Biophysical Journal Volume 107 July 2014 8–9 8

New and Notable

Don’t Flip Out: AChRs arePrimed to Catch and Hold YourAttention

Andrew J.R. Plested*Leibniz-Institut fur MolekularePharmakologie, Berlin, Germany andCluster of Excellence NeuroCure, Berlin,Germany

The muscle nicotinic receptor hascome a long way since Langley dousedit with nicotine and curare, dubbingit ‘‘the receptive substance’’, morethan a century ago (1). Neher and Sak-mann did the first biological single-molecule experiments on those samechannels (2), and similar work con-tinues to expose mechanisms thatunderlie Langley’s observations.

In this issue of Biophysical Journal,Purohit et al. (3) show that most pertur-bations around the agonist binding siteof the muscle nicotinic receptor have asimilar energetic effect on the low-affinity conformation of the bindingsite (referred to as ‘‘Catch’’), as theydo on the high-affinity conformation(the ‘‘Hold’’), which Langley waslooking at. The manipulations thatpermitted the relevant measurementshave been refined to an impressivedegree by Auerbach’s lab over theyears (e.g. ref. 4). One key recent inno-vation is a reliable measurement ofgating in the absence of ligand (5).Low-affinity binding and the gatingof bound channels are relatively easierto measure, so building a reversiblethermodynamic cycle combining bind-ing and gating reactions, in principle,reveals the high-affinity dissociationconstant (Fig. 1). A caveat to thisapproach is that, despite the hopesand dreams of many, cyclic modelscannot be fitted to most data—although a recent report suggests with

http://dx.doi.org/10.1016/j.bpj.2014.05.021

Submitted May 2, 2014, and accepted for

publication May 15, 2014.

*Correspondence: plested@fmp-berlin.de

Editor: Chris Lingle.

� 2014 by the Biophysical Society

0006-3495/14/07/0008/2 $2.00

simple data fits can work (6). The au-thors know this problem, and, to avoidit, fit only a linear scheme. The esti-mates from this fit allow calculationof the high-affinity dissociation con-stant for a series of mutants.

This simple result is profoundbecause it provides further proof thatconformational changes are embeddedwithin the process of binding. Over thelast 15 years, several groups havedared to dream that three separateobservations of intermediate statesbetween resting and open (eitherdirectly detected or inferred) mightcorrespond to something similar. First,the shut time distributions for therelated glycine receptor are toocomplicated in saturating agonist fora simple open and shut isomerization(7), which led to the flip model. Theseintermediates are accentuated withpartial agonists or by loss of functionmutations (8–10). Likewise, free-energy relations suggest that gating isa cascade through multiple brief con-formational intermediates (11), withtransitions necessarily beginning nearthe binding site. The gating behaviorof mutant nAChRs that are spontane-ously active is also too complicatedto be explained by a simple open-shutscheme. An elegant explanation forthis came when Mukhtasimova et al.(12) showed that ‘‘priming’’ of individ-ual binding sites could drive gating in adigital fashion. Although the exactphysical natures of the intermediatesremain unclear, these three explana-tions of early gating effects indicatedthat early conformational changesoccur at the binding site, separate, toa greater or lesser extent, from theopening of the channel gate.

Are these schemes mutually exclu-sive, or might they coalesce with thelatest concepts into a coherent visionof AChR gating? An important notionis to separate changes within one sub-unit from changes at distant bindingsites. The flip model, although prob-ably an oversimplification, has phys-ical appeal because it solves theproblem of globally increasing affinity,

including at sites that have not seenagonist, with a concerted conforma-tional change among all the subunits.The prime mechanism, on the otherhand, likely needs communication viathe channel domain to alter affinity atthe second ligand binding site on thisreceptor. In the article in hand, interac-tions between the distinct binding sitesare not addressed. As the authorsrightly point out, the idea of a water-tight cap on the binding site (onepossible interpretation of the primemechanism) is not consistent with thesimilar association rate predicted forthe high-affinity conformation. How-ever, there is no information into thespeed of binding reactions to the openchannel and any distinctions drawnbetween the proposals may prove tobe false dichotomies. Rather thancompeting for our attention, these phe-nomena might be separate aspects ofthe same conformational changes. Itis not hard to imagine the ‘‘hold’’ reac-tion comprising a flip into a globallyhigh-affinity conformation, primingall sites via the membrane domains(Fig. 1).

Discriminating these concepts will,in any case, be challenging, becausemodels incorporating multiple confor-mational changes cannot be fit whenthe number of free rate constants ex-ceeds what can be constrained evenby rich data sets (7–10,12). Globalfitting across multiple mutants mayallow the net to be cast wider,providing the biology behaves wellenough to satisfy the assumptions ofsuch fits. The rational malleability ofmuscle AChR gating suggests thatthe requisite mutant sets can be assem-bled (4).

What silent gears may grind beneathis largely hidden during these chan-nels’ everyday roles in synaptic trans-mission. Nature has whittled away theintermediates so that receptors appearto behave as simple on-off switches.However, intermediates hold the key

Catch

Agonist (e.g. ACh)

Hold

Prime

Flip

Low affinity binding

Closed

Open

Unl

igan

ded

gatin

g

Dili

gand

ed g

atin

g

High affinity binding

FIGURE 1 The grammar of nicotinic receptor gating. A receptor with two binding sites, like the

muscle nicotinic receptor, with possible congeners of the flip, prime, catch, and hold reactions around

the extracellular binding sites. All reactions are potentially independent from the opening of the chan-

nel (bottom row, green membrane domain). All binding sites are initially in the resting configuration,

ready to ‘‘catch’’ agonist. Within the context of this model incorporating the catch-and-hold idea,

‘‘Priming’’ may follow or initiate binding site transformation into the ‘‘hold’’ configuration. Likewise,

the phenomenon described as ‘‘Flip’’ might represent a concerted jump from the first to the third row.

Opacity indicates rough probability of occurrence, illustrating why linear schemes still approximate the

data well. Outer arrows indicate cycle of equilibrium constants used to calculate high-affinity binding.

To see this figure in color, go online.

AChR Binding 9

to partial agonists (8), and therefore,possibly, drug action. The extent towhich these results unify the field,and catalyze further studies, mightbe of equal importance. Hopefullyeveryone can now catch the idea thathigh-affinity binding sites can developindependently from channel gating,

from rearrangements of the agonistbinding sites. Hold tight!

REFERENCES

1. Langley, J. N. 1905. On the reaction of cellsand of nerve-endings to certain poisons,chiefly as regards the reaction of striated

muscle to nicotine and to curari. J. Physiol.33:374–413.

2. Neher, E., and B. Sakmann. 1976. Single-channel currents recorded from membraneof denervated frog muscle fibers. Nature.260:799–802.

3. Purohit, P., I. Bruhova, ., A. Auerbach.2014. Catch and hold activation of muscleacetylcholine receptors having transmitterbinding site mutations. Biophys. J. 107:88–99.

4. Jadey, S. V., P. Purohit, ., A. Auerbach.2011. Design and control of acetylcholinereceptor conformational change. Proc.Natl. Acad. Sci. USA. 108:4328–4333.

5. Purohit, P., and A. Auerbach. 2009. Unli-ganded gating of acetylcholine receptorchannels. Proc. Natl. Acad. Sci. USA. 106:115–120.

6. Purohit, P., and A. Auerbach. 2013. Loop Cand the mechanism of acetylcholine recep-tor-channel gating. J. Gen. Physiol. 141:467–478.

7. Burzomato, V., M. Beato,., L. G. Sivilotti.2004. Single-channel behavior of hetero-meric a1b glycine receptors: an attemptto detect a conformational change beforethe channel opens. J. Neurosci. 24:10924–10940.

8. Lape, R., D. Colquhoun, and L. G. Sivilotti.2008. On the nature of partial agonism inthe nicotinic receptor superfamily. Nature.454:722–727.

9. Lape, R., A. J. R. Plested,., L. G. Sivilotti.2012. The a1K276E startle disease mutationreveals multiple intermediate states in thegating of glycine receptors. J. Neurosci.32:1336–1352.

10. Plested, A. J., P. J. Groot-Kormelink, ., L.G. Sivilotti. 2007. Single-channel study ofthe spasmodic mutation a1A52S in recombi-nant rat glycine receptors. J. Physiol. 581:51–73.

11. Grosman, C., M. Zhou, and A. Auerbach.2000. Mapping the conformational waveof acetylcholine receptor channel gating.Nature. 403:773–776.

12. Mukhtasimova, N., W. Y. Lee, ., S. M.Sine. 2009. Detection and trapping of inter-mediate states priming nicotinic receptorchannel opening. Nature. 459:451–454.

Biophysical Journal 107(1) 8–9