Aromatic Residues ∈Trp-55 and δTrp-57 and the Activation of Acetylcholine Receptor Channels

The results indicate that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions.

Abstract

The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the ∈ subunit and Trp-57 in the δ subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the “allosteric” cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the ∈ subunit. (iii) In ∈ (but not δ) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Φ value analyses (of both tryptophan residues) show Φ ∼1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions. The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the ∈ subunit and Trp-57 in the δ subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the “allosteric” cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the ∈ subunit. (iii) In ∈ (but not δ) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Φ value analyses (of both tryptophan residues) show Φ ∼1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions. Acetylcholine receptor channels are allosteric proteins that “gate” the flow of ions at the vertebrate nerve-muscle synapse (1Karlin A. Methods Enzymol. 1977; 46: 582-590Crossref PubMed Scopus (17) Google Scholar, 2Sine S.M. Engel A.G. Nature. 2006; 440: 448-455Crossref PubMed Scopus (426) Google Scholar, 3Unwin N. J. Struct. Biol. 1998; 121: 181-190Crossref PubMed Scopus (77) Google Scholar). As with other members of this five-subunit (“Cys loop”) receptor family, the two AChR 2The abbreviations used are: AChR, acetylcholine receptor; ACh, acetylcholine; WT, wild type. agonist-binding sites are located in the extracellular domain of the protein, about 50 Å above the middle of the membrane. The occupancy of these sites by appropriate ligands alters the equilibrium constant for gating, which we define as the global and reversible isomerization of the protein between a stable, low affinity, nonconducting C conformation and a stable, high affinity, ion-conducting O conformation. Structures of the heteromeric Torpedo muscle-type AChR (4Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1417) Google Scholar), the homomeric ELIC (5Hilf R.J. Dutzler R. Nature. 2008; 452: 375-379Crossref PubMed Scopus (578) Google Scholar), GLIC (6Bocquet N. Nury H. Baaden M. Le Poupon C. Changeux J.P. Delarue M. Corringer P.J. Nature. 2009; 457: 111-114Crossref PubMed Scopus (591) Google Scholar), and acetylcholine-binding proteins (7Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1579) Google Scholar, 8Celie P.H. van Rossum-Fikkert S.E. van Dijk W.J. Brejc K. Smit A.B. Sixma T.K. Neuron. 2004; 41: 907-914Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar) show that each ligand-binding site contains several aromatic residues that are mostly conserved among these pentameric receptors (Fig. 1). In AChRs, residues Tyr-93, Trp-149, Tyr-190, and Tyr-198 are in the α∈ or αδ subunit (the positive face of the binding site), and residues Trp-55 and Trp-57 are in the complementary ∈ or δ subunit (the negative face), respectively. The foci of this report are these two minus-side tryptophan residues, which we will refer to as the W- amino acids. Affinity labeling studies of AChRs show that the two W- residues are located near the ACh-binding site and participate in the channel activation process. In Torpedo, γTrp-55 and δTrp-57 are sites of photo-incorporation of the competitive antagonist d-tubocurarine (9Chiara D.C. Cohen J.B. J. Biol. Chem. 1997; 272: 32940-32950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 10Xie Y. Cohen J.B. J. Biol. Chem. 2001; 276: 2417-2426Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and γTrp-55 is labeled by the agonist nicotine (11Chiara D.C. Middleton R.E. Cohen J.B. FEBS Lett. 1998; 423: 223-226Crossref PubMed Scopus (46) Google Scholar). Also, constitutively active AChRs are formed following the incorporation of a series of tethered quaternary ammonium derivatives at αTrp-149, αTyr-93, and γTrp-55/δTrp-57 (12Li L. Zhong W. Zacharias N. Gibbs C. Lester H.A. Dougherty D.A. Chem. Biol. 2001; 8: 47-58Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 13Stewart D.S. Chiara D.C. Cohen J.B. Biochemistry. 2006; 45: 10641-10653Crossref PubMed Scopus (11) Google Scholar). These experiments indicate that the W- residues are close to the agonist-binding site but more distant compared with αTrp-149 and αTyr-93 (for which shorter tethers were effective). Structures of acetylcholine-binding protein confirm this conclusion; the minus-side residue Trp-53 makes limited aromatic contacts with bound ligand, whereas the plus-side aromatic residues Trp-143 and, to a lesser extent, Tyr-185 and Tyr-192 (8Celie P.H. van Rossum-Fikkert S.E. van Dijk W.J. Brejc K. Smit A.B. Sixma T.K. Neuron. 2004; 41: 907-914Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar), form the main part of an “aromatic box” (14Lester H.A. Dibas M.I. Dahan D.S. Leite J.F. Dougherty D.A. Trends Neurosci. 2004; 27: 329-336Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar) that surrounds the ligand (Fig. 1, right). To what extents do the W- side chains influence ligand binding and channel gating? In Torpedo AChRs, a leucine substitution at either ∈Trp-55 or δTrp-57 increases the response to EC50 and increases the equilibrium dissociation constants for both agonists and antagonists (10Xie Y. Cohen J.B. J. Biol. Chem. 2001; 276: 2417-2426Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Replacement of Trp-54 in α7 neuronal AChRs (homologous to γTrp-55/δTrp-57 in muscle AChRs) by Phe, Ala, or His produces similar effects (15Corringer P.J. Galzi J.L. Eisele J.L. Bertrand S. Changeux J.P. Bertrand D. J. Biol. Chem. 1995; 270: 11749-11752Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), as do substitutions at homologous positions in other Cys loop receptors (16Buhr A. Baur R. Sigel E. J. Biol. Chem. 1997; 272: 11799-11804Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Sigel E. Baur R. Kellenberger S. Malherbe P. EMBO J. 1992; 11: 2017-2023Crossref PubMed Scopus (169) Google Scholar, 18Yan D. Schulte M.K. Bloom K.E. White M.M. J. Biol. Chem. 1999; 274: 5537-5541Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). At the level of rate and equilibrium constants (estimated by single channel kinetic analysis), the main effect of the mutations ∈W55F and δW57F in mouse AChRs is to slow the forward C → O rate constant, with more minor effects on the backward C ← O rate constant and the affinity of the C receptor for ACh (19Akk G. J. Physiol. (Lond.). 2002; 544: 695-705Crossref Scopus (36) Google Scholar). Here we extend these single channel studies of the two W- residues in recombinant (α1)2βδ∈ mouse AChRs. We examined mutants of these positions and were able to estimate binding, diliganded gating rate, and equilibrium constants for most. Furthermore, for some constructs we also measured the unliganded gating parameters, which allowed us to separately determine the functional effects of the side chain substitutions on the equilibrium dissociation constants of the C versus the O conformations. Detailed methods are given in Jha et al. (20Jha A. Cadugan D.J. Purohit P. Auerbach A. J. Gen. Physiol. 2007; 130: 547-558Crossref PubMed Scopus (77) Google Scholar). Mutant AChRs were transiently expressed in human embryonic kidney cells, and single channel currents were recorded in the cell-attached patch configuration at 23 °C. The bath and pipette solutions were Dulbecco’s phosphate-buffered saline containing (in mm) the following: 137 NaCl, 0.9 CaCl2, 2.7 KCl, 1.5 KH2PO4, 0.5 MgCl2, and 8.1 Na2HPO4 (pH 7.3). The currents were digitized at a sampling frequency of 50 kHz. Acetylcholine was added to the pipette solution at concentrations of 30, 100, 300, 500, 1000, 3000, and 5000 μm (Fig. 2). Usually the membrane potential (Vm) was approximately -100 mV, but in some experiments at high [ACh] the pipette potential was set to -70 mV (Vm approximately +40 mV) to relieve channel block by the agonist. This voltage perturbation was assumed only to increase the closing rate constant by 10-fold (21Auerbach A. Sigurdson W. Chen J. Akk G. J. Physiol. (Lond.). 1996; 494: 155-170Crossref Scopus (67) Google Scholar). Rate constant estimation (12 kHz bandwidth) was done by using QUB software. Clusters of individual channel, diliganded C ↔ O activity were usually selected by eye or by using a critical time of 50 ms (the minimum duration of the intervals flanking a cluster of openings). Clusters produced by ∈W55R had too low of an open probability for analysis. For all other mutants, the intra-cluster opening and closing rate constants (n ≤ 3 patches) were estimated from the interval durations by using a maximum likelihood algorithm (22Qin F. Auerbach A. Sachs F. Proc. Biol. Sci. 1997; 264: 375-383Crossref PubMed Scopus (241) Google Scholar) after imposing a dead time correction of, typically, 50 μs (2.5 samples). In some patches, an additional nonconducting state was connected to the conducting state, to accommodate a component associated with short lived desensitization. The diliganded opening rate constant (f2) was estimated from the saturation of the “effective” opening rate (f*) profile (Fig. 3). The fitting function was the logistic equation: f* = f2/(1 + es(x-i)), where s is the slope, x is [ACh], and i is the inflection concentration. The diliganded closing rate constant (b2) was estimated from the inverse of the open channel lifetime obtained at low ACh concentration (to avoid errors arising from channel block). The diliganded gating equilibrium constant was E2 = f2/b2. We could not estimate f2 for the ∈Trp-55 and constructs of saturation of the The ACh and dissociation rate constants were estimated by fitting intra-cluster conducting and nonconducting intervals two or ACh each W- was to influence only a single binding the kinetic used to the interval durations had two and binding where is the agonist in In the fitting f2 was to the value by the and and the dissociation rate constant were to the wild of and S. Auerbach A. J. Gen. Physiol. 2004; PubMed Scopus Google Scholar). The were and was estimated as the of the for gating, which is a of f2 versus E2 (Fig. was estimated as the of the of versus (Fig. The were estimated by an using We to unliganded gating agonist added to the in constructs Phe, and at ∈Trp-55 or at These mutations were expressed on a either (20Jha A. Cadugan D.J. Purohit P. Auerbach A. J. Gen. Physiol. 2007; 130: 547-558Crossref PubMed Scopus (77) Google Scholar, S. Auerbach A. J. Gen. Physiol. PubMed Scopus Google Scholar, P. Auerbach A. J. Gen. Physiol. 2007; 130: PubMed Scopus (46) Google Scholar) or S. Auerbach A. J. Gen. Physiol. PubMed Scopus Google Scholar, P. Auerbach A. J. Gen. Physiol. 2007; 130: PubMed Scopus (46) Google Scholar, C. S.M. Auerbach A. J. Gen. Physiol. PubMed Scopus Google Scholar), which by increase the unliganded gating equilibrium constant by a of or and, to of unliganded P. Auerbach A. Proc. Sci. S. A. 2009; PubMed Scopus Google Scholar). We were to on these for two W- only a high open probability and ∈W55R not to unliganded of of mutants and equilibrium constant measurements is in 1, We recorded single channel currents and were able to estimate both ACh binding and diliganded gating rate constants for W- mutants. of is in As the concentration of ACh the occupancy of and and the lifetime of the nonconducting component We could estimate a high concentration for this (the inverse of which is an estimate of the diliganded channel opening rate for ∈Trp-55 mutants and δTrp-57 mutants. The diliganded opening (f2) and closing (b2) rate and the diliganded gating equilibrium constant for these AChRs are in of the W- mutations In the were for the Ala, and substitutions and in δ the were for the and substitutions all of the substitution with to E2 was greater in ∈ in δ gating f2 diliganded opening rate diliganded closing rate E2 diliganded gating equilibrium constant = positive value that the O to are from are from S. Auerbach A. Proc. Sci. S. A. 2005; PubMed Scopus (67) Google in a The of the effects on E2 are compared with of other residues in the extracellular domain of the AChR, where side chain substitutions in (20Jha A. Cadugan D.J. Purohit P. Auerbach A. J. Gen. Physiol. 2007; 130: 547-558Crossref PubMed Scopus (77) Google Scholar, P. Auerbach A. J. Gen. Physiol. 2007; 130: PubMed Scopus Google Scholar) and ∈ K. M. N. S. P. S.M. Engel A.G. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) reduce diliganded gating by of the of the we could not a between side chain and the effect on In the ∈ subunit, the of E2 was and in the δ subunit the was At the level of rate the W- mutations E2 by the channel opening rate constant (f2) the channel closing rate constant The forward and backward gating rate in the form of a which is a of f2 versus E2 = are in The of this for ∈Trp-55 was similar to that for δTrp-57 These are also from for agonists C. M. Auerbach A. Nature. PubMed Scopus Google Scholar) or mutations of αTrp-149 P. Auerbach A. Proc. Sci. S. A. 2009; PubMed Scopus Google of is that a from to the of the gating of the residues to A. Proc. Sci. S. A. 2005; PubMed Scopus Google Scholar, Y. Auerbach A. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). this this suggests that the W- residues experience C versus O energy change with each as as with the agonist and residues in the positive face of the binding approximately at the of the channel opening process. The equilibrium dissociation constant for ACh binding to the C conformation is the of the rate constants We were able to estimate these for all of the mutants and (in δ) and and (in 2). of the side chain substitutions on by The effect was approximately in the ∈ and δ In the aromatic side chains and of an increase in compared with the side but this was not in The consequence of an substitution was approximately in both binding to the C conformation single site rate single site dissociation rate equilibrium dissociation constant to the C = positive value that the the affinity of the C transmitter binding site for are from are from are from are from S. Auerbach A. J. Gen. Physiol. 2004; PubMed Scopus Google are from G. J. Physiol. (Lond.). 2002; 544: 695-705Crossref Scopus (36) Google in a The effect of the mutations on was of a in We to the binding + C ↔ in the that we to the gating ↔ The for the ∈Trp-55 and δTrp-57 ACh binding are in as of versus The of these were for ∈Trp-55 and for the this suggests that at the state for ligand binding the two W- side chains are mostly in energy which that change energy relatively early in the + C ↔ binding process. In the series of we measured the unliganded gating equilibrium constant for some mutants by using a (Fig. These experiments avoid the associated with E2 in the of a high [ACh], which is by a in the single channel of channel block by the agonist. Also, analyses of unliganded gating allowed us to the to which the change in E2 on a change in versus the C versus O affinity The results of experiments agonist are in the W- substitutions not a effect on In both ∈ and only substitutions by The energy between the and of was for and in These energy are in with the effect of mutations of residue αTrp-149, where the energy between His versus Cys side chains was in P. Auerbach A. Proc. Sci. S. A. 2009; PubMed Scopus Google the W- unliganded gating energy changes were we that all ∈Trp-55 mutations whereas most mutations This that the of the O versus C conformation with a is at and more at compared with most other side gating unliganded opening rate unliganded closing rate unliganded gating equilibrium all mutations were expressed on a that by a of for which was expressed on a that by = positive value that the O to in a of the two W- residues ACh binding more channel gating, but in either the effects were not These aromatic side chains to play a relatively minor role in AChR activation. other residues to be this suggests that the plus-side of the binding site is the structural with to both ACh binding and channel As as energy is the used an site as being the between to antagonists S. H. S.M. P. Biochemistry. PubMed Scopus Google Scholar, A. M. J. J. Neuron. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), but may be appropriate in the of agonist of the ∈Trp-55 and δTrp-57 mutations for which rate and equilibrium constants were estimated E2 and which suggests that a at either is for the most binding and diliganded We the extents to which the of E2 in the mutants were by the effect of the substitution on the C versus O affinity The AChR is an allosteric protein in which ligand binding and the gating conformational change are J. J. Changeux J.P. J. Mol. Biol. PubMed Scopus Google Scholar). the W- mutations change only of the two binding sites the for E2 and and the value = P. Auerbach A. Proc. Sci. S. A. 2009; PubMed Scopus Google Scholar), we for mutants at the two W- In the ∈ subunit, the of the Ala, Phe, or on by and by 3). We that for these the in E2 mostly from a in the C versus O affinity the in for side chains was about that for aromatic side In the δ subunit the was more For mutants and the were approximately the as in the In the the value was the WT, and in the constructs and the affinity was the Also, in δ was between aromatic and side the effects of W- mutations on the affinity were greater in ∈ compared with and ACh binding to the O conformation the C versus O affinity = positive value that the R. equilibrium dissociation constant for ACh binding to the O energy positive value that the the affinity of the O transmitter binding site for in a In AChRs the equilibrium dissociation constant of the O conformation is P. Auerbach A. Proc. Sci. S. A. 2009; PubMed Scopus Google Scholar). We for the W- mutants from the of by using the = For both W- all of the mutations the The increases in were greater in for and where the in affinity were approximately in C and We that the main effect of W- mutations is to reduce the affinity of the O conformation of the AChR for the We the effects of W- mutations on ACh binding to C and O AChRs in of for ACh binding to the O conformation was calculated for aromatic versus side chain substitutions of At the consequence of the with an aromatic side chain was compared with with a side chain with to binding to the C conformation of the consequence of of with an aromatic side chain was compared with with a side chain and The of the energy between aromatic and was only slightly greater in compared with This that at the aromatic nature of the side chain a energy of approximately for ACh binding to and approximately for binding to These may from a with the ligand, from with residues on the side of the binding or This is not at all mutations the of ACh in O versus C was only and with that aromatic side chains more binding energy side in either the C or O conformation. To at both binding sites W- mutations a effect on unliganded gating, a effect on binding to the C conformation, and a relatively effect on binding to the O conformation. The ∈Trp-55 and in aromatic a more role in determining ACh affinity compared with be from the Φ value analyses of gating and AChR C ↔ O gating as a conformational in which which all residues gating structural changes approximately with to the affinity change at the binding site with the change in the A. Proc. Sci. S. A. 2005; PubMed Scopus Google Scholar, Purohit Auerbach A. 2008; PubMed Scopus Google Scholar). The are similar for the residues on both the of the subunit including ∈Trp-55 δTrp-57 and K. M. N. S. P. S.M. Engel A.G. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), where and P. Auerbach A. J. Gen. Physiol. 2007; 130: PubMed Scopus Google Scholar) where These results the that the structural boundaries of Φ may not be by the we that the dynamic elements of the conformational (the Φ the protein to binding, the high for both W- (Fig. are that the of ACh to the transmitter binding site is not in which perturbation of from changes in the dissociation rate constant the rate constant high were for a series of AChR residue M. at the of the Acetylcholine of and of Scholar), and for agonists of AChRs agonist are by the rate Y. Chen J. Auerbach A. J. Physiol. (Lond.). 1995; Scopus Google Scholar). for the that agonist binding a conformational change is that the rate constant is not The more the ACh Y. Chen J. Auerbach A. J. Physiol. (Lond.). 1995; Scopus Google Scholar). The that agonists and the residues are in energy at the state for binding that is a conformational change associated with ligand binding, and at this state for this the channel is the ligand is in with the The effects of W- mutations on binding and gating are with mutations of other AChR and in the of high of both the and neuromuscular AChR, we on the of the energy we we that in the be with structural and to the of the energy changes by W- In the mutants we may some to be with to the AChR We M. M. and M. for