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Contributions of Conserved Residues at the Gating Interface of Glycine Receptors*

  • Stephan A. Pless
    Correspondence
    To whom correspondence should be addressed: 2350 Health Science Mall, Vancouver, BC V6T 1Z3, Canada. Tel.: 604-827-4189
    Affiliations
    Departments of Anesthesiology, Pharmacology, and Therapeutics and the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada
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  • Ada W.Y. Leung
    Affiliations
    Departments of Anesthesiology, Pharmacology, and Therapeutics and the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada
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  • Jason D. Galpin
    Affiliations
    Departments of Anesthesiology, Pharmacology, and Therapeutics and the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada
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  • Christopher A. Ahern
    Affiliations
    Departments of Anesthesiology, Pharmacology, and Therapeutics and the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada
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  • Author Footnotes
    * This work was supported by Canadian Institutes of Health Research Grant 56858, the Heart and Stroke Foundation of Canada, the Michael Smith Foundation for Health Research (to C. A. A.), and a postdoctoral fellowship by the Heart and Stroke Foundation of Canada (to S. A. P.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.
Open AccessPublished:August 11, 2011DOI:https://doi.org/10.1074/jbc.M111.269027
      Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.

      Introduction

      The glycine receptor (GlyR)
      The abbreviations used are: GlyR
      glycine receptor
      pLGIC
      pentameric ligand-gated ion channel
      Akp
      2-amino-4-ketopentanoic acid
      GABAA and GABAC
      γ-aminobutyric acid, types A and C, respectively
      GluCl
      glutamate-gated chloride channel
      LBD
      ligand binding domain
      M1-M4
      transmembrane segments 1–4
      Nha
      nitrohomoalanine
      TMD
      transmembrane domain
      nAChR
      nicotinic acetylcholine receptor.
      chloride channel is a member of the Cys-loop receptor family, a subfamily of the pentameric ligand-gated ion channel (pLGIC) superfamily (
      • Lynch J.W.
      ). GlyRs mediate fast inhibitory neurotransmission in the nervous system, and recent studies have provided a wealth of insight into the structure and function of the GlyR and other pLGICs. One of the more studied regions, the N-terminal ligand binding domain (LBD), is composed of a 10-strand β-sheet sandwich interconnected by 9 loops and the ligand binding pocket situated at the interface between adjacent subunits (
      • Grudzinska J.
      • Schemm R.
      • Haeger S.
      • Nicke A.
      • Schmalzing G.
      • Betz H.
      • Laube B.
      ,
      • Pless S.A.
      • Millen K.S.
      • Hanek A.P.
      • Lynch J.W.
      • Lester H.A.
      • Lummis S.C.
      • Dougherty D.A.
      ,
      • Bocquet N.
      • Nury H.
      • Baaden M.
      • Le Poupon C.
      • Changeux J.P.
      • Delarue M.
      • Corringer P.J.
      ,
      • Hilf R.J.
      • Dutzler R.
      ,
      • Hilf R.J.
      • Dutzler R.
      ,
      • Pless S.A.
      • Hanek A.P.
      • Price K.L.
      • Lynch J.W.
      • Lester H.A.
      • Dougherty D.A.
      • Lummis S.C.
      ,
      • Hibbs R.E.
      • Gouaux E.
      ). The transmembrane domain (TMD) contains four α-helical segments (M1-M4), including the pore-lining M2 helices.
      How is the binding of an agonist molecule in the LBD communicated to the channel gate in the TMD, almost 60 Å away? This question has gained considerable attention in the past (
      • Dougherty D.A.
      ,
      • Lester H.A.
      • Dibas M.I.
      • Dahan D.S.
      • Leite J.F.
      • Dougherty D.A.
      ,
      • Miller P.S.
      • Smart T.G.
      ), and previous studies have identified a number of charged residues likely to couple the LBD to the TMD via electrostatic interactions in different pLGICs (
      • Kash T.L.
      • Dizon M.J.
      • Trudell J.R.
      • Harrison N.L.
      ,
      • Kash T.L.
      • Jenkins A.
      • Kelley J.C.
      • Trudell J.R.
      • Harrison N.L.
      ,
      • Lee W.Y.
      • Sine S.M.
      ,
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ,
      • Price K.L.
      • Millen K.S.
      • Lummis S.C.
      ,
      • Xiu X.
      • Hanek A.P.
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ). A number of critical residues have been identified at the interface of LBD and TMD of α1 GlyRs (
      • Absalom N.L.
      • Lewis T.M.
      • Kaplan W.
      • Pierce K.D.
      • Schofield P.R.
      ,
      • Cederholm J.M.
      • Absalom N.L.
      • Sugiharto S.
      • Griffith R.
      • Schofield P.R.
      • Lewis T.M.
      ,
      • Schofield C.M.
      • Jenkins A.
      • Harrison N.L.
      ,
      • Schofield C.M.
      • Trudell J.R.
      • Harrison N.L.
      ,
      • Crawford D.K.
      • Perkins D.I.
      • Trudell J.R.
      • Bertaccini E.J.
      • Davies D.L.
      • Alkana R.L.
      ), but evidence for direct electrostatic interactions is thus far missing. The interface of LBD and TMD in α1 GlyRs is of interest not only because of its proposed role in channel gating but also because inherited mutations of side chains near this interface cause hyperekplexia or startle disease (
      • Ryan S.G.
      • Sherman S.L.
      • Terry J.C.
      • Sparkes R.S.
      • Torres M.C.
      • Mackey R.W.
      ): Ala-52 in loop 2 (
      • Ryan S.G.
      • Buckwalter M.S.
      • Lynch J.W.
      • Handford C.A.
      • Segura L.
      • Shiang R.
      • Wasmuth J.J.
      • Camper S.A.
      • Schofield P.
      • O'Connell P.
      ), Leu-184 in loop 9 (
      • Chung S.K.
      • Vanbellinghen J.F.
      • Mullins J.G.
      • Robinson A.
      • Hantke J.
      • Hammond C.L.
      • Gilbert D.F.
      • Freilinger M.
      • Ryan M.
      • Kruer M.C.
      • Masri A.
      • Gurses C.
      • Ferrie C.
      • Harvey K.
      • Shiang R.
      • Christodoulou J.
      • Andermann F.
      • Andermann E.
      • Thomas R.H.
      • Harvey R.J.
      • Lynch J.W.
      • Rees M.I.
      ), Arg-218 in the pre-M1 domain (
      • Castaldo P.
      • Stefanoni P.
      • Miceli F.
      • Coppola G.
      • Del Giudice E.M.
      • Bellini G.
      • Pascotto A.
      • Trudell J.R.
      • Harrison N.L.
      • Annunziato L.
      • Taglialatela M.
      ), and Arg-271 and Lys-276 in the M2-M3 linker (
      • Rajendra S.
      • Lynch J.W.
      • Pierce K.D.
      • French C.R.
      • Barry P.H.
      • Schofield P.R.
      ,
      • Elmslie F.V.
      • Hutchings S.M.
      • Spencer V.
      • Curtis A.
      • Covanis T.
      • Gardiner R.M.
      • Rees M.
      ).
      Based on sequence alignment and structural data from other pLGICs, we have identified five highly conserved charged and aromatic amino acid side chains that line the interface of LBD and TMD: Glu-53 in loop 2, Phe-145 and Asp-148 in loop 7 (or Cys-loop), Phe-187 in loop 9, and Arg-218 in the pre-M1 domain (Fig. 1). These charged side chains are of particular interest because the equivalent side chain to Arg-218 has been proposed to interact with the equivalent residues to Glu-53 in GABAC receptors (
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ,
      • Price K.L.
      • Millen K.S.
      • Lummis S.C.
      ), whereas it is thought to interact with the side chain equivalent to Asp-148 in the β2 subunit of GABAA receptors (
      • Kash T.L.
      • Dizon M.J.
      • Trudell J.R.
      • Harrison N.L.
      ). We used natural and unnatural amino acid side-chain substitutions to probe the contributions of these side chains to channel gating and tested for possible electrostatic interactions between these residues.
      Figure thumbnail gr1
      FIGURE 1Sequence alignment and molecular model. A, shown is sequence alignment of various pLGICs. Shown are loops 2, 7, and 9 as well as the pre-M1 domain, with highly conserved aromatic (Phe-145, Phe-187), basic (Arg-218), and acidic (Glu-53, Asp-148) side chains highlighted in gray, blue, and red, respectively. B, a model of GluCl α (PDB code 3RHW) shows the LBD and the TMD. The inset shows the five side chains as highlighted in A. Note that the residues 53 and 145 have been mutated in silico to the corresponding α1 GlyR side chains with no further energy minimization (Glu-53 is a Val in GluCl α; Phe-145 is a Tyr in GluCl α).

      DISCUSSION

      The mechanistic nature of the linkage between the LBD and the TMD in pLGICs has garnered much attention, and significant progress has been made using structural (
      • Bocquet N.
      • Nury H.
      • Baaden M.
      • Le Poupon C.
      • Changeux J.P.
      • Delarue M.
      • Corringer P.J.
      ,
      • Hilf R.J.
      • Dutzler R.
      ,
      • Hilf R.J.
      • Dutzler R.
      ,
      • Hibbs R.E.
      • Gouaux E.
      ) and functional approaches (
      • Kash T.L.
      • Dizon M.J.
      • Trudell J.R.
      • Harrison N.L.
      ,
      • Kash T.L.
      • Jenkins A.
      • Kelley J.C.
      • Trudell J.R.
      • Harrison N.L.
      ,
      • Lee W.Y.
      • Sine S.M.
      ,
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ,
      • Price K.L.
      • Millen K.S.
      • Lummis S.C.
      ,
      • Xiu X.
      • Hanek A.P.
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ,
      • Purohit P.
      • Auerbach A.
      ,
      • Bouzat C.
      • Gumilar F.
      • Spitzmaul G.
      • Wang H.L.
      • Rayes D.
      • Hansen S.B.
      • Taylor P.
      • Sine S.M.
      ). However, in α1 GlyRs, little is known about possible electrostatic interactions that link the LBD to the TMD in other pLGICs. Here, we show that although individual cysteine mutations at positions 148 and 218 result in non-functional receptors, the double cysteine mutant Asp-148 → Cys,Arg-218 → Cys produces robust glycine-gated currents and displays functional properties that resemble those of WT receptors. This is a surprising finding given the severity of the side chain replacements in positions 148 and 218 (Asp to Cys and Arg to Cys, respectively), and the mutual rescue implies a strong physical coupling between the two cysteines. We thus propose that the two cysteine side chains are likely to form a disulfide bridge. As disulfide bridges can only form over short (2–3 Å) distances, our results suggest that the side chains in positions 148 and 218 may be in close (and likely static) proximity during channel gating. Moreover, as such short distances are ideal for salt bridge formation, we propose that Asp-148 and Arg-218 form in the native receptor. This notion is further supported by the requisite nature of charge at positions 148 and 218, negative and positive, respectively, for normal channel function in the α1 GlyR. The fact that the double charge reverse mutant Asp-148 → Arg,Arg-218 → Asp (as well as the single charge reverse mutants, Asp-148 → Arg and Arg-218 → Asp) did not result in functional channels could be due to the severe disruption of the highly conserved charge pattern of the interface between LBD and TMD (
      • Xiu X.
      • Hanek A.P.
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ), which shows a strong preference for negative charges in the LBD, whereas the TMD primarily contains positive charges. The idea of an electrostatic interaction between Asp-148 and Arg-218 is further supported by another study that suggested an interaction between the equivalent residues in the β2 subunit of GABAA receptors (
      • Kash T.L.
      • Dizon M.J.
      • Trudell J.R.
      • Harrison N.L.
      ). The proposed interaction between Asp-148 and Arg-218 in the α1 GlyR is of particular interest as mutations at side chains equivalent to α1 GlyR Arg-218 have been shown to be crucial for channel function in different pLGICs (
      • Kash T.L.
      • Dizon M.J.
      • Trudell J.R.
      • Harrison N.L.
      ,
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ,
      • Purohit P.
      • Auerbach A.
      ,
      • Vicente-Agullo F.
      • Rovira J.C.
      • Sala S.
      • Sala F.
      • Rodriguez-Ferrer C.
      • Campos-Caro A.
      • Criado M.
      • Ballesta J.J.
      ,
      • Keramidas A.
      • Kash T.L.
      • Harrison N.L.
      ,
      • Mercado J.
      • Czajkowski C.
      ) and especially because the α1 GlyR Arg-218 → Gln mutation can give rise to the inherited hyperekplexia or startle disease (
      • Castaldo P.
      • Stefanoni P.
      • Miceli F.
      • Coppola G.
      • Del Giudice E.M.
      • Bellini G.
      • Pascotto A.
      • Trudell J.R.
      • Harrison N.L.
      • Annunziato L.
      • Taglialatela M.
      ). The idea that Arg-218 contributes to a salt bridge crucial for channel gating in the native receptor provides an explanation for molecular basis for a pathophysiologically relevant mutant; the neutral Arg-218 → Gln mutation would prevent the formation of a strong salt bridge and, hence, give rise to the drastic effect on gating observed with this mutant. It further explains why Arg-218 → Gln results in functional receptors, albeit with severe functional impairments, whereas other replacements such as Cys and Asp do not; in the proposed salt bridge interaction with Asp-148, Arg-218 would (in addition to contributing a positive charge) act as a strong hydrogen bond donor. Although uncharged, Gln is a potent hydrogen bond donor, in contrast to Cys or Asp, and could interact with the hydrogen bond accepting Asp-148, although to a weaker extent than Arg.
      The emerging picture for the role of Arg-218 thus points to two crucial roles; first, Arg-218 contributes to an interaction with Asp-148 that is crucial for channel gating. This functionally important interaction can be restored by the proposed disulfide bond in the Asp-148 → Cys,Arg-218 → Cys double mutant. Second, and in agreement with previous studies on the equivalent residue of different nAChRs (
      • Purohit P.
      • Auerbach A.
      ,
      • Vicente-Agullo F.
      • Rovira J.C.
      • Sala S.
      • Sala F.
      • Rodriguez-Ferrer C.
      • Campos-Caro A.
      • Criado M.
      • Ballesta J.J.
      ) and α1 GlyR Arg-218 (
      • Castaldo P.
      • Stefanoni P.
      • Miceli F.
      • Coppola G.
      • Del Giudice E.M.
      • Bellini G.
      • Pascotto A.
      • Trudell J.R.
      • Harrison N.L.
      • Annunziato L.
      • Taglialatela M.
      ), α1 GlyR Arg-218 is important for channel expression. Our data show that in contrast to the functionally relevant salt bridge with Asp-148, which can be mimicked by the proposed disulfide bond, the positive charge of Arg-218 is required for efficient expression, as the Asp-148 → Cys,Arg-218 → Cys double mutant, although functionally similar to WT, shows dramatically reduced expression levels (Table 1).
      The available structural data suggest that the proposed salt bridge between Asp-148 and Arg-218 is flanked by Phe-145 and Phe-187. Interestingly, our data suggest that large hydrophobic side chains are required in positions 145 and 187, although aromaticity is not, thus ruling out contributions of the electrostatic surface potential of these side chains to channel gating. This finding not only excludes the possibility of a cation-π interaction between Arg-218 and either of the two aromatic side chains, but it also raises an intriguing possibility about the role of Phe-145 and Phe-187; given that salt bridges are significantly stronger in hydrophobic, compared with aqueous, environments (
      • Pless S.A.
      • Galpin J.D.
      • Niciforovic A.P.
      • Ahern C.A.
      ,
      • Gallivan J.P.
      • Dougherty D.A.
      ), it is possible that it is the primary role of Phe-145 and Phe-187 to provide/promote a hydrophobic environment for the formation of an energetically significant salt bridge between Asp-148 and Arg-218. Additionally, Phe-145 and Phe-187 could participate in hydrophobic interactions with Ile-51 (
      • Crawford D.K.
      • Perkins D.I.
      • Trudell J.R.
      • Bertaccini E.J.
      • Davies D.L.
      • Alkana R.L.
      ) and Leu-274 (
      • Lynch J.W.
      • Rajendra S.
      • Pierce K.D.
      • Handford C.A.
      • Barry P.H.
      • Schofield P.R.
      ), respectively. Furthermore, a recent study on the α1 GlyR Phe-145 equivalent in nAChRs proposed an interaction with the equivalent of α1 GlyR Pro-146 (Pro-136 in nAChR) (
      • Limapichat W.
      • Lester H.A.
      • Dougherty D.A.
      ). However, it should be noted that the same study also found a strong preference for aromaticity at the position equivalent to Phe-145 in nAChRs, a finding not in agreement with our data, suggesting that, although conserved, the same residues may support different functional roles in GlyRs and nAChRs. Although our data do not directly speak to a possible interaction with the adjacent Pro-146, we propose that the principal role of Phe-145 (and Phe-187) is to provide a hydrophobic framework for a strong electrostatic interaction between Asp-148 and Arg-218, a notion that is further supported by recent structural data from GluCl α (supplemental Fig. 2).
      Although the general importance of loop 2 for channel gating is uncontested in α1 GlyRs (
      • Cederholm J.M.
      • Absalom N.L.
      • Sugiharto S.
      • Griffith R.
      • Schofield P.R.
      • Lewis T.M.
      ,
      • Crawford D.K.
      • Perkins D.I.
      • Trudell J.R.
      • Bertaccini E.J.
      • Davies D.L.
      • Alkana R.L.
      ,
      • Pless S.A.
      • Lynch J.W.
      ,
      • Plested A.J.
      • Groot-Kormelink P.J.
      • Colquhoun D.
      • Sivilotti L.G.
      ) and other pLGICs (
      • Lee W.Y.
      • Sine S.M.
      ,
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ,
      • Price K.L.
      • Millen K.S.
      • Lummis S.C.
      ,
      • Aldea M.
      • Castillo M.
      • Mulet J.
      • Sala S.
      • Criado M.
      • Sala F.
      ,
      • Sala F.
      • Mulet J.
      • Sala S.
      • Gerber S.
      • Criado M.
      ,
      • Chakrapani S.
      • Bailey T.D.
      • Auerbach A.
      ,
      • McLaughlin J.T.
      • Fu J.
      • Rosenberg R.L.
      ,
      • Lee W.Y.
      • Free C.R.
      • Sine S.M.
      ), we propose that in α1 GlyRs there is no crucial contribution of the negative charge of Glu-53 to channel gating. Instead, it appears more likely that the side chain in position 53 of α1 GlyRs does not require a negative charge per se but a hydrophilic head group with no propensity to donate hydrogens, as hydrogens could result in steric and/or electrostatic clashes with other residues at the interface of LBD and TMD. It should be noted that Glu and Asp are the only naturally occurring side chains that fulfill these criteria and that both these side chains (
      • Absalom N.L.
      • Lewis T.M.
      • Kaplan W.
      • Pierce K.D.
      • Schofield P.R.
      ) as well as Nha and Akp (Fig. 5) are the only side chains that result in WT-like gating behavior at position 53 of the α1 GlyR. This is in agreement with a recent study that proposed an alcohol binding pocket that is flanked by loop 2, the pre-M1 domain, M2, and the M2-M3 linker (
      • Crawford D.K.
      • Trudell J.R.
      • Bertaccini E.J.
      • Li K.
      • Davies D.L.
      • Alkana R.L.
      ); odd-numbered residues in loop 2, such as Glu-53, are thought to point into this pocket (
      • Crawford D.K.
      • Perkins D.I.
      • Trudell J.R.
      • Bertaccini E.J.
      • Davies D.L.
      • Alkana R.L.
      ), possibly explaining the strict requirement for a hydrophilic side chain in position 53 as this would help create and maintain a likely water-filled allosteric ligand binding pocket. We thus propose that although an electrostatic interaction between the equivalent residues of α1 GlyR Glu-53 and Arg-218 has been proposed in GABAC receptors (
      • Wang J.
      • Lester H.A.
      • Dougherty D.A.
      ,
      • Price K.L.
      • Millen K.S.
      • Lummis S.C.
      ) and nAChRs (
      • Lee W.Y.
      • Sine S.M.
      ), such an interaction is not present in α1 GlyRs. However, the altered Hill coefficient for Nha at position 53 could indicate that the negative charge mildly contributes to cooperativity between adjacent subunits.
      In conclusion, we propose a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating in α1 GlyRs and has implications for the clinically relevant hyperekplexia mutant Arg-218 → Gln. The data further support a role for two highly conserved aromatic residues in loop 7 and loop 9 in shielding the proposed salt bridge between Asp-148 and Arg-218 from a polar environment, which would otherwise weaken the interaction. The study thus highlights important aspects of the α1 GlyR gating pathway and provides insight into the molecular mechanisms for a mutation that gives rise to a channelopathy.

      Acknowledgments

      We thank Dr. Ana Niciforovic for excellent technical assistance.

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