Why the International Olympic Committee Convened

Cyclic activation of endplate acetylcholine receptors

Tapan K. Nayaka and Anthony Auerbacha,1

aDepartment 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 On rare occasions, WT AChRs activate without a bound agonist active versus resting conformations of the protein. Activation can (10, 11). Many mutations throughout the protein increase sub- occur by either of two pathways that connect to form a cycle: stantially the frequency of constitutive openings (12), including some Agonists bind to resting receptors that then become active, or that occur naturally to cause disease (13). Hence, a second possible resting receptors activate and then bind agonists. We used muta- activation sequence is that an unliganded receptor switches on and A tions to construct endplate acetylcholine receptors (AChRs) having the agonist then binds (C ↔ O ↔ O). The equilibrium constants of “ ” only one functional neurotransmitter-binding site and single-channel the two steps of this gate-bind path are E0 (gating without any electrophysiology to measure independently binding constants for agonists) and 1/Jd (dissociation constant for binding to O). The four different agonists, to both resting and active conformations of agonist-independent, unliganded gating equilibrium constant E0 has each site. For all agonists and sites, the total free energy change in been measured for both fetal and adult AChRs (14, 15). each pathway was the same, confirming the activation cycle without These two agonist-activation pathways can be connected to external energy. Other results show that (i) there is no cooperativity form a cycle that, without external energy, must obey the prin- ciple of microscopic reversibility (MR; the total energy change between sites; (ii) agonist association is slower than diffusion for a complete transit around a cycle is zero). A secondary goal in resting receptors but nearly diffusional in active receptors; iii was to measure independently all of the rate constants in the ( ) whereas resting affinity is determined mainly by agonist associ- cycle to ascertain whether or not MR is satisfied in AChRs. If so, ation, active affinity is determined mainly by agonist dissociation; and “ ” iv the coupling constant, which is the factor by which an agonist ( ) at each site and for all agonists, receptor activation approxi- increases the gating equilibrium constant over the basal level mately doubles the agonist-binding free energy. We discuss a two- (E1/E0), will be equal to the equilibrium dissociation constant step mechanism for binding that involves diffusion and a local ratio (Kd/Jd)(SI Appendix, Eq. S1). conformational change (“catch”) that is modulated by receptor ac- Several observations are relevant regarding the mechanism of tivation. The results suggest that binding to a resting site and the agonist binding to adult AChRs. An equilibrium dissociation con- switch to high affinity are both integral parts of a single allosteric stant is the off/on rate constant ratio. The Kd values for agonists of transition. We hypothesize that catch ensures proper signal recog- similar size and charge differ substantially, mainly because of dif- nition in complex chemical environments and that binding site com- ferences in the association rate constant (kon) (16). Further, for all paction is a determinant of both resting and active affinity. agonists, kon is slower than expected from diffusion, and for some, it is temperature-dependent (17). These results suggest that the for- allosteric activation | ion channel | agonist binding | nicotinic mation of the low-affinity AC complex requires both diffusion to the target and a local conformational change of the binding site “ ” cetylcholine receptors (AChRs) are allosteric signaling ( catch ). An inference is that in binding to C, the agonist first forms an ultra-low-affinity “encounter complex” (18, 19) that then converts Aproteins that produce transient membrane currents by A switching globally between a resting C (closed-channel) confor- (via catch) to C. The encounter complex is too short-lived to be detected as a discrete shut interval in electrophysiology experiments. mation 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 Significance bound agonists, the newfound ligand-binding energy lowers the energy barrier between C and O and stabilizes the O conformation The binding of agonists to receptors is an essential event in cell so as to increase the activation rate and activation probability signaling. We propose a general mechanism for agonist bind- above their basal levels. ing based on a model allosteric protein, the neuromuscular Neuromuscular AChRs (∼300 kDa) have two α1 subunits and acetylcholine receptor. Binding constants were measured for one each of β, δ, and either e (adult-type) or γ (fetal-type), with different agonists, to both resting and active individual target two neurotransmitter-binding sites located at α–e/γ and α–δ sites. The results confirm a cyclic activation mechanism. Agonist subunit interfaces. The rate and equilibrium constants for bind- binding requires diffusion and a local conformational change, ing to the resting C conformation have been measured for many with receptor activation accelerating the latter so that associ- agonists in wild-type (WT) adult and fetal AChRs (1–4), but only ation becomes nearly diffusion-limited. At each site, receptor a few studies have addressed binding to the active O confor- activation approximately doubles the agonist-binding energy. mation (5, 6). Our primary goal was to compare agonist binding These results indicate that binding (“affinity”) and activation to C versus O at each kind of binding site. (“efficacy”), long considered to be independent processes, are Activation of receptors by agonists can be described by a re- linked obligatorily. We speculate that cyclic activation and action cycle (7–9). In this scheme (Fig. 1), C and O represent coupling between activation and binding are fundamental as- stable end states (energy wells) and the arrows represent un- pects of receptor operation. stable transition states (energy barriers). For a receptor with only one functional binding site, there are two activation pathways Author contributions: T.K.N. and A.A. designed research; T.K.N. performed research; that connect the unliganded resting state C with liganded active T.K.N. contributed new reagents/analytic tools; T.K.N. analyzed data; A.A. wrote the state AO (where the superscripted A is the agonist). The usual paper; and T.K.N. contributed to writing and critical assessment of the manuscript. sequence in WT receptors is that the agonist binds and the re- The authors declare no conflict of interest. ceptor then activates (C ↔ AC ↔ AO). The equilibrium constants This article is a PNAS Direct Submission. of this “bind-gate” path are 1/Kd (dissociation constant for Published under the PNAS license. 1 binding to C) and E1 (gating with one bound agonist). The free To whom correspondence should be addressed. Email: [email protected]. A energy difference, C to O, is the sum of the energy differences This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. for each step in the pathway. 1073/pnas.1711228114/-/DCSupplemental.

11914–11919 | PNAS | November 7, 2017 | vol. 114 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1711228114 Downloaded by guest on September 23, 2021 is “closing.” Likewise, “binding” is a complete passage between A A A unliganded and ligand-bound conformations (e.g., C and AC) that C C includes sojourns in the encounter complex. Forward binding is “association,” and backward binding is “dissociation.”“Affinity” is bind the inverse of an equilibrium dissociation constant. C K A α–e. We start by describing rate and equilibrium constants of the d A A C bind-gate pathway (C ↔ C ↔ O) at the adult AChR α–e site gate 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 Eo E1 functional. Because these receptors were activated by CCh at A A A only one site, we added background mutations (SI Appendix, O O Table S8) to increase the unliganded gating equilibrium constant CCh SI Appendix gate E0, to put E1 into a suitable range for analysis ( , Eq. S1). The background mutations had no effect on agonist binding (21) (SI Appendix, Fig. S10). A CCh O J O E1 was measured using 20 mM [CCh] to ensure that the d occupancy probability of the resting α–e site was >0.99. After ac- bind counting for a short-lived desensitized state and correcting for the effects of the background mutations and depolarization (which was Fig. 1. Activation pathways. Each receptor has two agonist-binding sites. A applied to reduce fast channel block by the agonist), we estimate CCh = − (superscript), agonist; C, resting state (low-affinity, closed-channel); O, active that E1 0.0026 at the standard condition ( 100 mV). We WT −7 state (high-affinity, open-channel). Vertical steps are gating, and others are know from experiments that E0 in adult AChRs is 7.6 × 10 (14), 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 ex- A B ternal 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 2 series of cholinergic agonists, Jd was approximately equal to Kd (16). This indicates that, on average, hold approximately doubles the agonist’s binding free energy. As a consequence, on a log-log C 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 dif- ferent agonists, and estimated the affinity correlation exponent at individual α–e, α–γ,andα–δ neurotransmitter-binding sites. In these experiments, we did not make assumptions regarding MR or binding site equivalence. The experiments confirm the cyclic acti- Fig. 2. Activation of α–e by CCh. The α–δ site of adult AChRs was disabled. vation mechanism without external energy, reveal differences in In all current traces, O is down (membrane potential = −100 mV except agonist action at each kind of binding site in C versus O, and where indicated). (A) Activation by the bind-gate pathway (background 10; suggestthattheC→ O transition reduces the contribution of the SI Appendix, Table S8). (Top) Low time-resolution currents showing clusters catch rearrangement to the apparent overall association rate con- of AC ↔ AO gating activity separated by silent desensitized periods. (Bottom) stant. They also confirm that binding and gating are linked ener- Interval-duration histograms and example clusters at different micromolar CCh getically, with agonist association/dissociation being the first/last values [CCh]. Kd was estimated from a global, cross-concentration fit with CCh step of the forward/backward allosteric transition. Possible struc- E1 fixed to its value at 20 mM [CCh] (red, C). (B) Activation by the gate- tural correlates of the binding process are discussed. bind pathway (background 13; SI Appendix, Table S8). (Top) Low time-res- olution currents without agonists showing clusters of C ↔ O gating activity Results separated by desensitized periods. *Long-duration opening. (Bottom)In- terval-duration histograms and example clusters at different nanomolar Definitions. Because there are short-lived intermediate states in values [CCh]. J CCh was estimated from a global, cross-concentration fit to both agonist-binding and receptor-isomerization reactions, we d the gate-bind activation sequence with E0 fixed (blue, C). In open dwell-time “ ” A define the following words. Gating is the global allosteric histograms, the O component (red) changes with [CCh], whereas the O transition, or a complete passage between C and O (with or (green) and long-lived (blue) components do not. (C) Rate and equilibrium without agonists). This isomerization involves structural changes constants measured independently for each step of the cycle (Fig. 1 and SI

at the binding sites (hold), extracellular domain, transmembrane Appendix, Tables S1 and S2), corrected to standard condition (adult-WT, BIOCHEMISTRY helices, and gate. Forward gating is “opening,” and backward gating −100 mV).

Nayak and Auerbach PNAS | November 7, 2017 | vol. 114 | no. 45 | 11915 Downloaded by guest on September 23, 2021 CCh so we immediately calculate the coupling constant, E1 /E0 = 3,364. (SI Appendix, Table S3). This result is further evidence that the From the natural logarithm of this number (SI Appendix,Eq.S2), we long-lived openings are not relevant to the cycle. estimate that the hold gating rearrangement at α–e stabilizes We also considered the possibility that the mutation we used the CCh receptor complex by −4.8 kcal/mol. to disable α–δ (δP123R) might have influenced the α–e site in a CCh Next, Kd at α–e was estimated by fitting globally intra- long-distance manner. To test this, we used α-conotoxin MI, a cluster interval durations from one-site AChRs activated by site-specific toxin that eliminates agonist binding only at α–δ micromolar [CCh]. In these experiments, constitutive openings (28). The results were the same as with δP123R (SI Appendix, and dissociation from O were infrequent, so the activation Fig. S2 and Table S5). Evidently, δP123R does not influence α–e, pathway was almost exclusively by bind-gate (P > 0.99). The and is as effective as this conotoxin in crippling α–δ. CCh result was Kd = 182 μM, which is the ratio of dissociation/ We extended this line of investigation by estimating both bind- CCh CCh −1 association rate constants (koff /kon ): 13,100 s /7.2 × gate and gate-bind constants at α–e (with δP123R) using ACh, 7 −1 −1 CCh 10 s ·M . Notice that kon is ∼50-fold slower than expected tetramethyammonium (TMA), or choline (Cho) as the agonist − − from diffusion (5 × 109–1010 M 1·s 1) (22–24). (SI Appendix, Figs. S3–S5 and Tables S1 and S2). SI Appendix, CCh From the natural log of Kd , we calculate that for this ag- Fig. S6 shows the normalized concentration–response curves for onist at α–e,theC→ AC binding free energy change is −5.1 kcal/mol, all four agonists at α–e. The Hill slopes were all <1, indicating CCh which is similar to the hold energy. From the logarithm of E1 , that only one site was active. we calculate that the AC → AO gating free energy change is Regarding gate-bind, the association rate constant to O was +3.5 kcal/mol. Adding these, we estimate that the overall energy similar for ACh, TMA, and CCh, namely, faster than to C and Cho change in the bind-gate activation pathway is −1.6 kcal/mol. near the diffusion limit, but jon was ∼100-fold slower. For all In the next experiments, we measured the CCh association and agonists, dissociation from O was slower than from C. The ag- j j dissociation rate constants to the O conformation ( on and off)in onist dependence of Kd was caused mainly by variations in as- the gate-bind pathway. When agonists are present, a resting re- sociation, but the agonist dependence of Jd was caused mainly by ceptor can either open constitutively (C → O) or bind a ligand variations in dissociation. As was the case with CCh, the α–e (C → AC). To ensure that activation almost always started with a coupling constants were the same measured either from binding constitutive opening, we added background mutations that in- or gating equilibrium constant ratios (SI Appendix, Table S4). creased the unliganded gating equilibrium constant substantially and applied very low (nanomolar) concentrations of CCh. Under α–γ and α–δ. We repeated the above experiments (four agonists, these conditions, the high affinity of O allowed CCh to bind to bind-gate and gate-bind) using fetal AChRs having only a func- constitutively active receptors, whereas the low-affinity resting of tional α–γ site (SI Appendix, Tables S1 and S2). Agonist binding C dictated that activation by bind-gate was negligible. Fig. 2B to α–γ was similar to α–e insofar as association to O was faster shows constitutive and agonist-induced single-channel activity than to C and near diffusion-limited, and dissociation from O generated by the C ↔ O ↔ AO gate-bind pathway. was slower and more variable than from C. At α–γ, the total Unliganded AChR gating is complex and generates both short- energy changes by the bind-gate and gate-bind paths were and long-lived open intervals (11, 12, 25). Respectively, these have equivalent (SI Appendix, Table S4). been interpreted as arising from AChRs in which either one or two There were two notable differences between α–γ and α–e. binding sites have been “primed” by a loop C rearrangement (26). First, as shown previously, the fetal α–γ site has a higher resting However, it was shown earlier that almost all mutations of aro- affinity for all four agonists, particularly ACh (29). Second, for matic residues at the binding site eliminate the long-lived openings, all agonists, at α–γ, association to O, which we propose to be without perturbing unliganded C ↔ O gating in most cases (27). diffusion-limited, is ∼10-fold faster than at α–e. We hypothesize These and other results (6) indicate that the short-lived openings that there are fixed charges on the γ subunit in the vicinity of the reflect the unliganded gating step of the cycle and that the long- binding pocket that serve to increase the local agonist concen- lived openings are not part of the cycle. tration, and thus increase the apparent association rate constant. We used an extended kinetic scheme to fit the constitutive, This hypothesis will be addressed experimentally, elsewhere. single-channel interval durations (SI Appendix). In gate-bind Next, we repeated the above experiments using adult AChRs activation, a component appeared in the open-interval dura- having only a functional α–δ site, with α–e disabled by the mu- tion histograms that increased in frequency with increasing tation eP121R (SI Appendix, Fig. S7 and Tables S1 and S2). We [CCh] (Fig. 2B). We interpret these intervals to represent so- measured the bind-gate parameters for all four agonists, but A journs in O. Also with increasing [CCh], the proportion of brief those for gate-bind were measured only for ACh, CCh, and A A shut intervals increased. We interpret these events as C → O TMA. The results show that the resting affinities of α–δ are transitions that occur because the liganded open receptor can approximately the same as at α–e for all agonists (CCh was about close and reopen before CCh dissociates. By fitting models to threefold lower). SI Appendix, Fig. S8 shows in graphical form interval durations obtained across a nanomolar range [CCh], we CCh the equilibrium and rate constants for all agonists and sites. estimate the open-state affinity of α–e to be Jd = 54 nM, −1 9 −1 −1 Fig. 3 summarizes the results regarding coupling constants. which is the ratio of joff/jon = 211 s /3.9 × 10 M ·s . Notice A CCh CCh Fig. 3 shows that the coupling constants calculated from the that jon approaches the diffusion limit and that Jd is ap- CCh gating equilibrium constant ratios and from the equilibrium proximately the square of Kd . dissociation constant ratios are the same. This demonstrates that The overall free energy change in gate-bind activation by CCh MR is satisfied for all agonists at all three sites. Fig. 3B compares at α–e is the sum of +8.3 kcal/mol (proportional to the natural the total coupling constant energy in adult- and fetal-type − log of E0) and 9.8 kcal/mol (proportional to the natural log of AChRs estimated using WT receptors (3) with the sum of the CCh − Jd ), or 1.6 kcal/mol. This is the same value estimated for the single-site energies, either α–e + α–δ for adult-type AChRs or bind-gate pathway. Similarly, the coupling constant calculated α–γ + α–δ for fetal-type AChRs (SI Appendix, Table S6). The two CCh CCh CCh from Kd /Jd (3,370) is the same as calculated from E1 /E0 estimates are the same, indicating that there is no measurable (3,364). This result demonstrates that the allosteric cycle for α–e interaction between the two binding sites. activation by CCh satisfies MR. We considered the possibility that the complexity of unli- Correlations. In this section, we describe correlations between ganded gating may have influenced the rate constant estimates. binding energies. Previously, we estimated that in WT adult The binding site mutation αY198F eliminates long-lived, con- AChRs, activation approximately doubles the agonist’s resting 1.92 ± 0.04 stitutive openings, with almost no effect on binding or gating binding energy [Jd ≈ Kd (16)]. To make this estimation, (27). We examined gate-bind activation by CCh at α–e with this Jd for each ligand was calculated from the other equilibrium mutation added (SI Appendix, Fig. S1). The rate and equilibrium constants assuming MR, and the two binding sites were assumed constants with αY198F were similar to those estimated without it to be equivalent. To avoid both of these assumptions, and to

11916 | www.pnas.org/cgi/doi/10.1073/pnas.1711228114 Nayak and Auerbach Downloaded by guest on September 23, 2021 A B form the encounter complex) and a catch conformational change to 1-site 2-site (to form the bound complex). The energy values are taken from the -3 -6 adult Cho fetal experimental rate constant measurements for CCh binding to α–e SI Appendix -8 TMA ( ). In binding to C (red line in Fig. 6), the catch barrier -5 CCh dominates so that this conformational change mainly determines -10 ACh k constants on, but in binding to O (blue line in Fig. 6), diffusion mainly de- -12 termines jon because the gating rearrangement lowers the catch free -7 2-sites combined slope=1.0 slope=1.1

from gating equilibrium from gating -7 -5 -3 -12 -10 -8 -6 -12 -10 -8 energy. As a consequence, the pseudotransition state for the com- from equilibrium dissociation sum of individual sites ‡ “ ” “ ” constants bined, two-step process ( in Fig. 6) shifts from late to early in C → O (from bound toward free) to reduce ϕbind. Fig. 3. MR is satisfied. (A) Coupling constant free energy (kcal/mol) calculated SI Appendix, Fig. S9 summarizes similar analyses for just the − from the ratio of equilibrium dissociation constants [abscissa; 0.59ln(Kd/Jd)] is catch step for different agonists and sites. At α–e, opening lowers the same as calculated from the ratio of gating equilibrium constants [ordi- the catch barrier below that for diffusion for ACh, CCh, and − nate; 0.59ln (E1/E0)] (SI Appendix,TableS1). The slope of the linear regression TMA, but not for the weak agonist Cho. For this reason, the is 1.0 ± 0.1. The color code of the symbols is shown in B.(B) Total receptor ϕbind value for Cho remains large in O-binding (Fig. 5, Bottom coupling constant free energy (two sites, combined) is the same whether esti- Left). At α–γ, for all agonists, the catch barrier is at or below that mated using WT AChRs (ordinate) or by adding single-site energies [abscissa; α–δ α–e + α–δ for adult (1.0 ± 0.05) and α–γ + α–δ for fetal (1.1 ± 0.08)]. of diffusion. At , this barrier with ACh and TMA is below that of diffusion, but TMA remains above. For all agonists and sites, the C → O gating transition not only increases affinity but ascertain if there is an affinity correlation at each kind of site also lowers free energy of the catch transition state. (including fetal α–γ, which was not probed previously), we Discussion plotted the experimental, single-site values of 1/Kd versus 1/Jd, log-log (Fig. 4). At all three kinds of site, the AC → AO transition Cycle. We investigated agonist binding to individual AChR neuro- approximately doubles the agonist-binding free energy. transmitter-binding sites in both their C and O conformations. For Bottom “ Fig. 4 ( ) shows log-log plots of Kd versus E1 [a binding- all agonists and at all sites, the free energy change in the physio- gating” plot (20)] for the agonists at each site (SI Appendix, Eq. logical activation pathway (bind-gate) and the mutation-induced S4 Top ). Whereas the 1/Kd-1/Jd plots in Fig. 4 ( ) were constructed activation pathway (gate-bind) was the same (Fig. 3A). The exper- from explicit measurements of these equilibrium constants in iments with the one-atom mutation αY198F show that long-lived bind-gate and gate-bind (with mutations added to generate constitutive openings are not part of the cycle. The results confirm Bottom constitutive activity), the binding-gating plots in Fig. 4 ( ) the cyclic mechanism without an additional energy source. The O are just from bind-gate and did not require a high level of con- state is the same, with respect to conductance, voltage dependence stitutive activity. The linear fit to a binding-gating plot provides (14), and energy (structure), with or without a bound agonist. “ ” − estimates of both the affinity correlation exponent m (from the Violations of MR have been observed in a Cl channel (30) and slope) and the unliganded gating equilibrium constant E0 (from a mutant NMDA receptor (31). In both instances, the external the y-intercept) (SI Appendix, Eq. S4). energy source is a permeating ion(s) that binds to a site(s) in the At all three AChR sites and for all agonists, 1/K (affinity) was d pore to alter the relative probability of subconductance levels. In correlated linearly with E1 (related to efficacy) on a log-log scale. From the slopes, we estimate m ∼ 2 at all sites, reflecting the fact these cases, the ion acts as a ligand (rather than as a charge carrier) that for all agonists, the global switch from ACtoAO approxi- to influence a local, conductance-changing protein rearrangement. mately doubles the ligand-binding energy at each site. Impor- The fact that MR is satisfied in AChRs (with a WT pore) indicates tantly, the E0 values for adult- and fetal-type AChRs estimated from the y-intercepts are in excellent agreement with those es- SI Ap- timated previously using completely different methods ( C vs O affinity ACh 11 pendix, Table S7). These correspondences confirm that Appendix, 8 ACh 8 ACh CCh 10 CCh S4 ) Eq. pertains to all three sites and show that it is possible to d 7 TMA 9 TMA estimate two agonist-independent constants, E0 and m, from a 7 CCh 6 8 log(1/J single-site binding-gating plot. Notice that E0 pertains to the Cho m=1.9±0.2 Cho 1.7±0.3 TMA 1.7±0.2 whole receptor, whereas m pertains to each binding site. How- 5 7 6 6 2.0 2.5 3.0 3.5 4.0 ever, m happens to be similar at α–δ, α–e, and α–γ. 345 3.0 3.5 4.0 log(1/K ) The second energy correlation we considered at each site is d binding vs gating 0 between the agonist association rate constant and the equilib- 0 0

rium dissociation constant, again on a log-log scale (Fig. 5). A ACh ACh ACh -2 ) -2 CCh -2 CCh log-log plot between a rate and an equilibrium constant is called 1 CCh TMA TMA -4 TMA a rate-equilibrium free energy relationship (REFER). REFERs Cho for AChR gating have been presented elsewhere (21); here, we log(E -4 Cho -4 Cho -6 report those for binding to both C and O. The linear slope of the m=1.9±0.1 2.0±0.1 1.9±0.0 -6 -6 ϕ -8 binding REFER for an agonist series ( bind) gives the relative -6 -4 -2 0 -6 -4 -2 0 -6 -4 -2 0 position of the effective binding transition state as being close to log(Kd) “bound” or to “free,” on a scale from 1 to 0. The top row of Fig. 5 shows REFERs for binding to resting Fig. 4. Correlations between equilibrium constants. Each column is a dif- ferent binding site. (Top) Log-log plot of resting affinity (1/K ) versus active sites. At all sites and for all agonists, log(kon) is almost perfectly d affinity (1/Jd) measured independently at individual sites. Solid lines indicate correlated with log(1/Kd)(ϕbind ∼ 1). This indicates that the A linear regression fit (slope m; SI Appendix,Eq.S3). At all sites and for all transition state is positioned near bound ( C). The bottom row 2 agonists, Jd ≈ Kd , indicating that binding energy approximately doubles of Fig. 5 shows REFERs for binding to active sites. For the A A j when the receptor switches from Cto O and that the coupling constant strong agonists ACh and CCh, log( on) is weakly correlated with energy is approximately equal to the resting binding energy (SI Appendix, log (J )(ϕ ∼ 0.25). This indicates that for these agonists, the d bind Eqs. S1 and S2). (Bottom) Binding-gating plots. Kd and E1 measured in- transition state is positioned closer to free (O). dependently at each site are correlated linearly on a log-log scale [with slope

Fig. 6 provides a rationale for the two apparent differences in (1 − m); SI Appendix,Eq.S4].TheWTE0 values calculated from the y-intercepts

agonist binding to C versus O, namely, slower association and larger (dashed lines and left arrowheads) are approximately the same as estimated BIOCHEMISTRY ϕbind. The overall binding process includes both a diffusion step (to previously using different methods (○; SI Appendix,TableS7).

Nayak and Auerbach PNAS | November 7, 2017 | vol. 114 | no. 45 | 11917 Downloaded by guest on September 23, 2021 A binding to C state C is always a constant fraction (1/m) of that of the high- 9 ACh ACh ACh affinity state. 8 8 CCh CCh Catch is the main determinant of resting affinity (1/Kd)(SI CCh ) 8 Appendix, Eq. S6), and hold is part of the gating equilibrium on TMA 7 TMA 7 SI Appendix TMA constant (E1)( , Fig. S11). The energy correlation log(k Cho Cho 7 Cho between these two binding site rearrangements indicates that ɸbind=1.06 ɸbind=0.91 6 ɸbind=1.01 6 binding and gating, two processes long considered to be in- 234345 234 log(1/Kd) dependent (33), are coupled. The catch-and-hold linkage sug- binding to O gests that agonist association (from the encounter complex) is

11 12 11 the first step in opening and that agonist dissociation (to the CCh ACh 10 TMA 11 ACh encounter complex) is the last step in closing. Binding to the 10 CCh ) ↔

on 9 10 resting state and the low high-affinity switch of the binding site 9 that occurs within gating are both integral parts of a single, log(j 8 ( ) 9 ( )TMA Cho ɸ =0.30 ɸ =0.20 ɸ =0.22 global allosteric transition. The effect of activation on catch (Fig. 7 bind bind 8 bind 8 6) is further evidence of this gating-binding linkage. 45678 678910116.0 6.5 7.0 7.5 8.0

log(1/Jd) Structure and Function. Binding free energy derives from a mixture of Fig. 5. Correlations between rate and equilibrium constants. (Top) Binding enthalpy (van der Waals, hydrogen bonds, electrostatic, dipole–dipole, ϕ ∼ to C. The linear slope is bind 1 at all three sites. Differences in Kd are due cation–π) and entropy (solvent, conformation, rotation–translation) exclusively to differences in kon (SI Appendix, Table S8). The effective bind- components. The energy doubling for agonists is curious because it is ing transition state to C is near bound (AC). (Bottom) Binding to O. The slope multiplicative rather than additive. It is easy to imagine a local hold ϕ ∼ is bind 0.25 at all three sites. Differences in Jd are due mainly to differences conformational change that adds a constant favorable energy but in joff. The effective binding transition state is near free (O). The symbols in more difficult to conjure one that increases the total energy from all parentheses were not included in the fits. sources by a constant factor. Hence, we speculate that in AChRs, one or a few of the above binding energy sources predominate. There is that neither ion binding nor the transmembrane current has a evidence suggesting that the entropic component of Kd is small in detectable effect on the energy balance of the cycle. adult AChRs (17). At all three binding sites, Kd is determined by cation–anion The total energy from affinity changes at two WT sites has –π ’ been estimated for several agonists (3, 29). In adult-type AChRs, (34) and cation (35, 36) interactions between the agonist s these energies reflect the coupling constant energy α–e plus α–δ, quaternary ammonium (QA) and a cluster of aromatic side and in fetal-type AChRs, they reflect α–γ plus α–δ. For all ago- chains. The free energy contribution from each of these has been estimated in adult AChRs (with ACh) in both C (37) and O (32) nists and in both fetal and adult receptors, the two-site energies α are the same as the sums of one-site energies (Fig. 3B). The conformations. In C, the interaction energy with Y190 is about AChR neurotransmitter-binding sites operate independently with regard to coupling constant and affinity.

Binding. Agonist binding to resting C versus active O appears to be on different. The association rate constant to C (kon) is relatively slow j off and below diffusion, whereas that to O ( on) is fast and close to the catch diffusion limit (SI Appendix,Fig.S8). Agonist differences in Kd are k caused by differences in on, whereas those in Jd are caused mainly ‡ ‡ by differences in joff (Fig. 5 and SI Appendix,Fig.S8). Despite these diffusion apparent differences, our proposed mechanism of binding is es- sentially the same for both resting and active sites (Fig. 6). In both instances, binding is a two-step process: diffusion and catch. The apparent differences are caused by the opening transition (specifi- cally the hold rearrangement of the binding site), which lowers the free encounter catch potential energy surface (PES). Consequently, whereas at resting sites, association is dominated by catch, at active sites, it is C or O complex A dominated by diffusion. The C → O transition also shifts the po- C sition of the overall effective transition state toward the free con- dition so as to reduce ϕbind. Our hypothesis is that agonist binding to C and to O occurs by the same essential mechanism, but over a PES that is modulated by gating. A Affinity Correlation. There is a linear correlation between resting O and active binding energies that generates a log-log correlation 5 kcal/mol between binding and gating equilibrium constants (Fig. 4). At all three kinds of AChR neurotransmitter-binding site, hold approxi- bound mately doubles the agonist resting binding energy, which is de- Fig. 6. Energy landscapes for agonist binding. PES for 20 mM CCh binding to termined mainly by catch. [This pertains to ligands that resemble α–e the neurotransmitter, but it does not pertain to all ligands. By the resting (red) and active (blue) site (Fig. 2). The three energy wells cor- definition, the binding energy of an antagonist either decreases or respond to the states of SI Appendix,Scheme1. Apparent association and ≥ ≤ dissociation (“on” and “off”) rate constants reflect both diffusion and the catch does not change with hold (Jd Kd); thus, for these ligands, m 1]. α–e SI Appendix conformational change. As shown in the gray area, at , the opening con- ,Fig.S11shows catch-and-hold energy landscapes formational change (hold) lowers the catch barrier free energy to make j > for a weak agonist and a strong agonist (32). The two binding site on kon. The position of the effective transition state for the overall binding process rearrangements comprise a linked, two-step reaction. Different (‡) moves from near the catch barrier in C (red) to near the diffusion barrier in O

agonists stabilize the high-affinity state to different extents, to (blue), to influence ϕbind (Fig. 5). For most agonists, gating causes the catch “tilt” the overall PES to different extents. Because of the barrier to change from being above to below the diffusion limit (SI Appendix, obligatory linkage, the degree of stabilization of the low-affinity Fig. S9).

11918 | www.pnas.org/cgi/doi/10.1073/pnas.1711228114 Nayak and Auerbach Downloaded by guest on September 23, 2021 twice that with αY198 and αW149. Further, the transition to O point of view of protein chemistry, the AC state seems unnecessary increases the αY190 interaction energy slightly more than the and a “waste” of binding energy. It is easy [one mutation (43)] to others (∼2.3-fold versus 1.7-fold). [In fetal receptors, γW55 also construct a receptor that is active constitutively with a high prob- makes a significant contribution to Kd, but the energy change ability, so that the neurotransmitter molecule could just diffuse into from this side chain in C → O has not been measured.] It is an active site, bind with high affinity, and increase open probability remarkable that in adult AChRs, hold not only approximately (PO). Part of the answer lies in the physiological requirement at the doubles binding energy at each site (Fig. 5) but does so nearly neuromuscular junction of having a fast-rising synaptic response – uniformly for each QA aromatic interaction. superimposed on a nearly silent (low PO) background. In order for A comparison of putative resting versus active structures of gate-bind activation to generate a rapid response to the neuro- other pentameric receptors shows that the binding sites are more transmitter, the basal activity level would have to be unacceptably compact in O versus C (38–42). In AChRs, a smaller pocket high. Another reason for catch may relate to chemical recognition. would be expected to decrease QA-ring separation (and perhaps A barrier to the formation of AC allows the receptor either to the effective dielectric constant) so as to strengthen all in- reject the ligand (by dissociation from the encounter complex) or teraction energies. Another C versus O structural parameter to to accept it (by crossing the catch barrier). At the neuromuscular consider is the position of the QA with regard to the pocket (34). synapse, AChRs must reject choline and accept ACh, but both A reduction in pocket volume and a deeper positioning of the ligands would be significant activators if allowed to diffuse into a QA caused by the hold rearrangement are consistent with higher constitutively O pocket. It is possible that the formation of ACand affinity of and slower dissociation from O for all agonists. the affinity correlation are mechanisms that ensure proper signal If a reduction in pocket volume is a structural correlate of hold, recognition in complex chemical environments that contain struc- then the results suggest that compaction lowers the catch energy turally similar molecules. barrier (Fig. 6). Further, the linear correlation in energy between catch and hold energies might indicate that catch, too, may involve Methods a reduction in pocket volume. It is possible that the movement of We performed single-channel, cell-attached, patch-clamp electrophysiology the agonist from the encounter complex into the aromatic cage using mouse AChRs having only one functional binding site. Currents were (catch) compacts the binding pocket, and that this process is in analyzed using QuB software to estimate rate constants, by fitting either some way arrested when the receptor achieves AC but continues A bind-gate or gate-bind activation sequences to intracluster interval dura- when the protein isomerizes to O. Perhaps the removal of the tions. A detailed description of the methods used is given in SI Appendix. arresting structure(s) (that occurs in AC → AO) also lowers the j catch energy barrier to allow on to approach the diffusion limit. ACKNOWLEDGMENTS. We thank M. Shero, M. Teeling, J. Jordan, and What is the selective advantage of having a local conformational C. Nicolai for technical assistance. This work was funded by the NIH change (catch) in the neurotransmitter-binding process? From the (Grant NS064969).

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