normalized to peak amplitudes at the maximal activation temperature [42.5°C for E600K (n = 7), 50°C for VR1 wild type (n = 6), right axis]. Heat activation as in A. Error bars represent SEM.

Figure 3

Mutations in E600 affect proton-dependent potentiation of heat-activated currents. (A) Titration analysis of proton-dependent potentiation of heat-activated currents in VR1 wild-type or E600D, E600H, or E600Q mutant channels. Channels were first sensitized by four subsequent heat pulses to 48°C (not shown). Upper bar shows the pH application protocol. Oocytes were perfused in bath solutions with a pH indicated above the bar for 1 min each (black), interrupted by perfusion at pH 7.5 (white). The lower bar shows the heat application protocol. For each pH, bath temperature was elevated from room temperature (white) to 42°C (black) for VR1 wild-type, E600H, and E600Q, or to 48°C for E600D. Both wild-type (WT)- and the E600Q-expressing cells were again tested at pH 7.5. The E600Q-expressing cells were subsequently perfused with standard solution containing 10 μM ruthenium red (RR). (B) Dose-response analysis of proton-mediated potentiation of heat-activated currents in oocytes expressing VR1 wild-type (n = 4), E600D (n = 3), E600H (n = 3), or E600Q (n = 3) channels. Recordings as in A. For VR1 wild type, E600D and E600H, currents before application of solution of the indicated pH were subtracted from heat-evoked peak currents. For E600Q, currents after ruthenium red block were subtracted from peak currents. Current values were normalized to currents at pH 5.0. Averaged currents are shown with error bars representing SEM.

Figure 4

Analysis of E600 mutants suggests direct titration by protons. (A) Proton dose-response curves recorded from transiently transfected HEK293 cells show a direct relationship between agonist potency and side-chain pK_a in wild-type, E600D, and E600H mutants. pH values producing half-maximal responses were 5.34 ± 0.01, 4.55 ± 0.02, and 5.58 ± 0.02, respectively. The Hill equation was used for curve fitting. Values represent the means ± SEM from 4–17 separate experiments. (B) Correlation of side-chain pK_a at position 600 with proton sensitivity. Dose-response curves for proton activation of E600 mutants with protonatable side chains recorded from Xenopus oocytes. Cells were perfused for 20 s with solutions of decreasing pH, with an intermittent wash at pH 7.5 for 40 s. Baseline-subtracted currents were normalized to steady-state currents at pH 3.5. Figure shows averaged data fitted with the Hill equation with average parameters obtained from fits to individual cells. For E600K, EC₅₀ = 5.5 ± 0.1 (n = 5), n_H = 2.1 ± 0.2; E600H, EC₅₀ = 5.2 ± 0.1 (n = 3), n_H = 2.0 ± 0.2; VR1 wild-type, EC₅₀ = 4.9 ± 0.1 (n = 9), n_H = 1.9 ± 0.1; E600D, EC₅₀ = 4.5 ± 0.1 (n = 3), n_H = 2.3 ± 0.3. Error bars represent SEM. Maximally activated current amplitudes at pH 3.5 were: VR1 wild type, 2.7 ± 0.2 μA; E600K, 4.4 ± 0.2 μA; E600H, 2.7 ± 0.1 μA; E600D, 2.4 ± 0.2 μA. (C) Dose-response curves for proton activation of E600 mutants with neutral side chains recorded from Xenopus oocytes. Fits as in B. For E600A, EC₅₀ = 5.0 ± 0.1 (n = 3), n_H = 1.7 ± 0.1; E600S, EC₅₀ = 4.7 ± 0.1 (n = 3), n_H = 1.4 ± 0.1; E600Q, EC₅₀ = 4.8 ± 0.1 (n = 4), n_H = 1.5 ± 0.1. Maximally activated current amplitudes at pH 3.5 were: E600Q, 2.8 ± 0.2 μA; E600S, 2.6 ± 0.1 μA; and E600A, 2.8 ± 0.1 μA.

Figure 5

Reduction in proton-activated currents in mutants at position 648. (A) Bar graph showing average ratios of proton- to capsaicin-activated steady-state amplitudes in oocytes expressing VR1 wild type (n = 5), or E600Q, D601N, E610Q, E648Q, or E648A (n = 4 each) channels. Cells were perfused for 20 s at pH 4.0, washed for 40 s, then perfused for 20 s with solution containing 5 μM capsaicin. Mutation of E648 to Ala drastically reduces proton-activated currents. Error bars represent SEM. (B) Representative proton- and capsaicin-activated currents in VR1 wild type (Left) and mutant E648A (Right) channels. Protocol as in Fig. 3A. (C) Dose-response analysis of heat-activated currents in E648 mutants resembles that of wild-type receptors. Performed as in Fig. 3 A and B.