HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 42, 32552-32558, October 20, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/42/32552    most recent M002391200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by García-Martínez, C. Articles by Ferrer-Montiel, A. Search for Related Content PubMed PubMed Citation Articles by García-Martínez, C. Articles by Ferrer-Montiel, A. Social Bookmarking                      What's this?

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

Carolina García-Martínez, Cruz Morenilla-Palao, Rosa Planells-Cases, Jaime M. Merino^§, and Antonio Ferrer-Montiel^¶

From the  Centro de Biología Molecular y Celular, Universidad Miguel Hernández, Elche (Alicante) 03202, Spain and ^§ Departamento Bioquímica y Biología Molecular, Universidad de Extremadura, Badajoz 06071, Spain

Received for publication, March 21, 2000, and in revised form, July 24, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 in Xenopus 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 Asp⁶⁴⁶ by 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1). VR1 is a nonselective cation channel with high Ca²⁺ permeability that integrates both types of pain-producing stimuli (1-5). These channels are activated by vanilloids such as capsaicin, the pungent ingredient of hot red peppers, and by temperatures higher than 40 °C (1, 2, 4). Recently, the lipid-based anandanamide was shown to be a potential endogenous VR1 agonist (6). Activation of the VR1 channel raises intracellular Ca²⁺ and excites a subset of dorsal root and trigeminal ganglion primary neurons (5). These neurons transmit noxious information to the central nervous system and release proinflammatory neuropeptides at peripheral terminals (5, 7). In addition to playing a role in nociception, the high Ca²⁺ permeability exhibited by VR1 strongly desensitizes capsaicin-operated responses (5, 7). This property partially accounts for the antinociceptive activity exhibited by vanilloids (5, 8, 9).

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 (1, 3, 10). 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 (1, 2, 4, 5, 7). Nonetheless, there is mounting evidence for molecular heterogeneity of vanilloid receptors, including the identification of a stretch-inactivating channel (11), a vanilloid receptor-like protein (VRl-1) (12), and an N-terminal splice variant of VR1 (VR.5'sv) (13).

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 (13, 14). 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 (15). 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) (1, 2, 4, 5, 16). 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 (17). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1) (kindly provided by Dr. David Julius). Site-directed mutagenesis was carried out by polymerase chain reaction as described (19, 20). Mutant receptors were confirmed by DNA sequencing. Capped cRNA was synthesized using the mMESSAGE mMACHINE^TM from Ambion (Austin, TX). 2-5 ng of cRNA was microinjected (V = 50 nl) into defolliculated oocytes (stages V and VI) as described (19). 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 (19, 20). 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 mM BaCl₂, 2.0 mM MgCl₂). Homomeric VR-1 channels were activated by application of 20 µM capsaicin 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 (21),

(Eq. 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 n is 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 (22). 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 (22, 23). As a divalent cation, we used Mg²⁺, because, at variance with Ca²⁺, this cation does not block or desensitize VR channels (1, 2). Reversal potentials (V_r) were obtained in the following external solutions: 125 mM sodium, 2 mM magnesium, 125 mM NaCl, 2.0 mM MgCl₂; 20 mM sodium, 1 mM magnesium, 20 mM NaCl, 1.0 mM MgCl₂, 105 mM N-methyl-D-glucamine (NMG); 20 mM sodium, 2 mM magnesium, 20 mM NaCl, 2.0 mM MgCl₂, 100 mM NMG; 20 mM sodium, 5 mM magnesium; 20 mM NaCl, 5.0 mM MgCl₂, 95 mM NMG; 20 mM sodium, 10 mM magnesium, 20 mM NaCl, 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 (24), 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 (21, 22),

(Eq. 2)

where P_K⁺/P_Na⁺ and P_Mg⁺/P_Na⁺ 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 ² in the fitting of the experimental data to the GHK equation. Activity coefficients () were taken as 0.25 and 0.75 for Mg²⁺ and monovalent cations (Na⁺ and K⁺), respectively (1). 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 mM sodium/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) (21). The goodness of fit was inferred from the ² test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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. 2A). Higher vanilloid concentrations accelerated receptor activation and concomitantly delayed agonist removal (Fig. 2B). These capsaicin-operated responses exhibited an EC₅₀ (concentration of agonist to activate half-maximal response) of ~0.5 µM, in agreement with other reports (1, 2). 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).

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Putative 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.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Neutralization 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, at Vh = 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. 2B). As illustrated in Fig. 2C, 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 Glu⁶⁴⁸ to 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 (Table I). In contrast, charge neutralization of Asp⁶⁴⁶ (D646N) generated channels that were significantly less sensitive to RR than wild type receptors (Fig. 2E). 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.

View this table: [in this window] [in a new window]   Table I Ruthenium red blockade efficacy of VR1 channels Experimental values were fitted to the logistic equation Iblocker/Iagonist = 1/(1+(blocker/IC50)n), where IC50 denotes the [blocker] to inhibit half of the maximal agonist response, and n 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 Glu⁶⁴⁸ and 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.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Amino 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 I). 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 (pH_o). 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 pH_o 6.4 (Table I) as compared with the neutral pH_o 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 pH_o 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 (25), as evidenced for Ca²⁺ channels and cyclic GMP-gated channels (26, 27). These studies could not be carried out at pH_o 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 pH_o 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 (28, 29). 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 (24); 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 (1, 2). 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 mM MgCl₂ are fairly linear, with a slight inward rectification at negative membrane potentials, and exhibit a reversal potential (V_r) of 36 ± 3 mV (Fig. 4A). A 10-fold increase in [Mg²⁺]_o shifted the reversal potential up to 25 mV toward more depolarizing potentials (Fig. 4A), 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. 4B). As shown, a 10-fold increase in [Mg²⁺]_o moved V_r ~10 mV toward positive potentials. This displacement of V_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 (Table II). Analysis of I-V relationships obtained varying the [Na⁺]_o suggested that the permeability to monovalent cations was unaffected by the mutations (Table II).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   D646N 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. The arrows 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 2 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. with N = 4.

View this table: [in this window] [in a new window]   Table II Reversal potentials of VR1 mutants Reversal potentials (Vr) were obtained from I-V relationships as the voltage at which the capsaicin-evoked ionic current was zero. Vr denotes the change in reversal potential as a result of the extracellular [Na+]o from 20 to 125 mM ([Na+]o) or the [Mg2+]o from 2 to 5 mM ([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.

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 (22, 23). Reversal potentials were plotted as a function of the external Mg²⁺ activities, and the experimental data were fitted to the GHK equation (Fig. 4C). For wild type channels, the parameters that best fit the data were P_K⁺/P_Na⁺ = 0.9 ± 0.1 and P_Mg²⁺/P_Na⁺ = 4.0 ± 0.3, which are in good agreement with those reported by others (1). 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 P_Mg²⁺/P_Na⁺ (1.0 ± 0.2) without significantly affecting the permeability to monovalent cations (P_K⁺/P_Na⁺ = 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 Asp⁶⁴⁶ residue is located in the sequence motif TXGMGD, which is virtually identical to the K⁺ channel signature sequence TXGYGD (15). 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. 5A). 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. 5C), suggesting that the amino acid at this position may modulate channel gating.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   A, mutation of Met644 to tyrosine produces functional capsaicin-gated ion channels. B and 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, at Vh = 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., with N = 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]_o shifted 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 P_K⁺/P_Na⁺ = 1.1 ± 0.1 and P_Mg²⁺/P_Na⁺ = 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 in shaker-like K⁺ channels (30).

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 (15). 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. 5B), 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. 5B), 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. 5C). 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. 5D, 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 in shaker-like K⁺ channels.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Homomeric VR1 channels expressed in Xenopus oocytes 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 (26, 27, 31-33). 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 shaker type 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 (15). 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 (15). 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 -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 (15). 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 (15, 34). A symmetric distribution of aspartic residues at the extracellular entryway would be essential to coordinate cationic molecules (25), 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 (30). Indeed, the tyrosine side chains point away from the pore and make interactions with residues from the helix pore (15). It is noteworthy that, in both VR1 and shaker-like K⁺ channels, mutations at this position markedly affect channel gating (30). 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 known shaker-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 (26). Likewise, a contribution of nonacidic pore residues cannot be ruled out (35). 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.

	FOOTNOTES

^* This work was supported by a joint grant from the Comisión Interministerial de Ciencia y Tecnología and the European Commission 1FD97-0662-C02-01, and La Fundación La Caixa Grant 98/027-00 (to A. F. M.). A preliminary account of these results was presented elsewhere (18).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Centro de Biología Molecular y Celular, Universidad Miguel Hernández, Avda Ferrocarril s/n, Elche (Alicante) 03202, Spain. Tel.: 34-966658727; Fax: 34-966658758; E-mail: aferrer@umh.es.

Published, JBC Papers in Press, August 7, 2000, DOI 10.1074/jbc.M002391200

	ABBREVIATIONS

The abbreviations used are: VR1, vanilloid receptor subunit 1; RR, ruthenium red; GHK equation, Goldman-Hodgkin-Katz equation for the constant field approximation; pH_o, extracellular pH; NMG, N-methyl-D-glucamine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Y. Y. Wang, R. B. Chang, H. N. Waters, D. D. McKemy, and E. R. Liman The Nociceptor Ion Channel TRPA1 Is Potentiated and Inactivated by Permeating Calcium Ions J. Biol. Chem., November 21, 2008; 283(47): 32691 - 32703. [Abstract] [Full Text] [PDF]

				D. S. K. Samways, B. S. Khakh, and T. M. Egan Tunable Calcium Current through TRPV1 Receptor Channels J. Biol. Chem., November 14, 2008; 283(46): 31274 - 31278. [Abstract] [Full Text] [PDF]

				A. Jara-Oseguera, I. Llorente, T. Rosenbaum, and L. D. Islas Properties of the Inner Pore Region of TRPV1 Channels Revealed by Block with Quaternary Ammoniums J. Gen. Physiol., November 1, 2008; 132(5): 547 - 562. [Abstract] [Full Text] [PDF]

				R. Xia, Z.-Z. Mei, H.-J. Mao, W. Yang, L. Dong, H. Bradley, D. J. Beech, and L.-H. Jiang Identification of Pore Residues Engaged in Determining Divalent Cationic Permeation in Transient Receptor Potential Melastatin Subtype Channel 2 J. Biol. Chem., October 10, 2008; 283(41): 27426 - 27432. [Abstract] [Full Text] [PDF]

				M. Mederos y Schnitzler, J. Waring, T. Gudermann, and V. Chubanov Evolutionary determinants of divergent calcium selectivity of TRPM channels FASEB J, May 1, 2008; 22(5): 1540 - 1551. [Abstract] [Full Text] [PDF]

				R. Xiao, J. Tang, C. Wang, C. K. Colton, J. Tian, and M. X. Zhu Calcium Plays a Central Role in the Sensitization of TRPV3 Channel to Repetitive Stimulations J. Biol. Chem., March 7, 2008; 283(10): 6162 - 6174. [Abstract] [Full Text] [PDF]

				S. Ryu, B. Liu, J. Yao, Q. Fu, and F. Qin Uncoupling Proton Activation of Vanilloid Receptor TRPV1 J. Neurosci., November 21, 2007; 27(47): 12797 - 12807. [Abstract] [Full Text] [PDF]