Cyclic activation of endplate acetylcholine receptors Tapan K. Nayaka and Anthony Auerbacha,1 a
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214
Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved September 20, 2017 (received for review June 21, 2017)
Agonists turn on receptors because they have a higher affinity for active versus resting conformations of the protein. Activation can occur by either of two pathways that connect to form a cycle: Agonists bind to resting receptors that then become active, or resting receptors activate and then bind agonists. We used mutations to construct endplate acetylcholine receptors (AChRs) having only one functional neurotransmitter-binding site and single-channel electrophysiology to measure independently binding constants for four different agonists, to both resting and active conformations of each site. For all agonists and sites, the total free energy change in each pathway was the same, confirming the activation cycle without external energy. Other results show that (i) there is no cooperativity between sites; (ii) agonist association is slower than diffusion in resting receptors but nearly diffusional in active receptors; (iii) whereas resting affinity is determined mainly by agonist association, active affinity is determined mainly by agonist dissociation; and (iv) at each site and for all agonists, receptor activation approximately doubles the agonist-binding free energy. We discuss a twostep mechanism for binding that involves diffusion and a local conformational change (“catch”) that is modulated by receptor activation. The results suggest that binding to a resting site and the switch to high affinity are both integral parts of a single allosteric transition. We hypothesize that catch ensures proper signal recognition in complex chemical environments and that binding site compaction is a determinant of both resting and active affinity. allosteric activation
| ion channel | agonist binding | nicotinic
A
cetylcholine receptors (AChRs) are allosteric signaling proteins that produce transient membrane currents by switching globally between a resting C (closed-channel) conformation and an active O (open-channel) conformation. Agonists are small molecules that bind to AChRs with higher affinity to O versus C. When a resting neuromuscular AChR activates with bound agonists, the newfound ligand-binding energy lowers the energy barrier between C and O and stabilizes the O conformation so as to increase the activation rate and activation probability above their basal levels. Neuromuscular AChRs (∼300 kDa) have two α1 subunits and one each of β, δ, and either e (adult-type) or γ (fetal-type), with two neurotransmitter-binding sites located at α–e/γ and α–δ subunit interfaces. The rate and equilibrium constants for binding to the resting C conformation have been measured for many agonists in wild-type (WT) adult and fetal AChRs (1–4), but only a few studies have addressed binding to the active O conformation (5, 6). Our primary goal was to compare agonist binding to C versus O at each kind of binding site. Activation of receptors by agonists can be described by a reaction cycle (7–9). In this scheme (Fig. 1), C and O represent stable end states (energy wells) and the arrows represent unstable transition states (energy barriers). For a receptor with only one functional binding site, there are two activation pathways that connect the unliganded resting state C with liganded active state AO (where the superscripted A is the agonist). The usual sequence in WT receptors is that the agonist binds and the receptor then activates (C ↔ AC ↔ AO). The equilibrium constants of this “bind-gate” path are 1/Kd (dissociation constant for binding to C) and E1 (gating with one bound agonist). The free energy difference, C to AO, is the sum of the energy differences for each step in the pathway. 11914–11919 | PNAS | November 7, 2017 | vol. 114 | no. 45
On rare occasions, WT AChRs activate without a bound agonist (10, 11). Many mutations throughout the protein increase substantially the frequency of constitutive openings (12), including some that occur naturally to cause disease (13). Hence, a second possible activation sequence is that an unliganded receptor switches on and the agonist then binds (C ↔ O ↔ AO). The equilibrium constants of the two steps of this “gate-bind” path are E0 (gating without any agonists) and 1/Jd (dissociation constant for binding to O). The agonist-independent, unliganded gating equilibrium constant E0 has been measured for both fetal and adult AChRs (14, 15). These two agonist-activation pathways can be connected to form a cycle that, without external energy, must obey the principle of microscopic reversibility (MR; the total energy change for a complete transit around a cycle is zero). A secondary goal was to measure independently all of the rate constants in the cycle to ascertain whether or not MR is satisfied in AChRs. If so, the “coupling” constant, which is the factor by which an agonist increases the gating equilibrium constant over the basal level (E1/E0), will be equal to the equilibrium dissociation constant ratio (Kd/Jd) (SI Appendix, Eq. S1). Several observations are relevant regarding the mechanism of agonist binding to adult AChRs. An equilibrium dissociation constant is the off/on rate constant ratio. The Kd values for agonists of similar size and charge differ substantially, mainly because of differences in the association rate constant (kon) (16). Further, for all agonists, kon is slower than expected from diffusion, and for some, it is temperature-dependent (17). These results suggest that the formation of the low-affinity AC complex requires both diffusion to the target and a local conformational change of the binding site (“catch”). An inference is that in binding to C, the agonist first forms an ultra-low-affinity “encounter complex” (18, 19) that then converts (via catch) to AC. The encounter complex is too short-lived to be detected as a discrete shut interval in electrophysiology experiments. Significance The binding of agonists to receptors is an essential event in cell signaling. We propose a general mechanism for agonist binding based on a model allosteric protein, the neuromuscular acetylcholine receptor. Binding constants were measured for different agonists, to both resting and active individual target sites. The results confirm a cyclic activation mechanism. Agonist binding requires diffusion and a local conformational change, with receptor activation accelerating the latter so that association becomes nearly diffusion-limited. At each site, receptor activation approximately doubles the agonist-binding energy. These results indicate that binding (“affinity”) and activation (“efficacy”), long considered to be independent processes, are linked obligatorily. We speculate that cyclic activation and coupling between activation and binding are fundamental aspects of receptor operation. Author contributions: T.K.N. and A.A. designed research; T.K.N. performed research; T.K.N. contributed new reagents/analytic tools; T.K.N. analyzed data; A.A. wrote the paper; and T.K.N. contributed to writing and critical assessment of the manuscript. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Published under the PNAS license. 1
To whom correspondence should be addressed. Email:
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ACA
C
Kd
bind AC
α–e. We start by describing rate and equilibrium constants of the
gate
Eo
E1
OA
AOA
gate
O
Jd
AO
bind Fig. 1. Activation pathways. Each receptor has two agonist-binding sites. A (superscript), agonist; C, resting state (low-affinity, closed-channel); O, active state (high-affinity, open-channel). Vertical steps are gating, and others are binding. On the front face, the activation cycle for one binding site shows two pathways connecting C with AO: bind-gate (red) and gate-bind (blue). Kd and Jd, resting and active equilibrium dissociation constants; E0 and E1, unliganded and monoliganded gating equilibrium constants. Without external energy, E1/E0 = Kd/Jd, where each ratio is the coupling constant.
In AChRs, the apparent Kd value is mainly determined by catch rather than by diffusion (SI Appendix, Eq. S6). As part of the global AC ↔ AO gating isomerization, there is a rearrangement of the binding pocket (“hold”) that serves to increase affinity. A second result of interest is that the high affinity of O is caused, in part, by an increased agonist association rate constant (jon) (5, 6). This observation suggests that the hold rearrangement does not generate high affinity by imposing a physical barrier, as this would be expected to decrease, rather than increase, jon. Third, for a series of structurally related cholinergic agonists and in WT adult AChRs, Kd and Jd (calculated assuming MR and two equivalent sites) are correlated exponentially. For a series of cholinergic agonists, Jd was approximately equal to Kd2 (16). This indicates that, on average, hold approximately doubles the agonist’s binding free energy. As a consequence, on a log-log scale, there is a linear correlation between Kd and E2, which is the gating equilibrium constant with two bound agonists (20). We have measured independently the rate and equilibrium constants for each step of each activation pathway for four different agonists, and estimated the affinity correlation exponent at individual α–e, α–γ, and α–δ neurotransmitter-binding sites. In these experiments, we did not make assumptions regarding MR or binding site equivalence. The experiments confirm the cyclic activation mechanism without external energy, reveal differences in agonist action at each kind of binding site in C versus O, and suggest that the C → O transition reduces the contribution of the catch rearrangement to the apparent overall association rate constant. They also confirm that binding and gating are linked energetically, with agonist association/dissociation being the first/last step of the forward/backward allosteric transition. Possible structural correlates of the binding process are discussed. Results Definitions. Because there are short-lived intermediate states in
both agonist-binding and receptor-isomerization reactions, we define the following words. “Gating” is the global allosteric transition, or a complete passage between C and O (with or without agonists). This isomerization involves structural changes at the binding sites (hold), extracellular domain, transmembrane helices, and gate. Forward gating is “opening,” and backward gating Nayak and Auerbach
is “closing.” Likewise, “binding” is a complete passage between unliganded and ligand-bound conformations (e.g., C and AC) that includes sojourns in the encounter complex. Forward binding is “association,” and backward binding is “dissociation.” “Affinity” is the inverse of an equilibrium dissociation constant. bind-gate pathway (C ↔ AC ↔ AO) at the adult AChR α–e site activated by the partial agonist carbamylcholine (CCh) (Fig. 2A and SI Appendix, Table S1). In these experiments, the α–δ site was disabled by the mutation δP123R so that only α–e was functional. Because these receptors were activated by CCh at only one site, we added background mutations (SI Appendix, Table S8) to increase the unliganded gating equilibrium constant E0, to put E1CCh into a suitable range for analysis (SI Appendix, Eq. S1). The background mutations had no effect on agonist binding (21) (SI Appendix, Fig. S10). E1CCh was measured using 20 mM [CCh] to ensure that the occupancy probability of the resting α–e site was >0.99. After accounting for a short-lived desensitized state and correcting for the effects of the background mutations and depolarization (which was applied to reduce fast channel block by the agonist), we estimate that E1CCh = 0.0026 at the standard condition (−100 mV). We know from experiments that E0WT in adult AChRs is 7.6 × 10−7 (14),
A
B
C
Fig. 2. Activation of α–e by CCh. The α–δ site of adult AChRs was disabled. In all current traces, O is down (membrane potential = −100 mV except where indicated). (A) Activation by the bind-gate pathway (background 10; SI Appendix, Table S8). (Top) Low time-resolution currents showing clusters of AC ↔ AO gating activity separated by silent desensitized periods. (Bottom) Interval-duration histograms and example clusters at different micromolar values [CCh]. KdCCh was estimated from a global, cross-concentration fit with E1CCh fixed to its value at 20 mM [CCh] (red, C). (B) Activation by the gatebind pathway (background 13; SI Appendix, Table S8). (Top) Low time-resolution currents without agonists showing clusters of C ↔ O gating activity separated by desensitized periods. *Long-duration opening. (Bottom) Interval-duration histograms and example clusters at different nanomolar values [CCh]. JdCCh was estimated from a global, cross-concentration fit to the gate-bind activation sequence with E0 fixed (blue, C). In open dwell-time histograms, the AO component (red) changes with [CCh], whereas the O (green) and long-lived (blue) components do not. (C) Rate and equilibrium constants measured independently for each step of the cycle (Fig. 1 and SI Appendix, Tables S1 and S2), corrected to standard condition (adult-WT, −100 mV).
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CA
so we immediately calculate the coupling constant, E1CCh/E0 = 3,364. From the natural logarithm of this number (SI Appendix, Eq. S2), we estimate that the hold gating rearrangement at α–e stabilizes the CCh receptor complex by −4.8 kcal/mol. Next, KdCCh at α–e was estimated by fitting globally intracluster interval durations from one-site AChRs activated by micromolar [CCh]. In these experiments, constitutive openings and dissociation from O were infrequent, so the activation pathway was almost exclusively by bind-gate (P > 0.99). The result was KdCCh = 182 μM, which is the ratio of dissociation/ association rate constants (koffCCh/konCCh): 13,100 s−1/7.2 × 107 s−1·M−1. Notice that konCCh is ∼50-fold slower than expected from diffusion (5 × 109–1010 M−1·s−1) (22–24). From the natural log of KdCCh, we calculate that for this agonist at α–e, the C → AC binding free energy change is −5.1 kcal/mol, which is similar to the hold energy. From the logarithm of E1CCh, we calculate that the AC → AO gating free energy change is +3.5 kcal/mol. Adding these, we estimate that the overall energy change in the bind-gate activation pathway is −1.6 kcal/mol. In the next experiments, we measured the CCh association and dissociation rate constants to the O conformation (jon and joff) in the gate-bind pathway. When agonists are present, a resting receptor can either open constitutively (C → O) or bind a ligand (C → AC). To ensure that activation almost always started with a constitutive opening, we added background mutations that increased the unliganded gating equilibrium constant substantially and applied very low (nanomolar) concentrations of CCh. Under these conditions, the high affinity of O allowed CCh to bind to constitutively active receptors, whereas the low-affinity resting of C dictated that activation by bind-gate was negligible. Fig. 2B shows constitutive and agonist-induced single-channel activity generated by the C ↔ O ↔ AO gate-bind pathway. Unliganded AChR gating is complex and generates both shortand long-lived open intervals (11, 12, 25). Respectively, these have been interpreted as arising from AChRs in which either one or two binding sites have been “primed” by a loop C rearrangement (26). However, it was shown earlier that almost all mutations of aromatic residues at the binding site eliminate the long-lived openings, without perturbing unliganded C ↔ O gating in most cases (27). These and other results (6) indicate that the short-lived openings reflect the unliganded gating step of the cycle and that the longlived openings are not part of the cycle. We used an extended kinetic scheme to fit the constitutive, single-channel interval durations (SI Appendix). In gate-bind activation, a component appeared in the open-interval duration histograms that increased in frequency with increasing [CCh] (Fig. 2B). We interpret these intervals to represent sojourns in AO. Also with increasing [CCh], the proportion of brief shut intervals increased. We interpret these events as AC → AO transitions that occur because the liganded open receptor can close and reopen before CCh dissociates. By fitting models to interval durations obtained across a nanomolar range [CCh], we estimate the open-state affinity of α–e to be JdCCh = 54 nM, which is the ratio of joff/jon = 211 s−1/3.9 × 109 M−1·s−1. Notice that jonCCh approaches the diffusion limit and that JdCCh is approximately the square of KdCCh. The overall free energy change in gate-bind activation by CCh at α–e is the sum of +8.3 kcal/mol (proportional to the natural log of E0) and −9.8 kcal/mol (proportional to the natural log of JdCCh), or −1.6 kcal/mol. This is the same value estimated for the bind-gate pathway. Similarly, the coupling constant calculated from KdCCh/JdCCh (3,370) is the same as calculated from E1CCh/E0 (3,364). This result demonstrates that the allosteric cycle for α–e activation by CCh satisfies MR. We considered the possibility that the complexity of unliganded gating may have influenced the rate constant estimates. The binding site mutation αY198F eliminates long-lived, constitutive openings, with almost no effect on binding or gating (27). We examined gate-bind activation by CCh at α–e with this mutation added (SI Appendix, Fig. S1). The rate and equilibrium constants with αY198F were similar to those estimated without it 11916 | www.pnas.org/cgi/doi/10.1073/pnas.1711228114
(SI Appendix, Table S3). This result is further evidence that the long-lived openings are not relevant to the cycle. We also considered the possibility that the mutation we used to disable α–δ (δP123R) might have influenced the α–e site in a long-distance manner. To test this, we used α-conotoxin MI, a site-specific toxin that eliminates agonist binding only at α–δ (28). The results were the same as with δP123R (SI Appendix, Fig. S2 and Table S5). Evidently, δP123R does not influence α–e, and is as effective as this conotoxin in crippling α–δ. We extended this line of investigation by estimating both bindgate and gate-bind constants at α–e (with δP123R) using ACh, tetramethyammonium (TMA), or choline (Cho) as the agonist (SI Appendix, Figs. S3–S5 and Tables S1 and S2). SI Appendix, Fig. S6 shows the normalized concentration–response curves for all four agonists at α–e. The Hill slopes were all kon. The position of the effective transition state for the overall binding process (‡) moves from near the catch barrier in C (red) to near the diffusion barrier in O (blue), to influence ϕbind (Fig. 5). For most agonists, gating causes the catch barrier to change from being above to below the diffusion limit (SI Appendix, Fig. S9).
Nayak and Auerbach
twice that with αY198 and αW149. Further, the transition to O increases the αY190 interaction energy slightly more than the others (∼2.3-fold versus 1.7-fold). [In fetal receptors, γW55 also makes a significant contribution to Kd, but the energy change from this side chain in C → O has not been measured.] It is remarkable that in adult AChRs, hold not only approximately doubles binding energy at each site (Fig. 5) but does so nearly uniformly for each QA–aromatic interaction. A comparison of putative resting versus active structures of other pentameric receptors shows that the binding sites are more compact in O versus C (38–42). In AChRs, a smaller pocket would be expected to decrease QA-ring separation (and perhaps the effective dielectric constant) so as to strengthen all interaction energies. Another C versus O structural parameter to consider is the position of the QA with regard to the pocket (34). A reduction in pocket volume and a deeper positioning of the QA caused by the hold rearrangement are consistent with higher affinity of and slower dissociation from O for all agonists. If a reduction in pocket volume is a structural correlate of hold, then the results suggest that compaction lowers the catch energy barrier (Fig. 6). Further, the linear correlation in energy between catch and hold energies might indicate that catch, too, may involve a reduction in pocket volume. It is possible that the movement of the agonist from the encounter complex into the aromatic cage (catch) compacts the binding pocket, and that this process is in some way arrested when the receptor achieves AC but continues when the protein isomerizes to AO. Perhaps the removal of the arresting structure(s) (that occurs in AC → AO) also lowers the catch energy barrier to allow jon to approach the diffusion limit. What is the selective advantage of having a local conformational change (catch) in the neurotransmitter-binding process? From the
point of view of protein chemistry, the AC state seems unnecessary and a “waste” of binding energy. It is easy [one mutation (43)] to construct a receptor that is active constitutively with a high probability, so that the neurotransmitter molecule could just diffuse into an active site, bind with high affinity, and increase open probability (PO). Part of the answer lies in the physiological requirement at the neuromuscular junction of having a fast-rising synaptic response superimposed on a nearly silent (low PO) background. In order for gate-bind activation to generate a rapid response to the neurotransmitter, the basal activity level would have to be unacceptably high. Another reason for catch may relate to chemical recognition. A barrier to the formation of AC allows the receptor either to reject the ligand (by dissociation from the encounter complex) or to accept it (by crossing the catch barrier). At the neuromuscular synapse, AChRs must reject choline and accept ACh, but both ligands would be significant activators if allowed to diffuse into a constitutively O pocket. It is possible that the formation of AC and the affinity correlation are mechanisms that ensure proper signal recognition in complex chemical environments that contain structurally similar molecules.
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Methods We performed single-channel, cell-attached, patch-clamp electrophysiology using mouse AChRs having only one functional binding site. Currents were analyzed using QuB software to estimate rate constants, by fitting either bind-gate or gate-bind activation sequences to intracluster interval durations. A detailed description of the methods used is given in SI Appendix.
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ACKNOWLEDGMENTS. We thank M. Shero, M. Teeling, J. Jordan, and C. Nicolai for technical assistance. This work was funded by the NIH (Grant NS064969).
