Published online November 26, 2007
doi:10.1085/jgp.200709858
The Journal of General Physiology, Vol. 130, No. 6, 569-579
The Rockefeller University Press, 0022-1295 $30.00
© 2007 Purohit et al.
Acetylcholine Receptor Gating: Movement in the 
-Subunit Extracellular Domain
Prasad Purohit and
Anthony Auerbach
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214
Correspondence to Anthony Auerbach: auerbach{at}buffalo.edu
Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains ("
blocks") move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the 
-subunit, the first -block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the ß-core of the -subunit. Y127 is at the boundary between the first and second blocks, at a subunit interface. Y127 mutations cause large changes in the gating equilibrium constant and with a characteristic -value ( = 0.77) that places this residue in the second -block. We also examined the effect on gating of mutations in neighboring residues 
I43 ( = 0.86), 
N39 (complex kinetics), I49 (no effect) and in residues that are homologous to Y127 on the , ß, and subunits (no effect). The extent to which Y127 gating motions are coupled to its neighbors was estimated by measuring the kinetic and equilibrium constants of constructs having mutations in Y127 (in both subunits) plus residues D97 or I43. The magnitude of the coupling between D97 and Y127 depended on the Y127 side chain and was small for both H (0.53 kcal/mol) and C (–0.37 kcal/mol) substitutions. The coupling across the single – subunit boundary was larger (0.84 kcal/mol). The -value for K145 (0.96) indicates that its gating motion is correlated temporally with the motions of residues in the first -block and is not synchronous with those of Y127. This suggests that the inner and outer sheets of the -subunit ß-core do not rotate as a rigid body.
Abbreviations used in this paper: AChBP, ACh binding protein; AChR, acetylcholine receptor; ECD, extracellular domain; REFER, rate-equilibrium free energy relationship; TMD, transmembrane domain; wt, wild type.
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INTRODUCTION
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The diliganded gating isomerization of the acetylcholine receptor (AChR), between C(losed) and O(pen) structures, is a conformational "wave" that links a change in affinity for ligands at the transmitter binding sites with a change in the ionic conductance of the pore. In the -subunit, the first group of amino acids to undergo a C
O structural change is near the transmitter binding sites (Akk et al., 1996
; Corringer et al., 2000; Chakrapani et al., 2003). Homologous residues in the ACh binding protein (AChBP) have been shown to change conformation as a result of agonist binding (Brejc, 2001; Celie et al., 2004; Hansen et al., 2005). The second group of residues to move in gating is near the base of the extracellular domain (ECD), in loops 2 and 7 (the "cys-loop") (Chakrapani et al., 2004). These ECD movements subsequently propagate into the transmembrane domain (TMD), toward an equatorial "gate" in M2 that regulates the conductance of the pore.
The relative timing of a residue's gating motion can be inferred from the rate constants of the diliganded C
O gating reaction (Auerbach, 2007). The slope of a log–log plot of the opening rate constant vs. the equilibrium constant for a series of mutations of a single residue is thought to give the relative time in the channel-opening process that the mutated residue converts from C to its O structure. In the extracellular region of the -subunit, values decrease from the transmitter binding site ( = 0.93), to the cys-loop and loop 2 ( = 0.78), to the M2–M3 linker ( = 0.64).
Currently, there is a model for the structure of closed-unliganded Torpedo AChRs, 
4-Å resolution (Unwin, 2005), in which the ß cores of the ECD of the two subunits are rotated with respect to those in the three non– subunits. This observation led to the proposal that during diliganded CO gating (as opposed to agonist binding) there is a symmetry-restoring rotation of the inner ß-sheet of the -subunit ECD.
Here we report the results of mutations on gating of two -subunit residues that are near the top of the inner (strands 1, 2, 3, 5, 6, and 8) and outer (strands 4, 7, 9, and 10) ß sheets of the -subunit, Y127 (on ß-strand 6), and K145 (on ß-strand 7). Y127 lies at a boundary between the first two blocks, with its atoms <4 Å from residues in both D97 in loop A ( = 0.93) in the Torpedo AChR structure (Unwin, 2005). However, Y127 and D97 are 9 Å apart in the mouse -subunit ECD fragment structure (2qc1.pdb, Dellisanti et al., 2007). Y127 is located at or near the C terminus of ß-strand 6, one position from the C128–C142 disulfide bond that defines the cys-loop (loop 7) of the eukaryote pentameric receptor superfamily (Fig. 1). Y127 also is at a subunit interface and faces either the (
in embryonic AChRs) or subunit, and for this reason the structure of this residue is poorly resolved in the monomeric ECD fragment (Dellisanti et al., 2007). Mukhtasimova and Sine (2007) found that the mutation Y127T substantially decreases Keq, as do the mutations N39A and N41A in nearby residues in these non– subunits. Moreover, the effects of these perturbations were not independent, which suggests that these positions are coupled energetically and are a link for the intersubunit propagation of the gating conformational cascade.
In both the Torpedo AChR and ECD fragment structures, K145 is <4 Å from two residues whose mutation significantly changes Keq, D200 (in loop C) and Y93 (in loop A) (Akk et al., 1996; Akk, 2001). Although rate constants for only a few mutations of each of these positions have been measured, the values are consistent with a -value near 1, which places these neighboring amino acids in the first, = 0.93 block. M144, next to K145 in sequence, was measured to have a -value of 0.84 ± 0.05. Mukhtasimova et al. (2005) found that substitution of A, Q, and E side chains at K145 all reduce Keq substantially and that the effect of K145E and D200N mutations are not energetically independent, and proposed that interactions between K145–D200 vs. K145–Y190 (based on structure) stabilize the C vs. O conformation, respectively.
We have extended these studies regarding Y127 and K145 by more extensive -value analysis, and have related the results to the ECD rotation hypothesis for gating. First, we measured rate constants from single-channel currents and estimated for Y127 (all 20 natural amino acid side chains) and its neighbor in the -subunit, I43. Second, we estimated the magnitude of the energetic coupling between Y127 and either I43 or D97, in six different constructs. Third, we examined the kinetic behavior of mutations to residues in the , , and ß subunits that are homologous to Y127. Fourth, we measured the diliganded gating rate constants of four mutants of position K145. The results show that a point side chain substitution at Y127 can change Keq by a factor of 290,000, that Y127 is a member of the second -block, that the coupling between Y127 and D97 or I43 is measurable but small (<1 kcal/mol). Regarding K145, mutations alter the channel opening rate (relative to the change in Keq) to a greater extent than for Y127, which suggests that these two residues do not move synchronously in the gating reaction.
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MATERIALS AND METHODS
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For the details of mutagenesis, expression, electrophysiology, rate constant determination, and -value analysis, see Jha et al. on page 547 of this issue. In brief, mouse AChR subunits were transiently expressed in HEK 293 cells and recordings were from cell-attached patches (22°C, –100 mV membrane potential). Agonist was added to the pipette solution (500 µM ACh, 5 mM carbamylcholine, or 20 mM choline). Currents were analyzed with QUB software (www.qub.buffalo.edu). Opening and closing rate constants were estimated from interval durations by using a maximum-interval likelihood algorithm (Qin et al., 1997) after imposing a dead time of 25 µs. was estimated as the slope of the rate-equilibrium free energy relationship (REFER), which is a plot of log ko vs. log Keq. Each point in the plot represents the mean of at least three different patches.
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RESULTS
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Mutations of Y127 and its Homologues
In vertebrate 1 subunits, position 127 is always a Y but in non–1 subunits it is never a Y (but is, rather, S, A, T, or V). A tyrosine at position 127 is a specific marker for the vertebrate neuromuscular 1-subunit. The location of Y127 in the Torpedo AChR structure is shown in Fig. 1.
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Figure 1. Location of Y127 in Torpedo AChRs. (A) A Cartoon of the / subunits viewed from the exterior of the AChR. Only the (left) and subunits are shown; the horizontal lines mark, approximately, the membrane. In the three blocks that link the transmitter binding site with the gate are color coded as purple ( = 0.93, W149, K145), orange ( = 0.78, Y127), green ( = 0.65, S269), and red ( = 0.31, L251). (B and C) Expansion of boxed region in A. K145 (purple) is on ß-strand 7 and Y127 (orange) at the / (B) and / (C) subunit interface. Y127 is <4 Å from residues D97 and N94 in loop A (purple), Q48 in loop 2 (orange), and N39/I43 in ß-strand 1 (black). Structures were displayed by using PYMOL (DeLano Scientific).
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Fig. 2 and Table I show the results of single-channel kinetic analyses of wild-type AChRs plus all 19 natural amino acid substitutions at Y127.
16 of the mutations decreased Keq (D by 4,900-fold) while the three aromatic side chains H, W, or F increased Keq (F by 59-fold). There was no correlation between side chain hydrophobicity or volume and the change in Keq. The change in Keq in AChRs having D vs. F at position 127 (in both subunits) was 290,000-fold, which represents an energy difference of 7.4 kcal/mol. For comparison, the maximum fold-changes in Keq caused by mutations of some other -subunit residues are shown in Table II.
In our hands, Y127 is the most sensitive position ever reported for a point side chain substitution in both subunits. The substantial changes in Keq indicate that the energetic consequences of the mutations are substantially different in C vs. O, which implies that Y127 changes its structure, environment, or both (i.e., moves) in the gating reaction.
The mutation-induced changes in Keq at Y127 arose mainly from changes in the channel opening rate constant (ko). Fig. 3 shows a REFER analysis (a log–log plot of ko vs. Keq) of the mutational series at Y127.
Each 10-fold change in Keq arose, on average, from an 6.2-fold change in ko and an 1.6-fold change in kc. The slope of this relationship, , was 0.77 ± 0.02. Notice that the results for AChRs activated by different agonists scatter about the same line and that the estimate was similar regardless of whether the AChRs were activated by acetylcholine (0.85 ± 0.04), carbamylcholine (0.75 ± 0.2), or choline (0.75 ± 0.04) (Fig. 3).
The -value for Y127 is the same as those for several residues in loop 2 and the cys-loop ( = 0.80 ± 0.05 and 0.78 ± 0.03) (Jha et al., 2007) and R209 in the pre-M1 linker (0.74 ± 0.02, on an E45A background) (Purohit and Auerbach, 2007), but is different from those for the transmitter binding site (0.93 ± 0.02) (Grosman et al., 2000) and residue D97 in loop A (0.93 ± 0.03) (Chakrapani et al., 2003). This result suggests that position 127 moves relatively early in the diliganded channel-opening process and that its gating motions are correlated temporally with other residues in the second ( = 0.78) gating block, but that these occur after those in the first ( = 0.93) gating block.
We measured the single-site association and dissociation rate constants (k+ and k–) and equilibrium dissociation constant (k+/k– = Kd) for ACh binding to the closed conformation in one mutant construct, Y127C (Fig. 4).
In this mutant Kd = 144 µM, which is in the range of previous measurements for wild-type AChRs exposed to 140 mM NaCl (100–150 µM) (Akk and Auerbach, 1996; Chakrapani et al., 2003). Similarly, the association and dissociation rate constants in the mutant, k+ = 2.108 M–1s–1 and k– = 3.0 x 104 s–1, were similar to wt values.
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Figure 4. The mutation Y127C does not alter the closed-channel equilibrium dissociation constant. Left, open and closed interval duration histograms at different ACh concentrations. The solid lines are calculated from the globally optimized rate constants. Number of events analyzed at various concentrations of ACh were: 100 µM, 3391; 300 µM, 2244; and 500 µM, 6940. Right, example clusters from each concentration. The optimal rate constants were: k+ (single-site association) = 205 µM s–1, k– (single-site dissociation) = 29604 s–1, ko = 2089 s–1, and kc = 5032 s–1. We calculate Kd (=k–/k+) = 144 µM for the mutant. For comparison, the wt estimates are k+ = 167 µM s–1 and k- = 24,745 s–1, KD = 148 µM (Chakrapani and Auerbach, 2005). There is no significant effect of this mutation on ACh binding to closed AChRs and we speculate that Y127 mutations that change Keq do so by changing the unliganded gating equilibrium constant rather than the closed/open affinity ratio. Calibration bars for single channel traces: (horizontal scale bar = 100 ms, vertical scale bar = 6 pA).
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We also probed the effects on gating of mutations to residues in the ß, , and subunits that are homologous to Y127. In the non– subunits, which are homologous in both sequence and structure to the subunits in the vicinity of Y127, the residue in question (ßS127, T127, or S129) immediately preceded in sequence the extracellular disulfide bond. Seven mutations of these three positions all yielded AChRs having wt-like gating behaviors (Table IV).
I49, I43, and N39
We next examined the gating properties of AChRs having mutations of residues that are close to Y127 (Fig. 1 B). I49 is at the N terminus of ß-strand 2, 5 Å from Y127. The gating kinetics for three mutants of this position, C, V, and Y, did not change Keq by greater than threefold (Table I). Thus, we have no evidence that the I49 side chain moves relative to its local environment between C and O conformations.
N39 or I43 are neighbors of Y127 in the companion, non– subunit. A REFER analysis of position I43 is shown in Fig. 5.
All four of the tested substitutions decreased Keq, with = 0.86 ± 0.10. Although this result indicates that I43 moves early in the reaction, we are unable to distinguish this -value from those of the first (0.93; agonist and loops A, B, and C) and second (0.77; Y127, loop 2, and cys-loop) blocks of the -subunit. At N39, F and D substitutions caused a small (less than threefold) change in Keq, and the substitution of an Ile at this position also generated currents having wt-like kinetic behavior (when activated by 30 µM ACh). The substitution of an H increased the cluster open probability relative to the wt, but the kinetics of these intracluster intervals was complex, with at least two conducting and two nonconducting states apparent. Therefore, unambiguous values of ko and kc could not be estimated. These results suggest that N39 moves during gating, but we were unable to estimate a -value for this position.
Coupling of Y127 Gating Motions within and between Subunits
In the -subunit, two residues in loop A, part of which contributes to the transmitter binding site, may be close to Y127: D97 and N94. Mutation of D97 causes a substantial change in Keq and has a -value that is different from that of Y127 (0.93 vs. 0.77). We therefore tested whether an interaction between Y127 and D97 couples the gating motions (energy transfer) between the transmitter binding site (in the first -block) and the cys-loop (in the second -block).
We probed a D97Y127 interaction by measuring the gating kinetics of AChRs having a mutation (in both subunits) at both of these positions (Table III
). Six pairwise combinations were tested, with two different side chains at Y127 (H and C) and three different side chains at D97 (M, Y, and H). By themselves, the mutations at position 127 either reduced Keq (C, by 201-fold) or increased Keq (H, by 7.3-fold), while those at position 97 always increased Keq (M, Y, or H, by 5.5-, 20-, and 7.3-fold, respectively). The hallmark of energetic coupling between Y127 and D97 is a fold-change in Keq with both sites mutated that is not equal to the product of the fold-changes for each site mutated.
With Y127H (activated by choline), the observed values of Keq for the three D97 mutants were, on average, modestly (2.5-fold) smaller than predicted assuming independence (Table III). With the Y127C constructs (activated by ACh), the observed values of Keq for the three D97 mutants were close to those predicted assuming independence. The average coupling energy was 0.53 kcal/mol for the Y127H background and –0.37 kcal/mol for the Y127C background. These results suggest that the magnitude of the coupling energy can vary with the side chain substitution. However, the coupling energy was small for both of the two tested backgrounds, especially when one considers that this coupling energy is spread between two Y127–D97 pairs (two subunits). Overall, the results suggest that although a large magnitude of energy change is associated with positions D97 and Y127 when examined individually, a D97Y127 perturbation in combination is not an important component of energy transfer within the transition state of diliganded gating. The assumption that the residues may be interacting at the -block boundaries, however, is based on the proximity of the two residues in the Torpedo AChR structure. Two problems with this assumption are that Y127 and D97 are >9 Å apart in the mouse -subunit fragment structure, and that neither structure reflects a ligand-bound AChR. There is a reason to suspect that loop A moves as a consequence of agonist binding (in addition to channel gating), so we do not know the separation between these residues in fully liganded AChRs.
We next measured the extent of coupling between Y127H (7.3-fold increase in Keq) and I43H (13.8-fold decrease). Together, these mutations caused a 2.2-fold increase in Keq, whereas if they were independent we would expect a 1.9-fold decrease in Keq. This approximately fourfold effect indicates that there is modest degree of coupling between the Y127 and I43 side chains (+0.84 kcal/mol; Table IV). Note that this interaction occurs at a single subunit interface and should therefore be considered to be substantially greater than the Y127–D97 interaction.
K145
We measured the gating rate constants for four different mutations of K145, which is on ß-strand 6 (Fig. 1). In the unliganded Torpedo structure, this residue is within 4 Å D200 and loop A residue Y93, two residues that have been shown to move during diliganded C-O gating. K145 is also likely to be close to moving-residue Y190 (Chen et al., 1995) when the transmitter binding site is occupied by an agonist (Celie et al., 2004). Finally, K145 is near T202, a residue that has not yet been probed at the rate constant level.
All four of the mutations of K145 (C, A, R, and D) decreased Keq, by up to 282-fold (Table V).
The causes of these decreases were, in all cases, almost exclusively due to decreases in ko. Fig. 6 shows the REFER for K145. The -value was 0.96 ± 0.04.
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DISCUSSION
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Comparison with Previous Results
Mukhtasimova and Sine (2007) studied the kinetic behavior of two Y127 mutants (F and T) plus N39A and N41A. Further, they measured the coupling between three pairs and two triplet combinations of these mutants. Although they studied human AChRs activated by ACh in 142 mM KCl and we studied mouse AChRs activated by ACh or choline in 140 mM NaCl, both sets of results are in general agreement. Mutations to Y127 have a profound effect on channel gating (Keq), and this residue is a site where gating motions are coupled between subunits.
The main difference in the two sets of results is in relation to the Y127F mutation. We measured a much larger increase in Keq for Y127F (58.7-fold vs. 2.2-fold increase). We speculate that this difference can be traced to an immeasurably fast opening rate constant for this construct in the experiments where the mutant AChRs were activated by ACh. In wt AChRs the difference in Keq for different agonists is manifest almost exclusively as a difference in the opening rate constant ( = 0.93; Grosman et al., 2000). Assuming that this pattern pertains to the Y127F mutant, then ko with ACh should be 400 times larger than ko with choline (Chakrapani and Auerbach, 2005). In this case, our measurement for ko with choline (2853 s–1) translates to an opening rate of ko with ACh of >106 s–1, which is too fast to be detected experimentally. Perhaps the brief gaps observed in the experiments with human AChRs (Fig. 2 and Table II in MS) did not arise from CO gating but rather from channel block by the agonist or some other process. Our results do not agree with the proposal that aromatic side chains can be substituted at position Y127 without consequence.
Mukhtasimova et al. (2005) also measured the gating rate constants for E, Q, and A mutants of K145. They report that these mutations decrease ko but leave kc essentially unchanged is consistent with our estimated value of 0.96 for this position.
Structure–Function
A D-to-F side chain substitution at Y127 changes Keq by nearly 290,000-fold. The magnitude of this change is substantially greater than that caused by any other ECD side chain substitution observed so far, even considering the fact that both subunits carried the mutation. (The change in Keq would be 540-fold if the energy difference between C and O was equally distributed between the two subunits).
The relationship between a change in structure and the magnitude of the change in Keq is complex. Although we measured Keq for all 20 natural side chains at Y127 and for four side chains at K145, we are nonetheless unable to draw strong conclusions about the chemical natures of the forces behind the Y127 gating motions. We note, however, that the mutations of Y127 that increased Keq are aromatic and flat. There is no apparent correlation between side chain volume or hydrophobicity and the magnitude of the change in Keq. Also, the charged side chains D, K, R, and E all reduced Keq at Y127 (by 4847-, 1282-, 553-, and 104-fold, respectively), and D and R reduced Keq at K145 (by 282- and 60-fold, respectively), so the sign of the charge at both of these positions appears not to be an important determinant of Keq.
The gating motion of K145 (as evidenced by the mutation-induced change in Keq) occurs approximately synchronously (same -value) as other residues near the transmitter binding site, in loops A, B, and C. The movement of K145 is correlated temporally with the movement of its close neighbors D200 and Y93. The movement of Y127 occurs after the movement of K145, and approximately synchronously with residues in the cys-loop and loop 2.
Rotation Hypothesis
The mutation-induced changes in Keq at positions K145 and Y127 are consistent with the proposal that gating entails a rotation of the -subunit ß-sandwich core (Unwin et al., 2002). However, some observations of AChR function appear to be inconsistent with this hypothesis. (a) A substituted cysteine accessibility study of residues between L36 and I53 in strands ß1 and ß2 in the 7 AChR showed that the rates of reaction with MTSEA in the presence of ACh varied significantly (McLaughlin et al., 2007). However, the rate of reaction decreased and increased, respectively, for the closely apposed residues M40 and N52, a result that is unexpected for a rigid body rotation of the ß-core. (b) The effects of mutations on Keq have been measured for seven different residues that are in the inner ß strands of the ECD core: L40A (in strand 1), I49C, V, and Y, V54L, R55A and W (in strand 2), and A122L, S126V and A, and Y127 (in strand 6). Of these constructs, only the Y127 mutants changed Keq by greater than threefold and, hence, gave a clear indication of motion. Although the lack of change in Keq does not unequivocally indicate a lack of gating motion, it would be surprising if a rotation altered the energetic environment only around Y127. More residues (Celie et al., 2004) and mutations in both the inner and outer leaflets of the ß-core need to be tested to test the energetic consequences of such a rotation. (c) The asynchrony of motion (different values) for Y127 and K145 is unexpected if the ß-core rotation was that of a rigid body motion. In summary, the results suggest that the hypothesis of a ß-sandwich core rotation in the gating reaction is, at best, incomplete. Because the rotation hypothesis arose from a comparison of the structures of vs. non– subunits in unliganded AChRs, we speculate that such movements may occur upon ligand binding rather than channel gating.
Map
Fig. 7 shows the map of superimposed on the mouse -subunit fragment structure (2qc1.pdb, Dellisanti et al., 2007).
The values for the purple residues are 0.93, those for the orange residues are 0.77, and the white residues show no indication of a gating motion (
Keq < threefold). This pattern suggests that the diliganded gating motions in the -subunit mainly propagate along the - (-) subunit interface.
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Figure 7. Gating movements occur mainly along the /() subunit interface. The structure is the mouse -subunit ECD fragment (2qc1.pdb). The diliganded gating rate constants have been measured for the residues shown as spheres. White: mutations resulted in little or no change in Keq (less than threefold), hence these residues show no indication of undergoing a gating motion (positions 20, 29, 31, 35, 40, 49, 54, 55, 56, 65, 110, 116, 120, 141, 143, 146, 207, and 208). Colored: mutations significantly changed Keq. Purple,  0.93 (positions 93, 95–100, 144, 145, 149, 152, 153, 190, 192, 198, and 200). Orange, 0.77 (positions 45–48, 127, 132, 133, 134, 135, 137, 138, 140, and 209). The channel opening gating motions in the -subunit ECD appear to propagate mainly along the interface with the (or ) subunit, with residues near the binding site moving before those near the ECD–TMD interface.
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changes significantly (by 0.16 units) between D97 and Y127 (which are within 4 Å in 2bg9.pdb and 9 Å in 2qc1.pdb), whereas is the same for residues that are separated by much larger distances. For example, residues separated by >20 Å and that have similar values are Y127 and F135 ( 0.78) and D97 and D152 ( 0.93). These results, along with similar comparisons elsewhere in the AChR, suggest that residues are grouped into contiguous domains within which members all have approximately the same -value, and that can change abruptly in space as expected from discrete boundaries. However, it is important to mention again that we cannot be certain that D97 and Y127 are as closely apposed in the state of our reaction (the agonist-bound closedopen) as they are in the unliganded Torpedo structure or the toxin-bound -subunit fragment. The results indicate that first gating -block extends at least to K145, and perhaps to M144, which has a -value of 0.84 ± 0.05 (Chakrapani et al., 2004).
Coupling
Our results indicate that there is only a small amount of energetic coupling (<0.6 kcal/mol) between Y127 and D97 even though these side chains are close, are mutation-sensitive, and have different values (Fig. 1). It is therefore unlikely that an interaction between these two residues is an important link in the propagation of the AChR gating conformational wave.
Mukhtasimova and Sine (2007) found large coupling coefficients between the intersubunit pairs Y127T/N39A (1.7 kcal/mol) and Y127T/N41A (3.8 kcal/mol). Our estimate of coupling for the Y127H/I43H pair was somewhat smaller (0.84 kcal/mol) but still larger than for the Y127/D97 pair. Our results support the idea that Y127 is a site where the gating conformational cascade in the - subunit is linked to that in the or subunits. The - value of I43 (0.86 ± 0.10) cannot be distinguished from those of either D97 (0.93 ± 0.01) or Y127 (0.77 ± 0.02). Thus, we are unable to use -value analysis to determine if the -subunit motions are synchronous with those of , or, if not, which subunit precedes the other.
The Framework for AChR Gating
The results presented here and in the two companion papers support the idea that the framework for understanding the mechanism of diliganded AChR gating is that it is "brownian conformational wave." All of the 29 newly probed positions have values that are similar to those previously reported for other amino acids in the extracellular region of the AChR -subunit, and with magnitudes as expected based on location. There is little doubt that in the AChR, the map of is highly organized and that residues are clustered into blocks. Whatever mechanisms are proposed for AChR gating, and whatever physical interpretation is applied to (relative timing, fractional side chain structure, multiple pathways), these must account for this highly ordered map of values that has been derived from an extensive array of experiments.
The results do not support the notion that there is a single, rate-limiting structural transition that is the intersection of the C and O conformational ensembles. If there is a rotation of the -subunit ß-core, it is unlikely to be as a rigid body because K145 on the outer sheet and Y127 on the inner sheet belong to two different blocks. Although R209 and E45 both move and make a substantial energy contribution to the TR, these energy changes apparently do not arise from the perturbation of a salt bridge between this pair. The movement of the M2–M3 linker is an important TR event, but a full, cis–trans isomerization of the P272 or G275 backbone is not necessary for efficient gating. Rotations, electrostatic forces, changes in backbone bond angles, and hydrophobic interaction may occur in various regions of the protein, but each of these structural transitions contributes only a fraction to the total energy to the TR barrier.
Rather than conceiving of the energy barrier separating C from O as the point intersection of two parabolas, the experimental results suggest that this TR barrier is a broad, corrugated, flat plateau (Auerbach, 2005). The map and range of values, the spatially distributed effects of mutations on Keq, and the rather weak coupling energies that we have observed between specific pairs of moving residues all suggest that the barrier for diliganded gating arises from the motions of many different metastable intermediate structures that are separated, sequentially, by small energy barriers. This energy distribution is certainly not isotropic, because some moving residues make larger energy contributions than others.
Several important regions of the AChR have not yet been mapped for , including most of M1, the upper half of M2, and some regions of the ECD in the -subunit, and many regions of the non– subunits. This map of the TR, along with high resolution structures of the diliganded C and O end state ensembles, should serve as a guide for understanding the details of the structural transitions that constitute AChR gating.
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ACKNOWLEDGMENTS
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We would like thank Mary Merritt and Mary Teeling for technical assistance.
Olaf S. Andersen served as editor.
Submitted: 17 July 2007
Accepted: 8 November 2007
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REFERENCES
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P. Purohit and A. Auerbach
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