Tài liệu Báo cáo khoa học: Activation of the Torpedo nicotinic acetylcholine receptor The contribution of residues aArg55 and cGlu93 ppt

Activation of the Torpedo nicotinic acetylcholine receptor The contribution of residues aArg55 and cGlu93 Ankur Kapur, Martin Davies, William F Dryden and Susan M.J Dunn Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada Keywords acetylcholine; loop D; mutagenesis; nicotinic receptor; oocytes Correspondence S.M.J Dunn, Department of Pharmacology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada Fax: +780 4924325 Tel: +780 4923414 E-mail: susan.dunn@ualberta.ca (Received 22 October 2005, revised 13 December 2005, accepted 23 December 2005) doi:10.1111/j.1742-4658.2006.05121.x The Torpedo nicotinic acetylcholine receptor is a heteropentamer (a2bcd) in which structurally homologous subunits assemble to form a central ion pore Viewed from the synaptic cleft, the likely arrangement of these subunits is a–c–a–d–b lying in an anticlockwise orientation High affinity binding sites for agonists and competitive antagonists have been localized to the a–c and a–d subunit interfaces We investigated the involvement of amino acids lying at an adjacent interface (c–a) in receptor properties Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used to investigate the consequences of mutating aArg55 and cGlu93, residues that are conserved in most species of the peripheral nicotinic receptors Based on homology modeling, these residues are predicted to lie in close proximity to one another and it has been suggested that they may form a salt bridge in the receptor’s three-dimensional structure (Sine et al 2002 J Biol Chem 277, 29 210–29 223) Although substitution of aR55 by phenylalanine or tryptophan resulted in approximately a six-fold increase in the EC50 value for acetylcholine activation, the charge reversal mutation (aR55E) had no significant effect In contrast, the replacement of cE93 by an arginine conferred an eight-fold increase in the potency for acetylcholine-induced receptor activation In the receptor carrying the double mutations, aR55E-cE93R or aR55F-cE93R, the potency for acetylcholine activation was partially restored to that of the wild-type The results suggest that, although individually these residues influence receptor activation, direct interactions between them are unlikely to play a major role in the stabilization of different conformational states of the receptor The muscle-type nicotinic acetylcholine receptor (nAChR) is the prototype of the Cys-loop ligand-gated ion channel (LGIC) super-family that includes the neuronal nicotinic, c-aminobutyric acid (GABA) type A, 5-hydrotryptamine type (5-HT3) and glycine receptors This is largely a consequence of the abundance of this receptor in Torpedo electric organ, which facilitated its early purification and characterization The Torpedo nAChR is a pentameric transmembrane protein complex in which four structurally related subunits (a, b, c, d) in a stoichiometry of : : : assemble to form a central cation-selective ion channel [1,2] The a and b subunits of the Torpedo receptor referred to in this report correspond to the a1 and b1 subunits in the nomenclature recommended by the International Union of Pharmacology [3] Radioligand binding studies have demonstrated that, under equilibrium conditions, the nAChR carries two high affinity Abbreviations 5-HT3A receptor, serotonin type A receptor; a-BgTx, alpha-bungarotoxin; ACh, acetylcholine; AChBP, acetylcholine binding protein; dTC, d-tubocurarine; GABA, c-aminobutyric acid; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; PTMA, phenyltrimethylammonium; WT, wild-type 960 FEBS Journal 273 (2006) 960–970 ª 2006 The Authors Journal compilation ª 2006 FEBS A Kapur et al homologous dW57 (lying in what is now referred to as the loop D domain) as specific sites of ligand incorporation [5,15-17] In the present study, we have investigated the effects of mutations of the equivalent residue (aR55) lying in loop D of the a-subunit, i.e at the opposite side of the subunit from residues (in loops A– C) that have previously been implicated in agonist binding (Fig 1) Within the LGIC family, this residue in the peripheral nAChR is unique; whereas almost all subunits in the family have an aromatic residue at this position, a positively charged arginine residue is conserved in all peripheral a-subunits (see Fig 1) Previous comparative modeling studies have revealed that E93 of the c-subunit (lying in putative binding loop A) may lie in close proximity to aR55, leading to the proposal that an ionic interaction between these two residues may stabilize receptor conformation [18,19] This A 53 55 56 57 58 59 60 N N N N T Y V α1 α1 γ δ β2 A T.Ca_nAChR H_nAChR T.Ca_nAChR T.Ca_nAChR Rat_GABAA Rat_5-HT3 AChBP 54 V V V V M I F R R W W Y W W L L I M F Y Q R K E D Q R Q Q Q I H Q Q T Q Q Q A A F T W W W W W W W B β - D + δ α h - F D γ D AC + E F C h B A AC binding sites for agonists and competitive antagonists [4,5] It is now generally agreed that these sites lie at the interfaces between the a–c and the a–d subunits [6] Labeling and mutational studies have identified several key amino acids lying in discrete noncontiguous ‘loops’ of the a-subunits (designated as loops A–C, the ‘primary component’), together with amino acids in the neighboring c and d subunits (lying in loops D–F, the ‘secondary component’) that participate in forming these binding pockets [7–9] Although none of the ligand-gated ion channel family to which the nAChR belongs has been crystallized, the published structure of a related protein [10], the acetylcholine binding protein (AChBP), lends credence to current ideas of high affinity binding site location The AChBP, which is secreted by the glial cells of the snail, Lymnaea stagnalis, is a truncated homologue of the extracellular amino terminal domains of the nAChR[see 10] Inspection of its structure has reinforced earlier predictions that the residues involved in forming the binding sites occur at subunit–subunit interfaces and that the stretches of amino acids that have been implicated in binding are arranged in looplike structures The structural homology of all subunits in the LGIC family suggests that each of the five subunit–subunit interfaces contributes to ligand binding and ⁄ or the conformational changes that are involved in the transduction mechanism(s) that link agonist binding to channel opening This is particularly true of the homopentameric receptors, e.g the a7 neuronal nAChR and the 5-HT3A homomeric receptor In these receptors, there are five identical interfaces that presumably play equivalent roles in ligand recognition and receptor function In the heteromeric receptors, the roles of all five subunit interfaces are less clear Due to structural homology, all subunits carry all putative binding loops (A–F), suggesting that each interface has the potential to form a binding site, albeit with a distinct affinity arising from the nonequivalence of the intersubunit contacts within the pentamer Alternatively, these homologous loops at each interface may contribute to receptor assembly and ⁄ or play a role in the conformational changes that result in channel activation or receptor desensitization In the case of the Torpedo nAChR, the importance of the a-subunit in ligand binding has long been recognized [11-15], but the involvement of non a-subunit residues has become clear only more recently The first direct evidence for the contribution of the c and d subunits to ligand recognition came from photoaffinity labeling studies using [3H]nicotine and [3H]d-tubocurarine (dTC), which identified residues cW55 and the Role of a–c subunit interface in nAChR function C E B + - D A α Fig Loop D of the LGIC family (A) Amino acid sequence alignments of residues lying in loop D of the a1, c and d subunits from Torpedo californica (T Ca) nAChR, human (H) a1 nAChR subunit, b2 of rat GABAA receptor, rat 5-HT3A subunit and AChBP Numbering shown is for the Torpedo nAChR a1 subunit The positively charged R55 residue is unique to the peripheral nAChR a1 subunit since other members of the LGIC family have an aromatic amino acid in this position (B) Schematic representation of the subunit arrangement of the Torpedo nAChR showing the ‘six binding loop’ model of high affinity ligand binding sites Also represented is loop D of the a-subunit (not previously implicated in ligand binding), which lies at the b-a and c–a subunit interfaces FEBS Journal 273 (2006) 960–970 ª 2006 The Authors Journal compilation ª 2006 FEBS 961 Role of a–c subunit interface in nAChR function A Kapur et al residue is also conserved in peripheral nAChR c (and e) subunits We therefore also investigated the effects of a charge reversal mutation of this residue (cE93R) both alone and in combination with the aR55 mutations Our results demonstrate that mutations of these residues, which lie at an interface (c–a) that has not previously been implicated in receptor function, can have significant effects on ligand binding and ⁄ or channel gating However, we conclude that a direct interaction between aR55 and cE93 is unlikely to make a major contribution to nAChR properties Receptor Results Functional effects of aR55 mutations The functional responses of wild-type (WT) or mutant receptors expressed in Xenopus oocytes were studied using two-electrode voltage clamp techniques Figure shows the concentration-effect curves for ACh-mediated responses The WT nAChR receptor has an EC50 value for ACh-induced activation of $24 lm with an estimated Hill coefficient of 1.6 The substitution of aArg55 with glutamic acid (aR55E) or lysine (aR55K) resulted in a statistically insignificant shift in the EC50 values for ACh activation to 29 and 47 lm, respectively, and had no significant effect on the cooperativity of receptor activation In contrast, the aR55F and aR55W mutations caused a five- to six-fold shift in the EC50 for ACh activation to 112 and 151 lm, respectively In addition, the Hill coefficients for Ach-induced Fig ACh activation of wild-type (WT) and aR55 mutant receptors Concentration-effect curves obtained from oocytes expressing WT (n), aR55E (h), aR55K (n), aR55W (e) and aR55F (s) nAChR Data are normalized to Imax for each individual point The data represent the mean ± SEM from at least three oocytes The data obtained from curve-fitting are summarized in Table 962 Table Concentration-effect data for ACh activation of wild-type (WT) and mutant receptors expressed in Xenopus oocytes Data represent the mean ± SEM Values for log EC50 and Hill coefficient (nH) were determined from concentration-effect curves using GRAPHPAD PRISM software Log EC50 and Hill coefficients from individual curves were averaged to generate final mean estimates The values in parentheses are the number of oocytes used for each receptor type Statistical analysis was performed by comparing the log EC50 and nH of the mutant receptors to the WT nAChR (a p