Advertisement

Identification of an Aspartic Residue in the P-loop of the Vanilloid Receptor That Modulates Pore Properties*

      Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed inXenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu648 and Glu651 rendered minor effects on VR1 pore attributes, while Glu636 slightly modulated pore blockade. In contrast, replacement of Asp646by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg2+ with respect to Na+ without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp646 is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.
      VR1
      vanilloid receptor subunit 1
      RR
      ruthenium red
      GHK equation
      Goldman-Hodgkin-Katz equation for the constant field approximation
      pHo
      extracellular pH
      NMG
      N-methyl-d-glucamine
      The molecular mechanism underlying chemical and thermal nociception is starting to be understood, thanks to the cloning of a capsaicin-operated neuronal receptor referred to as the vanilloid receptor subunit 1 (VR1)1(
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ). VR1 is a nonselective cation channel with high Ca2+permeability that integrates both types of pain-producing stimuli (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ,
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • Rosen T.A.
      • Gilvert H.
      • Skinner K.
      • Raumann B.E.
      • Basbaum A.I.
      • Julius D.
      ,
      • Caterina M.J.
      • Julius D.
      ,
      • Nagy I.
      • Rang H.P.
      ,
      • Szallasi A.
      • Blumberg P.M.
      ). These channels are activated by vanilloids such as capsaicin, the pungent ingredient of hot red peppers, and by temperatures higher than 40 °C (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ,
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • Rosen T.A.
      • Gilvert H.
      • Skinner K.
      • Raumann B.E.
      • Basbaum A.I.
      • Julius D.
      ,
      • Nagy I.
      • Rang H.P.
      ). Recently, the lipid-based anandanamide was shown to be a potential endogenous VR1 agonist (
      • Zygmunt P.
      • Peterson J.
      • Andersson D.
      • Chuang H.
      • Sørgård M.
      • Di Marzo V.
      • Julius D.
      • Högestätt E.
      ). Activation of the VR1 channel raises intracellular Ca2+ and excites a subset of dorsal root and trigeminal ganglion primary neurons (
      • Szallasi A.
      • Blumberg P.M.
      ). These neurons transmit noxious information to the central nervous system and release proinflammatory neuropeptides at peripheral terminals (
      • Szallasi A.
      • Blumberg P.M.
      , 7). In addition to playing a role in nociception, the high Ca2+permeability exhibited by VR1 strongly desensitizes capsaicin-operated responses (
      • Szallasi A.
      • Blumberg P.M.
      , 7). This property partially accounts for the antinociceptive activity exhibited by vanilloids (
      • Szallasi A.
      • Blumberg P.M.
      ,
      • Sterner O.
      • Szallasi A.
      ,
      • Williams M.
      • Kowaluk E.A.
      • Arneric S.P.
      ).
      VR1 subunits are membrane proteins with a predicted relative molecular mass of 95 kDa that show similarity to the family of putative store-operated calcium channels (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ,
      • Caterina M.J.
      • Julius D.
      ,
      • Birnbaumer L.
      • Zhu X.
      • Jiang M.
      • Boulay G.
      • Peyton M.
      • Vannier B.
      • Brown D.
      • Platano D.
      • Sadeghi H.
      • Stefani E.
      • Birnbaumer M.
      ). Although the molecular composition and stoichiometry of neuronal VR1 channels is undetermined, heterologous expression of VR1 subunits gives rise to homomeric receptors that recapitulate most of the reported physiological properties (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ,
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • Rosen T.A.
      • Gilvert H.
      • Skinner K.
      • Raumann B.E.
      • Basbaum A.I.
      • Julius D.
      ,
      • Nagy I.
      • Rang H.P.
      ,
      • Szallasi A.
      • Blumberg P.M.
      , 7). Nonetheless, there is mounting evidence for molecular heterogeneity of vanilloid receptors, including the identification of a stretch-inactivating channel (
      • Suzuki M.
      • Sato J.
      • Kutsuwada K.
      • Ooki G.
      • Imai M.
      ), a vanilloid receptor-like protein (VRl-1) (
      • Caterina M.J.
      • Rosen T.A.
      • Tominaga M.
      • Brake A.J.
      • Julius D.
      ), and an N-terminal splice variant of VR1 (VR.5′sv) (
      • Schumacher M.A.
      • Moff I.
      • Sudanagunta S.P.
      • Levine J.D.
      ).
      Structurally, VR1 subunits display a hydrophilic intracellular N terminus domain containing three conserved ankyrin repeats and several kinase consensus sequences (Fig. 1). This protein domain might also contain the vanilloid binding site (
      • Schumacher M.A.
      • Moff I.
      • Sudanagunta S.P.
      • Levine J.D.
      ,
      • Jung J.
      • Hwang S.W.
      • Kwak J.
      • Lee S.-Y.
      • Kang C.-J.
      • Kim W.B.
      • Kim D.
      • Oh U.
      ). Hydrophobicity analysis of the protein reveals the presence of six putative transmembrane-spanning segments (S1 through S6) and a stretch linking the fifth and sixth segments that contains an amphipathic fragment denoted as the P-loop (Fig. 1). By analogy with shaker-like ion channels, this protein motif could critically contribute to structure the channel permeation pathway (
      • Doyle D.A.
      • Cabral J.M.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • McKinnon R.
      ). Because this is a newly identified channel family, the molecular determinants that specify its pore attributes are yet unrecognized. Thus, their elucidation is a target of intense research. Amino acid sequence analysis of the VR1 putative pore region reveals the presence of four acidic residues, Glu636, Asp646, Glu648, and Glu651 (Fig. 1), that may play an important role defining the channel ion selectivity and blockade by noncompetitive antagonists such as ruthenium red (RR) (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ,
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • Rosen T.A.
      • Gilvert H.
      • Skinner K.
      • Raumann B.E.
      • Basbaum A.I.
      • Julius D.
      ,
      • Nagy I.
      • Rang H.P.
      ,
      • Szallasi A.
      • Blumberg P.M.
      ,
      • Dray A.
      • Forbes C.A.
      • Burgess G.M.
      ). Recent evidence shows that mutation of Glu648 significantly reduced proton-activated currents without altering heat- or capsaicin-evoked responses or without eliminating the ability of protons to potentiate responses to these stimuli (
      • Jort S.-V.
      • Tominaga M.
      • Julius D.
      ). Here, we report that site-specific neutralization of these negatively charged positions generated functional, capsaicin-operated ion channels and show that the amino acid at position 646 modulates the RR inhibition efficacy. Furthermore, our results show that neutralization of Asp646 significantly reduced the permeability of divalent cations with respect to Na+ without affecting that characteristic of monovalent cations. Taken together, these experiments support the tenet that Asp646 is a structural determinant of the VR1 pore-forming region.
      Figure thumbnail gr4
      Figure 4D646N mutant receptors are less permeable to Mg2+. I-V characteristics are shown of ionic currents elicited by 20 μm capsaicin in Mg2+-Ringer's solution containing 1 and 10 mm[Mg2+]o for wild type channels (A) and D646N mutant receptors (B). Each trace is representative of at least three oocytes. Oocytes were held at −80 mV in the appropriate buffer and depolarized to 20 mV in 4 s (25 mV/s) using a ramp protocol. Leak currents were obtained in the absence of agonist and subtracted from the capsaicin-evoked ionic currents. Thearrows indicate reversal potentials (I = 0).C, reversal potentials for VR1 wild type and D646N mutant receptors plotted as a function of the extracellular Mg2+activity. Solid lines depict the best fit to the GHK equation using a nonlinear square algorithm. The goodness of the fit to the GHK equation was assessed by the χ2 test. Relative permeabilities of K+ and Mg2+ were 0.9 ± 0.1 and 4.0 ± 0.3 for wild type channels and 0.8 ± 0.2 and 1.0 ± 0.2 for D646N mutant receptors. Reversal potential values are given as mean ± S.E. withN = 4.
      Figure thumbnail gr1
      Figure 1Putative molecular determinants of the permeation properties of ionotropic VR1 receptors. The proposed molecular model for VR1 subunits consists of (a) an N-terminal domain containing three ankyrin repeats (●), (b) six transmembrane-spanning segments and a large stretch connecting the fifth and sixth membrane segments that contains a short amphipathic fragment (curved arrow), and (c) an intracellular C-terminal domain. On top is depicted the deduced amino acid sequence for the proposed pore-forming region (P-loop, boxed amino acid sequence). Acidic residues are in boldface type and underlined. Numbers on top denote the amino acid number in the deduced primary sequence.

      EXPERIMENTAL PROCEDURES

       Site-directed Mutagenesis, cRNA Preparation, and Microinjection into Xenopus Oocytes

      VR1 is a cDNA clone encoding a functional capsaicin-operated channel from dorsal root ganglion (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ) (kindly provided by Dr. David Julius). Site-directed mutagenesis was carried out by polymerase chain reaction as described (
      • Ferrer-Montiel A.V.
      • Montal M.
      ,
      • Ferrer-Montiel A.V.
      • Montal M.
      ). Mutant receptors were confirmed by DNA sequencing. Capped cRNA was synthesized using the mMESSAGE mMACHINE™ from Ambion (Austin, TX). 2–5 ng of cRNA was microinjected (V = 50 nl) into defolliculated oocytes (stages V and VI) as described (
      • Ferrer-Montiel A.V.
      • Montal M.
      ). Oocytes were functionally assayed 3–5 days after cRNA injection.

       Electrophysiological Characterization of the VR1 Mutants

      Capsaicin-evoked whole cell currents were measured under voltage clamp (Turbo TEC 10CD; NPI Electronics, Tamm, FRG) with a two-microelectrode voltage clamp (
      • Ferrer-Montiel A.V.
      • Montal M.
      ,
      • Ferrer-Montiel A.V.
      • Montal M.
      ). Oocytes were transferred to the recording chamber (V = 0.2 ml) and were perfused (1 ml/min) with the appropriate Ringer's solution in the absence and/or presence of capsaicin as VR1 agonist.
      Dose-response relationships for RR inhibition were obtained in Mg2+-Ringer's solution (10 mm Hepes, pH 7.4, 115 mm NaCl, 3.0 mm KCl, 0.1 mmBaCl2, 2.0 mm MgCl2). Homomeric VR-1 channels were activated by application of 20 μmcapsaicin in the absence or presence of increasing concentrations of RR at a holding potential (V h) of −80 mV. Responses were normalized with respect to that evoked in the absence of channel blocker. Dose-response curves were fitted to a Michaelis-Menten binding isotherm (
      • Ferrer-Montiel A.V.
      • Sun W.
      • Montal M.
      ),
      IImax=11+[blocker]IC50n
      Equation 1


      Where IC50 is the inhibition constant and denotes the concentration of blocker to inhibit half of the maximal response (I max) recorded in its absence, and nis the steepness of the inhibition curve.
      I-V characteristics were recorded using a ramp protocol (PULSE/PULSEFIT; HEKA, FRG); oocytes were depolarized from −80 mV 20 mV in 4 s (25 mV/s) unless otherwise indicated. Leak currents were measured in the absence of agonist in the external bath medium and subtracted from the ionic current recorded in its presence. Additional details concerning recording and procedures are as described elsewhere (
      • Ferrer-Montiel A.V.
      • Sun W.
      • Montal M.
      ). All measurements were performed at 23 ± 2 °C.

       Calculation of the Relative Ionic Permeabilities Using the Goldman-Hodgkin-Katz (GHK) Equation

      To determine the relative ionic permeabilities of monovalent and divalent cations with respect to Na+, we applied the constant field approximation using the GHK equation (
      • Ferrer-Montiel A.V.
      • Sun W.
      • Montal M.
      ,
      • Lewis C.A.
      ). As a divalent cation, we used Mg2+, because, at variance with Ca2+, this cation does not block or desensitize VR channels (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ,
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • Rosen T.A.
      • Gilvert H.
      • Skinner K.
      • Raumann B.E.
      • Basbaum A.I.
      • Julius D.
      ). Reversal potentials (V r) were obtained in the following external solutions: 125 mm sodium, 2 mmmagnesium, 125 mm NaCl, 2.0 mmMgCl2; 20 mm sodium, 1 mmmagnesium, 20 mm NaCl, 1.0 mmMgCl2, 105 mm N-methyl-d-glucamine (NMG); 20 mmsodium, 2 mm magnesium, 20 mm NaCl, 2.0 mm MgCl2, 100 mm NMG; 20 mm sodium, 5 mm magnesium; 20 mmNaCl, 5.0 mm MgCl2, 95 mm NMG; 20 mm sodium, 10 mm magnesium, 20 mmNaCl, 10 mm MgCl2, 85 mm NMG. All Ringer's solutions contained 10 mm Hepes, pH 7.4, and 3.0 mm KCl.
      Since Mg2+ does not activate the endogenous Ca2+-activated, voltage-dependent chloride conductance (
      • Miledi R.
      • Parker I.
      ), the contribution of Cl permeability to the reversal potentials was considered negligible. The GHK equation modified to include the contribution of the permeability to divalent cations is as follows (
      • Ferrer-Montiel A.V.
      • Sun W.
      • Montal M.
      ,
      • Ferrer-Montiel A.V.
      • Sun W.
      • Montal M.
      ),
      Vr=RTF1n(Na+) o+PK+PNa+(K+) o+4PMg2+PNa+ (Mg2+) o1+expFVrRT(Na+) i+PK+PNa+(K+) i
      Equation 2


      where PK+/ PNa+ and PMg+/ PNa+denote the relative permeabilities of K+ and Mg2+ with respect to Na+ and (x)i and (x)o refer to the intracellular and extracellular activities of the permeant ions (Na+, K+, and Mg2+).RT/F is 25.3 mV at 20 °C; [Na+]i = 10 mm, [K+]i = 120 mm, [K+]o = 3.0 mm, and [Mg2+]i = 0. The intracellular ion concentration of 130 mm produced the minimal χ2 in the fitting of the experimental data to the GHK equation. Activity coefficients (γ) were taken as 0.25 and 0.75 for Mg2+ and monovalent cations (Na+ and K+), respectively (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ). The junction potential between the ground electrode and bath consequent to changing the extracellular ionic conditions from 20 mm sodium/1 mm magnesium to 20 mmsodium/10 mm magnesium was 2 mV.V r values were corrected accordingly, plotted as a function of the extracellular ionic activity, and fitted to the GHK equation with a nonlinear least-squares regression algorithm using the MicroCal ORIGIN version 5.0 (Microcal, Amherst, MA) (
      • Ferrer-Montiel A.V.
      • Sun W.
      • Montal M.
      ). The goodness of fit was inferred from the χ2 test.

      RESULTS

       Neutralization of Asp646 Reduces Ruthenium Red Sensitivity of VR1 Channels

      To study the functional role played by negatively charged residues located within or nearby the proposed P-loop of VR1 channels (Fig. 1), we neutralized these acidic amino acids. Wild type and mutant receptors were expressed in Xenopus oocytes for functional characterization. Heterologous expression of VR1 transcripts in frog oocytes generated capsaicin-elicited ionic currents that, at concentrations ≤10 μm, activated slowly to reach a relatively stable plateau level and subsided rapidly to base line upon agonist washout (Fig. 2 A). Higher vanilloid concentrations accelerated receptor activation and concomitantly delayed agonist removal (Fig. 2 B). These capsaicin-operated responses exhibited an EC50(concentration of agonist to activate half-maximal response) of ∼0.5 μm, in agreement with other reports (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ,
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • Rosen T.A.
      • Gilvert H.
      • Skinner K.
      • Raumann B.E.
      • Basbaum A.I.
      • Julius D.
      ). Neutralization of acidic residues in the pore region did not significantly modify capsaicin efficacy (EC50 ∼0.2–0.5 μm for E636Q, D646N, E648Q, and E651Q mutants); nor did it change dramatically the maximal response to capsaicin instillation with respect to wild type channels (see legend to Fig. 2). Likewise, the kinetic profile of capsaicin-elicited inward currents was not altered by the mutations performed (data not shown).
      Figure thumbnail gr2
      Figure 2Neutralization of acidic residues modulates receptor sensitivity to RR inhibition. Ionic currents evoked by 10 μm (A) and 20 μm (B) capsaicin from Xenopus oocytes expressing cRNA transcripts of VR1. C–F, ruthenium red blockade on wild type and mutant receptor. Oocytes were bathed in Mg2+-Ringer's solution and activated with 20 μm capsaicin in the absence or presence of increasing blocker concentrations. Membrane currents were recorded in the whole-cell voltage clamp configuration, atV h = −80 mV. Capsaicin and blocker were applied for the duration indicated by the horizontal bars. Maximal currents elicited by capsaicin were as follows: 390 ± 678 nA (number of oocytes (N) = 128) for wild type; 319 ± 222 (N = 61) for E636Q; 574 ± 644 (N= 74) for D646N; 410 ± 503 (N = 37) for E648Q; 118 ± 123 (N = 20) for E651Q. Values are given as mean ± S.D. p < 0.1 as compared with wild type.
      To investigate whether the mutated amino acids modulate pore properties, we used as a sensitive pore probe the positively charged ruthenium red, a VR1 channel blocker. These studies were performed at saturating vanilloid concentrations to ensure complete and fast channel opening. We choose 20 μm capsaicin because it evoked a rapid activation to a plateau level that readily declined to the original base line upon agonist washout (Fig. 2 B). As illustrated in Fig. 2 C, oocytes expressing wild type channels exhibited capsaicin-operated ionic currents that were rapidly blocked in a concentration-dependent manner by micromolar RR concentrations applied extracellularly. RR inhibition of VR1 channels was washable and weakly voltage-dependent (data not shown). Neutralization of Glu636 and Glu648to glutamine gave rise to functional channels that responded to capsaicin and displayed RR sensitivity similar to that characteristic of wild type receptors (Fig. 2, D and F). Similar results were obtained for E651Q (TableI). In contrast, charge neutralization of Asp646 (D646N) generated channels that were significantly less sensitive to RR than wild type receptors (Fig.2 E). Whereas VR1 channels were blocked by 80% with 1 μm RR, capsaicin-elicited responses from D646N mutants were only reduced by 25%. Complete blockade of D646N channels required RR concentrations as high as 50 μm.
      Table IRuthenium red blockade efficacy of VR1 channels
      SpeciespH 7.4pH 6.4
      IC50nIC50n
      μmμm
      VR10.14 ± 0.090.9 ± 0.101.3 ± 0.071.3 ± 0.1
      E636Q0.04 ± 0.010.7 ± 0.050.8 ± 0.201.0 ± 0.2
      D646N1.7 ± 0.201.6 ± 0.33.0 ± 0.401.7 ± 0.4
      E648Q0.25 ± 0.061.4 ± 0.31.5 ± 0.251.3 ± 0.3
      E651Q0.16 ± 0.031.1 ± 0.21.3 ± 0.231.1 ± 0.2
      M644Y0.20 ± 0.120.7 ± 0.1ND
      E636K/K639E0.17 ± 0.060.9 ± 0.08ND
      Experimental values were fitted to the logistic equationI blocker/I agonist = 1/(1+(blocker/IC50)n), where IC50 denotes the [blocker] to inhibit half of the maximal agonist response, andn is the steepness of the relationship. E636K and D646K did not exhibit capsaicin-evoked ionic currents. Data are given as mean ± S.E., with N ≥ 3. ND, not determined.
      RR sensitivity of VR1 species was quantified from dose-response relationships (Fig. 3, top, and Table I). RR blocked VR1 wild type channels with an IC50 of 0.14 ± 0.09 μm and a steepness,n, of 0.9 ± 0.3. Neutralization of Glu648and Glu651 did not significantly alter RR blockade efficacy (IC50 = 0.25 ± 0.06 μm for E648Q, and IC50 = 0.16 ± 0.03 μm for E651Q). Replacement of E636 by glutamine, however, increased the RR blockade efficacy by ∼3-fold (IC50 = 0.04 ± 0.01 μm). In contrast, mutation of Asp646 to asparagine reduced RR sensitivity by ∼10-fold (IC50 = 1.7 ± 0.2 μm). These data suggest that Asp646 is a molecular determinant of RR sensitivity and that the residue at position 636 modulates pore blockade. Replacement of the acidic residues by lysine at these positions (E636K and D646K) or simultaneous neutralization of both residues, E636Q/D646N, did not produce functional capsaicin-operated channels, precluding a detailed study of the functional interplay of these two protein positions.
      Figure thumbnail gr3
      Figure 3Amino acid at position 646 appears to be a molecular determinant of RR blockade efficacy. Dose-response curves for ruthenium red blockade of the capsaicin-evoked currents of VR1 and mutant receptors at pHo 7.4 (top) and 6.4 (bottom). Solid lines depict theoretical fits to the logistic equation (Table ). Values are given as means ± S.E., N ≥ 6.

       Acidification of the Extracellular Medium Modulates RR Blockade Efficacy

      The stability of a complex between RR and pore acidic residues will be determined by their degree of protonation and the spatial arrangement of carboxylic groups. Accordingly, protonation of acidic groups involved in RR binding should weaken its blockade efficacy. We tested this prediction by obtaining the RR inhibition efficacy for all VR1 species at acidic extracellular pH (pHo). As illustrated in Fig. 3 (bottom), VR1 wild type and E636Q, E648Q, and E651Q mutant receptors exhibited ⩾10-fold lower sensitivity to RR blockade at pHo 6.4 (Table I) as compared with the neutral pHo 7.4. In marked contrast, extracellular acidification reduced by only 2-fold RR blockade efficacy of D646N mutants (Table I and Fig. 3, bottom). These observations suggest that carboxylate groups involved in RR binding become protonated at pHo 6.4, resulting in a reduced RR blocking sensitivity. That carboxylate groups exhibit rather neutral pK a values is not surprising, since pK a of acidic groups buried inside proteins can vary several units depending on the dielectric environment (
      • Fersht A.
      ), as evidenced for Ca2+ channels and cyclic GMP-gated channels (
      • Seifert R.
      • Eismann E.
      • Ludwing J.
      • Baumann A.
      • Kaupp U.B.
      ,
      • Klöckner
      • Mikala G.
      • Schwartz A.
      • Varady G.
      ). These studies could not be carried out at pHo ≤6.0 because we observed activation of endogenous currents that were insensitive to RR.
      Collectively, these results imply that protonation of acidic residues implicated in RR binding modulates blockade efficacy. The observation that RR inhibition of D646N channels is largely insensitive to pHo acidification supports the notion that this residue is a structural determinant of the high affinity RR binding site. The 2-fold reduction of RR inhibition efficacy observed in the D646N mutant may result from the reported strong delocalization in the amide group of asparagine that makes this group capable of associating with protons and possibly with cations (

      Bash, H., and Tora, H. (1992) The Chemistry of Acid Derivatives, Suppl. B, Vol. 2, pp. 1–50, John Wiley & Sons, Inc., New York.

      ,

      Zalaweski, R. I. (1992) The Chemistry of Acid Derivatives, Suppl. B, Vol. 2, pp. 305–369, John Wiley & Sons, Inc., New York.

      ). Alternatively, an electrostatic influence exerted by the three other neighboring glutamate residues can not be ruled out.

       D646N Mutant Channels Display Lower Permeability to Mg2+

      Since Asp646 appears to be an important structural determinant of VR1 channel blockade, it is conceivable that this residue also contributes to define the ionic permeability, especially to divalent cations. To test this hypothesis, we investigated the relative ionic permeability of wild type and D646N mutant channels. We focused on Mg2+ for two reasons: (a) Mg2+ is not an activator of the endogenous calcium-activated chloride conductance (
      • Miledi R.
      • Parker I.
      ); and (b) at variance with Ca2+, Mg2+ does not block nor desensitize VR1 channels at millimolar concentrations. Indeed, attempts to measure the relative Ca2+ permeability failed due to the large blockade and desensitization of VR1 channels provoked by this cation, consistent with previous observations (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ,
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • Rosen T.A.
      • Gilvert H.
      • Skinner K.
      • Raumann B.E.
      • Basbaum A.I.
      • Julius D.
      ). Capsaicin-evoked ionic currents from VR1 wild type channels in the presence of [Ca2+]o ≥ 1 mm were ≤10 nA, preventing the accurate measurement of reversal potentials.
      For VR1 wild type channels, I-V relationships obtained at low [sodium]o (20 mm) and 1 mmMgCl2 are fairly linear, with a slight inward rectification at negative membrane potentials, and exhibit a reversal potential (V r) of −36 ± 3 mV (Fig.4 A). A 10-fold increase in [Mg2+]o shifted the reversal potential up to 25 mV toward more depolarizing potentials (Fig. 4 A), indicating that VR1 channels are permeable to Mg2+. Mutation of Asp646 to asparagine slightly affected the I-V characteristics but significantly altered the permeability to Mg2+ (Fig. 4 B). As shown, a 10-fold increase in [Mg2+]o moved V r ∼10 mV toward positive potentials. This displacement ofV r was 15 mV smaller than that observed for VR1 channels, suggesting that the D646N mutant receptor exhibits lower permeability to Mg2+. At variance with the D646N mutant, neutralization of the other negatively charged residues did not significantly affect the apparent Mg2+ permeability, as evidenced by the similar shift in V r consequent to changing the extracellular ionic conditions (TableII). Analysis of I-V relationships obtained varying the [Na+]osuggested that the permeability to monovalent cations was unaffected by the mutations (Table II).
      Table IIReversal potentials of VR1 mutants
      SpeciesV rΔV r
      125 mm = Na, 2 mm Mg20 mm = Na, 2 mm Mg20 mm = Na, 5 mm MgΔ [Na+]oΔ [Mg2+]o
      mVmV
      VR16 ± 4−30 ± 5−20 ± 13610
      E636Q4 ± 3−31 ± 6−21 ± 43510
      D646N−2 ± 4−33 ± 3−29 ± 331 4
      p < 0.05.
      E648Q7 ± 5−28 ± 4−19 ± 335 9
      E651Q3 ± 4−36 ± 5−24 ± 43912
      M644Y1 ± 2−26 ± 2−19 ± 227 7
      p < 0.05.
      E636K/K639E3 ± 1−31 ± 3−22 ± 2349
      Reversal potentials (V r) were obtained fromI-V relationships as the voltage at which the capsaicin-evoked ionic current was zero. ΔV rdenotes the change in reversal potential as a result of the extracellular [Na+]o from 20 to 125 mm(Δ[Na+]o) or the [Mg2+]o from 2 to 5 mM (Δ[Mg+2]o). Ionic currents were elicited by 20 μm capsaicin. Ramps were evoked from −80 to +20 mV in 4 s as described in Fig. 4. External ionic composition was as described under “Experimental Procedures.” Data are given as mean ± S.E., N ≥ 3.
      2-a p < 0.05.
      To further underscore that Asp646 is a molecular determinant of VR1 ionic selectivity, we determined the relative permeabilities to K+ and Mg2+ with respect to Na+, using the GHK equation modified to include the contribution of divalent cations (
      • Ferrer-Montiel A.V.
      • Sun W.
      • Montal M.
      ,
      • Lewis C.A.
      ). Reversal potentials were plotted as a function of the external Mg2+ activities, and the experimental data were fitted to the GHK equation (Fig.4 C). For wild type channels, the parameters that best fit the data were PK+/ PNa+ = 0.9 ± 0.1 and PMg2+/ PNa+ = 4.0 ± 0.3, which are in good agreement with those reported by others (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • Rosen T.A.
      • Levine J.D.
      • Julius D.
      ). This result implies that VR1 channels display a similar selectivity to K+ and Na+ and a preferential permeability for Mg2+ over Na+. Neutralization of Asp646 with asparagine decreased 4-fold the PMg2+/ PNa+(1.0 ± 0.2) without significantly affecting the permeability to monovalent cations ( PK+/ PNa+ = 0.8 ± 0.2). Thus, these findings using Mg2+ as a divalent cation hint that Asp646 contributes to define the permeability to divalent cations.

       Mutation of Met644 to Tyrosine Appears Not to Be Essential to Define Pore Properties

      The Asp646residue is located in the sequence motif TXGMGD, which is virtually identical to the K+ channel signature sequence TXGYGD (
      • Doyle D.A.
      • Cabral J.M.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • McKinnon R.
      ). To further investigate the role of this amino acid sequence in pore properties, we mutated Met644 to tyrosine. The M644Y mutant channel was functional and exhibited capsaicin-operated ionic currents in Xenopus oocytes (Fig.5 A). The capsaicin EC50 was not changed by the mutation (∼0.3 μm). Likewise, the incorporation of a tyrosine in Met644 did not alter the RR sensitivity (Table I). However, this mutant channel exhibited a remarkably slow kinetics of the capsaicin-operated responses (Fig. 5 C), suggesting that the amino acid at this position may modulate channel gating.
      Figure thumbnail gr5
      Figure 5A, mutation of Met644 to tyrosine produces functional capsaicin-gated ion channels. Band C, rescue of the nonfunctional phenotype of the single mutant E636K by the double mutant E636K/K639E. Ionic currents were elicited by 20 μm capsaicin. Membrane currents were recorded in the whole-cell voltage clamp configuration, atV h = −80 mV. Capsaicin was applied for the duration indicated by the horizontal bars. D, blockade of M644Y and E636K/K639E VR1 mutants by RR. Capsaicin-operated ionic currents were recorded in the absence and presence of 1 μm RR in the external bath. Capsaicin responses were normalized with respect to that in the absence of antagonist. Data are given as mean ± S.E., withN = 3.
      We also studied the relative ionic permeability of K + and Mg2+ to Na+. As illustrated in Table II, a 6-fold increase in the [Na]oshifted the reversal potential by 29 mV, while a 2.5-fold increment in the [Mg2+]o changed V r up to 7 mV toward depolarizing potentials. These data suggest that replacement of Met644 by tyrosine slightly modified the permeation properties. To further support this notion, we determined the relative ionic permeabilities to K+ and Mg2+ with respect to Na+ using the GHK approximation. The estimated ionic permabilities were PK+/ PNa+ = 1.1 ± 0.1 and PMg2+/ PNa+ = 2.9 ± 0.4. These results display a modest 20% increase and 30% decrease in K+ and Mg2+ permeability, respectively. Collectively, these data indicate that the residue at position 644 plays a marginal role in modulating the ionic permeability, similar to the function assigned to this amino acid inshaker-like K+ channels (
      • Heginbotham L.
      • Lu Z.
      • Abramson T.
      • MacKinnon R.
      ).

       Mutation of Lys639 to Glutamic Rescues Channel Activity of the E636K Mutant

      The proposed similar modular organization of VR1 and shaker-like channels implies a comparable pore structure composed of a selectivity filter and a pore helix (
      • Doyle D.A.
      • Cabral J.M.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • McKinnon R.
      ). The pore helix would encompass residues from Asn625 to Phe640 and contain residue Glu636, which could form an intrahelical salt bridge with Lys639 (Fig. 1). Incorporation of a positively charged residue at position Glu636 (E363K) renders nonfunctional VR1 channels (Fig.5 B), presumably by disrupting this interaction. Should be this the case, mutation of Lys639 to Glu in the mutant E636K can rescue channel function. We tested this hypothesis, and, in contrast to E636K (Fig. 5 B), heterologous expression of the double mutant in Xenopus oocytes gave rise to capsaicin-operated ionic currents that closely resemble those of wild type channel (Fig. 5 C). Indeed, the capsaicin EC50 for this mutant is analogous to wild type channels (data not shown). Furthermore, the E636K/K639E double mutant exhibits RR sensitivity (Fig. 5 D, Table I) and ionic permeability (Table II) similar to VR1 wild type. Thus, the E636K/K639E double mutant recapitulates the functional pore properties of wild type channels. This result is consistent with existence of a helix in the pore region of VR1 channels akin to that present inshaker-like K+ channels.

      DISCUSSION

      Homomeric VR1 channels expressed in Xenopusoocytes gave rise to capsaicin-activated ionic currents sensitive to RR inhibition and to receptors permeable to divalent cations. Site-specific neutralization of acidic residues in the putative pore-forming region influenced the pore attributes of homomeric VR1 channels. The most salient contribution of these studies is the identification of the amino acid at position 646 (Fig. 1) as a molecular determinant of pore properties such as blockade by ruthenium red and Mg2+ permeability. Replacement of Asp646 by asparagine created channels exhibiting lower sensitivity to RR blockade and reduced Mg2+ permeability with respect to Na+. Charge neutralization of Glu648 and Glu651 residues did not alter these VR1 pore properties, while mutation of Glu636 weakly modulated pore blockade. Modulation of both channel blockade and Mg2+ permeability by Asp646 suggests a direct interaction of RR and the divalent cation with this residue and implies that the spatial arrangement of carboxylic groups structures a high affinity binding site for cationic molecules in the pore, similar to that described for Ca2+-permeable channels (
      • Seifert R.
      • Eismann E.
      • Ludwing J.
      • Baumann A.
      • Kaupp U.B.
      ,
      • Klöckner
      • Mikala G.
      • Schwartz A.
      • Varady G.
      ,
      • Ellinor P.T.
      • Yang J.
      • Sather W.A.
      • Zhang J.-F.
      • Tsien W.A.
      ,
      • Premkumar L.S.
      • Auerbach A.
      ,
      • Beck C.
      • Woolmuth L.P.
      • Seeburg P.H.
      • Sakmann B.
      • Kuner T.
      ). It should be noted, however, that neutralization of Asp646 neither prevented completely RR blockade nor drastically changed the ionic selectivity, suggesting the contribution of other amino acids determining these pore properties. Consistent with this view, incorporation of a glutamine at position 636 increased ∼3-fold RR inhibition efficacy, suggesting a role of this amino acid in modulating pore blockade, perhaps by tuning the geometry of the blocker binding site. The double mutant E636Q/D646N, which could have further assisted in understanding the interplay of these two protein positions defining pore function did not generate capsaicin-activated channels, precluding any functional characterization.
      Although caution must be exercised when inferring protein structure from functional assays using site-directed mutagenesis, our data identify the P-loop on VR1 as a basic pore module that governs key properties of ion permeation and pore blockade. The proposed molecular model for VR1 channel resembles that established for shakertype channels; namely they encompass six transmembrane-spanning segments, representing the S5-P-S6 region of the pore module (Fig. 1). The high resolution structure of a bacterial K+channel (KcsA) from Streptomyces lividans, formed by only two transmembrane segments and a connecting amphipathic loop, has provided fundamental insights into the mechanisms underlying pore function (
      • Doyle D.A.
      • Cabral J.M.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • McKinnon R.
      ). Our functional observations endorse the tenet that VR1 channels exhibit a similar modular organization as K+channels, consistent with a model in which a ring of presumably four P-loops forms the inner core of the channel. This claim is further underscored by comparison of the amino acid sequences of KcsA and VR1 channels. In particular, there are seven residues in the C-end half of the P-loop that are virtually identical in both channel types, including the signature sequence motif TXGYGD that in VR1 channels is TXGMGD, thus suggesting a similar pore organization. A central question arises: is the spatial location of acidic residues in the VR1 channel consistent with their role in pore function? In analogy to the KcsA channel, the pore in the VR1 channel would be composed of a turret, pore helix, and selectivity filter (
      • Doyle D.A.
      • Cabral J.M.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • McKinnon R.
      ). This architecture would place Glu636 in the pore helix, Asp646 at the pore entrance, and Glu648 and Glu651 would be nearby the extracellular entryway. The finding that the nonfunctional phenotype of the E636K mutant can be efficiently rescued by the additional mutation of Lys639 to glutamic acid (E636K/K639E) is compatible with the tenet that these charged residues form an intrahelical salt bridge and, in turn, suggests that the segment comprising from Asn625 to Phe640 may have α-helical secondary structure (Fig. 1). Accordingly, the location of Glu636 would be consistent with a role providing stability to the channel selectivity filter and/or contributing to hold the putative tetramer together (
      • Doyle D.A.
      • Cabral J.M.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • McKinnon R.
      ). In contrast, Asp646 could structure a high affinity cation binding site right at the pore vestibule by strategically positioning a ring of carboxylate groups. In support of this view, substituted cysteine accessibility studies in the carboxyl half of the P region of shaker-like K+ channels, together with the crystallographic structure of KcsA, reveal that the acidic residue in the sequence motif TXGYGD is exposed at the outer mouth of the channel (
      • Doyle D.A.
      • Cabral J.M.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • McKinnon R.
      ,
      • Pascual J.M.
      • Shieh C.-C.
      • Kirsch G.E.
      • Brown A.M.
      ). A symmetric distribution of aspartic residues at the extracellular entryway would be essential to coordinate cationic molecules (
      • Fersht A.
      ), thus modulating ion permeation and blockade in this channel family. Positioning two additional acidic residues, Glu648 and Glu651, close to the permeation pathway will ensure a strong negative electrostatic potential required to raise the local concentration of positively charged molecules such as ruthenium red and cations.
      To further substantiate the proposed pore organization, we mutated Met644 in the VR1 sequence motif TXGMG to tyrosine (Fig. 1). The M644Y mutant channel exhibited capsaicin-operated ionic currents that were sensitive to RR block. The relative ionic permeability to K+ and Mg2+ with respect to Na+ was slightly lower than that characteristic of wild type channels, implying a marginal role of this residue in modulating ionic selectivity. This finding is in good agreement with studies in shaker-like K+ channels, showing that nonconservative mutations of the aromatic residue at this position (TXGYGD) leave the K+ selectivity intact (
      • Heginbotham L.
      • Lu Z.
      • Abramson T.
      • MacKinnon R.
      ). Indeed, the tyrosine side chains point away from the pore and make interactions with residues from the helix pore (
      • Doyle D.A.
      • Cabral J.M.
      • Pfuetzner R.A.
      • Kuo A.
      • Gulbis J.M.
      • Cohen S.L.
      • Chait B.T.
      • McKinnon R.
      ). It is noteworthy that, in both VR1 and shaker-like K+ channels, mutations at this position markedly affect channel gating (
      • Heginbotham L.
      • Lu Z.
      • Abramson T.
      • MacKinnon R.
      ). Additional work is necessary to understand the role of this amino acid in channel function.
      Collectively, our findings imply that the underlying pore-forming region of VR1 shares structural features of the well knownshaker-like K+ channels and indicate that Asp646 modulates VR1 pore properties. Nonetheless, the amino acid at this position is not sufficient to account for all pore properties of VR1, therefore suggesting the existence of additional structural determinants. Accordingly, it is plausible that amino acid residues at the extracellular end of S5 and S6 located near the membrane-water interface as well as amino acids in the linkers that connect these segments with the P-loop modulate pore attributes, as reported for cyclic GMP-gated ion channels (
      • Seifert R.
      • Eismann E.
      • Ludwing J.
      • Baumann A.
      • Kaupp U.B.
      ). Likewise, a contribution of nonacidic pore residues cannot be ruled out (
      • Williamson A.V.
      • Sather W.A.
      ). Further studies are needed to identify additional molecular determinants and to decipher the molecular mechanisms implicated in VR1 pore function. We have initiated a substituted cysteine accessibility reporter strategy to unveil the pore structure at the inner and outer surfaces. The proposed model should provide a testable hypothesis that may contribute to outline a molecular blueprint for the pore-forming region of this newly identified channel family.

      Acknowledgements

      We are indebted to David Julius for providing the VR1 subunit cDNA; to Remedios Torres for technical assistance with cRNA preparation, oocyte manipulation, and injection; to Remedios Galiana-Gregori for assisting in site-specific mutagenesis; and to Gregorio Fernández-Ballester for technical assistance. We thank José M. González-Ros and Marco Caprini for insightful comments on the manuscript.

      REFERENCES

        • Caterina M.J.
        • Schumacher M.A.
        • Tominaga M.
        • Rosen T.A.
        • Levine J.D.
        • Julius D.
        Nature. 1997; 389: 816-824
        • Tominaga M.
        • Caterina M.J.
        • Malmberg A.B.
        • Rosen T.A.
        • Gilvert H.
        • Skinner K.
        • Raumann B.E.
        • Basbaum A.I.
        • Julius D.
        Neuron. 1998; 21: 531-543
        • Caterina M.J.
        • Julius D.
        Curr. Opin. Neurobiol. 1999; 9: 525-530
        • Nagy I.
        • Rang H.P.
        J. Neurosci. 1999; 19: 10647-10665
        • Szallasi A.
        • Blumberg P.M.
        Pharmacol. Rev. 1999; 51: 159-211
        • Zygmunt P.
        • Peterson J.
        • Andersson D.
        • Chuang H.
        • Sørgård M.
        • Di Marzo V.
        • Julius D.
        • Högestätt E.
        Nature. 1999; 400: 452-457
        • Sterner O.
        • Szallasi A.
        Trends Pharmacol. Sci. 1999; 20: 459-465
        • Williams M.
        • Kowaluk E.A.
        • Arneric S.P.
        J. Med. Chem. 1999; 42: 1481-1500
        • Birnbaumer L.
        • Zhu X.
        • Jiang M.
        • Boulay G.
        • Peyton M.
        • Vannier B.
        • Brown D.
        • Platano D.
        • Sadeghi H.
        • Stefani E.
        • Birnbaumer M.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202
        • Suzuki M.
        • Sato J.
        • Kutsuwada K.
        • Ooki G.
        • Imai M.
        J. Biol. Chem. 1999; 274: 6330-6335
        • Caterina M.J.
        • Rosen T.A.
        • Tominaga M.
        • Brake A.J.
        • Julius D.
        Nature. 1999; 398: 436-441
        • Schumacher M.A.
        • Moff I.
        • Sudanagunta S.P.
        • Levine J.D.
        J. Biol. Chem. 2000; 275: 2756-2762
        • Jung J.
        • Hwang S.W.
        • Kwak J.
        • Lee S.-Y.
        • Kang C.-J.
        • Kim W.B.
        • Kim D.
        • Oh U.
        J. Neurosci. 1999; 19: 529-536
        • Doyle D.A.
        • Cabral J.M.
        • Pfuetzner R.A.
        • Kuo A.
        • Gulbis J.M.
        • Cohen S.L.
        • Chait B.T.
        • McKinnon R.
        Science. 1998; 280: 68-77
        • Dray A.
        • Forbes C.A.
        • Burgess G.M.
        Neurosci. Lett. 1990; 110: 52-59
        • Jort S.-V.
        • Tominaga M.
        • Julius D.
        Proc. Natl. Acad. Sci. 2000; 97: 8134-8139
        • Ferrer-Montiel A.V.
        • Montal M.
        Methods Companion Methods Enzymol. 1994; 6: 60-69
        • Ferrer-Montiel A.V.
        • Montal M.
        Methods Mol. Biol. 1999; 128: 167-178
        • Ferrer-Montiel A.V.
        • Sun W.
        • Montal M.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8021-8025
        • Ferrer-Montiel A.V.
        • Sun W.
        • Montal M.
        Biophys. J. 1996; 71: 749-758
        • Lewis C.A.
        J. Physiol. 1979; 286: 501-527
        • Miledi R.
        • Parker I.
        J. Physiol. 1985; 357: 173-183
        • Fersht A.
        Enzyme Structure and Mechanism. Freeman & Co., New York1985
        • Seifert R.
        • Eismann E.
        • Ludwing J.
        • Baumann A.
        • Kaupp U.B.
        EMBO J. 1999; 18: 119-130
        • Klöckner
        • Mikala G.
        • Schwartz A.
        • Varady G.
        J. Biol. Chem. 1996; 271: 22293-22296
      1. Bash, H., and Tora, H. (1992) The Chemistry of Acid Derivatives, Suppl. B, Vol. 2, pp. 1–50, John Wiley & Sons, Inc., New York.

      2. Zalaweski, R. I. (1992) The Chemistry of Acid Derivatives, Suppl. B, Vol. 2, pp. 305–369, John Wiley & Sons, Inc., New York.

        • Heginbotham L.
        • Lu Z.
        • Abramson T.
        • MacKinnon R.
        Biophys. J. 1994; 66: 1061-1067
        • Ellinor P.T.
        • Yang J.
        • Sather W.A.
        • Zhang J.-F.
        • Tsien W.A.
        Neuron. 1995; 15: 1121-1132
        • Premkumar L.S.
        • Auerbach A.
        Neuron. 1996; 16: 869-880
        • Beck C.
        • Woolmuth L.P.
        • Seeburg P.H.
        • Sakmann B.
        • Kuner T.
        Neuron. 1999; 22: 559-570
        • Pascual J.M.
        • Shieh C.-C.
        • Kirsch G.E.
        • Brown A.M.
        Neuron. 1995; 14: 1055-1063
        • Williamson A.V.
        • Sather W.A.
        Biophys. J. 1999; 77: 2575-2589