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

Open AccessPublished:October 20, 2000DOI:https://doi.org/10.1074/jbc.M002391200
      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.

      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.

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