© 2005 Nature Publishing Group
Cis–trans isomerization at a proline opens the pore
of a neurotransmitter-gated ion channel
Sarah C. R. Lummis1*, Darren L. Beene2*, Lori W. Lee2, Henry A. Lester3, R. William Broadhurst1
& Dennis A. Dougherty2
5-Hydroxytryptamine type 3 (5-HT3) receptors are members of
the Cys-loop receptor superfamily1. Neurotransmitter binding in
these proteins triggers the opening (gating) of an ion channel by
means of an as-yet-uncharacterized conformational change. Here
we show that a specific proline (Pro 8*), located at the apex of
the loop between the second and third transmembrane helices
(M2–M3)2,3, can link binding to gating through a cis–trans iso-
merization of the protein backbone. Using unnatural amino acid
mutagenesis,aseriesofprolineanalogues withvarying preference
for the cis conformer was incorporated at the 8* position. Proline
functional channels. Among the functional mutants there was a
strong correlation between the intrinsic cis–trans energy gap of
the proline analogue and the activation of the channel, suggesting
that cis–trans isomerization of this single proline provides the
on an M2–M3 loop peptide reveal two distinct, structured forms.
Our results thus confirm the structure of the M2–M3 loop and the
critical role of Pro 8* in the 5-HT3receptor. In addition, they
suggest that a molecular rearrangement at Pro 8* is the structural
mechanism that opens the receptor pore.
The neurotransmitter binding site in the Cys-loop superfamily of
ion channels is located about 60A˚from the channel pore, presenting
a conundrum as to the molecular events that link binding and
gating1,3. Evidence implicates the M2–M3 loop, and in low-
resolution structural studies this region interacts with loops 2 and
7 of the extracellular domain (Fig. 1)4–8. In the cation-selective
nicotinic acetylcholine (nACh) and 5-HT3receptors, the apex of
the M2–M3 loop contains a conserved proline (Pro8*) that is ideally
placed to provide a hinge for movement of the channel-lining M2
helix. This proline is essential for receptor function in the 5-HT3
Lys or Asn gave receptors that were trafficked to the membrane
properly and displayed wild-type binding properties for a radio-
labelled antagonist (Supplementary Information); however, all
receptors were non-functional2. This indicates that mutations at
Pro 8* affect receptor function by altering gating, not ligand binding
or folding/assembly, and that a unique characteristic of proline is
absolutely required for the gating of the receptor.
To explore the possible role of Pro8* in channel gating, we used
nonsense suppression in Xenopus oocytes to incorporate a series of
technique involves inserting a nonsense codon (TAG) in place of
the proline codon into the DNA encoding the receptor subunit,
synthesizing messengerRNA,andinjecting thisintoXenopus oocytes
along with a transfer RNA that both recognizes the TAG codon and
has the appropriate proline analogue attached at the 3
resulting mutant receptors were examined using voltage-
clamp analysis of 5-HT-induced whole-cell responses and
When incorporated into a protein, proline disrupts main-chain
hydrogen bonding, because it lacks a backbone NH moiety10.
Figure 1 | Overall layout of the 5-HT3receptor showing the extracellular
(predominantly b-sheet) and transmembrane (a-helical) regions. Shown
are two adjacent subunits from a homology model of the 5-HT3receptor30
built from AChBP (extracellular domain)19and the nACh receptor
purple; loop 7 is red; loop 2 is green; the conserved Leu thought to
region corresponding to the peptide studied by NMR (Fig. 4), comprising
the M2–M3 loop and parts of M2 and M3, is shown in blue, with Pro 8* in
1Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.2Division of Chemistry and Chemical Engineering, and3Division of Biology,
California Institute of Technology, Pasadena, California 91125, USA.
*These authors contributed equally to this work.
Vol 438|10 November 2005|doi:10.1038/nature04130
© 2005 Nature Publishing Group
Previously, we probed a highly conserved Pro in the middle of the
first transmembrane helix (M1) by substitution with an a-hydroxy
acid such as Vah (a-hydroxyvaline; Fig. 2a), which similarly lacks an
NH11. In M1 Vah substitutes effectively for Pro; however, at position
8* Vah produces non-functional receptors. Similarly, the N-methyl
amino acids Sar (sarcosine) and N-Me-Leu give non-functional
cyclic proline analogues at position 8* yielded mixed results: some
produced functional receptors and others did not (Fig. 2a). Thus,
key to the proper functioning of Pro at 8*.
Proline is also unusual in forming cis peptide bonds at a frequency
(,5%) much higher than any other naturally occurring amino acid
(,0.1%)12, reflecting an intrinsic diminution of the energy gap
between cis and trans forms in proline10. The analogues 2,4-CH2-Pro
and 2-Me-Pro show a reduced preference for a cis conformation
relative to Pro13, and these produced non-functional receptors (that
is, no current was seen in response to normally saturating concen-
trations of serotonin). Immunocytochemical experiments revealed
that these receptors did reach the cell surface (Supplementary
Of the proline analogues that give functional receptors, t-4F-Pro
and c-4F-Pro give near-wild-type EC50(effector concentration for
half-maximum response) values (1.38 ^ 0.06 and 1.15 ^ 0.12mM,
respectively), and also show intrinsic cis–trans preferences similar
to native proline. Other analogues, however, show much greater
propensities to adopt the cis conformer. Most extreme is Dmp
(5,5-dimethylproline), which strongly favours the cis form
(Table 1). Incorporation of this amino acid at position 8* resulted
in a 60-fold decrease in EC50. Furthermore, in oocytes that expressed
Dmp-containing receptors, agonist exposure led to partially irrever-
sible inactivation, as evidenced by a continual increase in standing
current after initial receptor activation; this current could be sub-
stantially reversed by the addition of the channel blocker TMB-8
(ref. 11) (Fig. 3). Dmp and other analogues with high cis preference
showed low and variable expression levels in Xenopus oocytes. This
could reflect difficulties in folding the protein when a key proline is
present in an unconventional conformation. Overall, we found three
types of behaviours for incorporation of proline analogues. Pro
but did express and were inserted into the membrane. Intermediate
derivatives produced responses with a range of EC50values. The
most cis-producing residue produced highly sensitive receptors that
displayed partially irreversible activation.
An evaluation of a series of Pro analogues that exhibit a range of
intrinsic cis–transenergy gapsrevealed aremarkabletrend.As shown
in Fig. 2b, a linear relationship was observed between the cis–trans
energy gap of the proline analogue (DDG(c–t)) and activation of the
EC50, the functional output we measure, reflects a composite of
binding and gating events. However, as noted above, substitution at
Pro 8* by natural amino acids does not change antagonist binding
affinity. In addition, the apparent affinity of the 5-HT3receptor
competitive antagonist MDL72222 is not significantly altered when
Information). These data show that for amino acids with a wide
range of cis preferences (,0.1–71%) at Pro8*, the binding site is
unperturbed. We therefore consider the variations in EC50to reflect
changes in receptor gating, not binding14, justifying the free energy
treatment of Fig. 2b.
The slopeoftheline inFig. 2bis 1,withinexperimental error.This
indicates that the full energetic perturbation of the proline cis–trans
Together, these observations provide a compelling link between
the conformational isomerization at residue 8* and the gating of
To provide further support that a proline in the M2–M3 loop can
serve as a structural switch, we used nuclear magnetic resonance
Figure 2 | Unnatural amino acid mutagenesis. a, Residues used in this
study. As noted in the text, the conventional amino acids Gly, Ala, Cys, Val,
Lys and Asn also give non-functional channels. b, Linear energy correlation
between cis–trans isomerization and receptor activation. Data are from
Aze they are partially obscured by the marker.
Table 1 | Influence of proline isomerism on receptor function
Residue Per cent cis* EC50
5 1.29 ^ 0.07
0.75 ^ 0.06
0.42 ^ 0.03
0.030 ^ 0.024
0.021 ^ 0.009
*Determined from studies of model peptide systems reported previously21–23. Because
variations are seen depending on methodology and exact model system, all values are
referenced to the cis–trans ratio seen for Pro in the same study, and Pro is set to 5%, the
value obtained from statistical surveys of protein structures.
†Values are ^s.e.m.
‡Values are relative to proline.
NATURE|Vol 438|10 November 2005
© 2005 Nature Publishing Group
(NMR) spectroscopy to evaluate the structure of a 20-amino-acid
peptide spanning the M2–M3 loop (SDTLPATAIGTPLIGVYFVV;
Pro 8* is italic). When solubilized in a micellar solution of SDS,
this relatively short sequence is structured, with two segments
(residues 6–10 and 13–20 of the peptide; see Supplementary Infor-
mation). Thus, key features of the M2–M3 loop, as reported in the
low-resolution nACh receptor structure8,15(see also Fig. 1), are
recapitulated in the model peptide. Interestingly, two conformers
are clearly evident in the peptide structure, with the major form
about five times more prevalent than the minor form (Fig. 4). In the
major conformer Pro8* was assigned the trans conformation based
onobservednuclear Overhausereffect connections. Some featuresof
the minor form cannot be resolved due to overlapping resonances,
but the TOCSY (total correlated spectroscopy) spectrum clearly
shows that the two glycines that flank Pro8* (positions 10 and 15
in the peptide; 6* and 11* in the protein) are in different environ-
ments in the two conformers, with both pairs of diastereotopic Ha
nuclei giving separate resonances in the minor conformer (Fig. 4).
These results are consistent with two conformations at Pro8*
producing two conformers of the peptide.
We propose the following mechanism for gating in the 5-HT3
receptor. In the closed state of the channel, Pro8* is in the trans
Figure 4 |1H NMR of a model peptide. The HN-Hafingerprint region of a
TOCSY spectrum for the 20-residue peptide discussed in the text resolves
signals for the major (blue) and minor (red) conformers in a micellar
solution of SDS at 298K. Integration indicates that 13–20% of the minor
conformer is present. Signals arising from distinct Haor Hbenvironments
within a particular residue are connected with vertical lines.
Figure 3 | Current traces in voltage-clamp experiments (at approximately
EC50and approximately Imax) for receptors expressing unnatural amino
acids. For Dmp at position 8* (residue 308 of 5-HT3), a full dose–response
range is given along with the TMB-8 block of standing currents seen after
receptor activation. Bars represent 5-HTapplication.
Figure 5 | A proposed gating mechanism. A single 5-HT3receptor subunit
is depicted, illustrating how trans–cis isomerization at Pro8* (blue space-
filling model) could function as a hinge for movement of M2 (green) during
gating, moving the occluding Leu9
region. The open structure on the right was generated by manually
converting Pro308 to the cis conformation and treating M2 as a rigid body.
Top: side view; bottom: top view, showing only M2, M3 and the M2–M3
0residue (yellow) away from the channel
NATURE|Vol 438|10 November 2005
© 2005 Nature Publishing Group
conformation. Binding of ligand initiates a structural change that
crucial pivot point reorients the M2 transmembrane helix, opening
the channel. We have crudely modelled the change that would occur
if Pro8* isomerized from the trans to the cis conformation (Fig. 5),
treating the M2 a-helix as a rigid body. Because of the hinge location
of Pro8*, the structurally modest cis–trans isomerization at this
single residue propagates to a substantial structural effect more than
adequate to gate the channel.
Our model also suggests a natural coupling between the agonist
binding site and the conformational change associated with gating.
Several workers have noted the proximity of loops 2 and 7 (the
eponymous Cys loop) to the M2–M3 loop4–8,16. Although the image
ofFig. 1is notbasedonatomic-resolutiondata,Pro8*inits position
at the apex of M2–M3 is in close proximity to both loops 2 and 7. It
conformation8. Expanding upon this model, we propose that loops 2
and 7 act as a caliper to immobilize Pro8* in a trans conformation.
Arrival of agonist causes a movement of the key binding site
tryptophan (Trp183 in the 5-HT3receptor; Trp149 in the nACh
receptor)17–20. This Trp is directly linked to loop 7 by a six-amino-
acid stretch of sheet structure termed b7 (dark grey in Fig. 1). Thus,
the binding site Trp maybe theactuator that, perhaps viab7,releases
the clamp on M2–M3. Pro8* can then undergo a spontaneous (or
protein aided) cis–trans isomerization, gating the channel.
The per cent cis values of Table 1 are based on model peptide
systems21–23; in any particular context the equilibrium could be
perturbed. It may well be that structural features of the wild-type
receptor act to favour the cis conformer of the native proline at
position 8*, once the structural change associated with agonist
binding has occurred. There is also an issue of timescale. Estimates
of the opening rate for the 5-HT3receptor are in the 10–100s21
within a factor of ten of this regime, and structural features of the
receptor could act to accelerate significantly the isomerization rate.
Protein prolyl isomerases are well known, and even a simple hydro-
gen bond to the proline amide nitrogen can accelerate isomerization
,260-fold26. It is possible that the receptor has incorporated struc-
tural features that facilitate cis–trans isomerization. Therefore, prolyl
cis–trans isomerization is a kinetically viable candidate for the gating
Wehaveestablished aclearcorrelation betweentheconformation-
al preference of a single, specific proline residue and the gating
efficiency of a Cys-loop receptor. Proline has unique structural and
conformational properties, and it is found with anomalously high
frequency in the transmembrane regions of ion channels and
transporters, suggesting a key role in structural changes associated
with transmembrane signalling27,28. Theunnatural amino acid muta-
genesis approach has allowed an exquisitely detailed probe of the
Pro8* site, yielding an experimentally based model of channel
opening involving a precise structural change at the amino acid
level that induces gating. The model is consistent with all available
structural data, and it suggests much future work. It will be inter-
esting to perform the same ‘proline scan’ on the nACh receptor,
which also contains a proline at position 8*, but which displays a
much faster opening rate. In contrast, other Cys-loop receptors do
not contain this proline, and the possibility of a cis–trans isomeriza-
tionatanon-proline siteoracompletelydifferentgating mechanism
should be explored.
Mutagenesis and preparation of cRNA and oocytes. Mutant 5-HT3Areceptor
subunits were developed using pcDNA3.1 (Invitrogen) containing the complete
cells as previously described29. For nonsense suppression, the proline codon at
308 was replaced by TAG as previously described9. Wild type and mutant
receptor subunit coding sequences were then subcloned into pGEMHE. This
was linearized with Nhe1 (New England Biolabs) and cRNA synthesized using
the T7 mMESSAGE mMACHINE kit (Ambion). Oocytes from Xenopus laevis
were prepared and maintained as described previously9.
Synthesis of tRNA and dCA-amino acids. Unnatural amino acids were
chemically synthesized as nitroveratryloxycarbonyl (NVOC) protected cyano-
methyl esters and coupled to the dinucleotide dCA, which was then enzymati-
cally ligated to 74-mer THG73 tRNACUAas detailed previously9. Immediately
before co-injection with mRNA, tRNA-aa was deprotected by photolysis.
Typically, 5ng mRNA and 25ng tRNA-aa were injected into stage V–VI oocytes
in a total volume of 50nl. For control experiments, mRNA was injected in the
absence of tRNA, and with the THG73 74-mer tRNA. Experiments were
performed 18–36h after injection.
Characterization of mutant receptors. 5-HT-induced currents were recorded
from individual oocytes using two-electrode voltage clamp with either a
GeneClamp 500 amplifier or an OpusXpress system (Molecular Devices Axon
Instruments). All experiments were performed at 22–258C. Serotonin (creati-
nine sulphate complex, Sigma) was stored as 25mM aliquots at 2808C, diluted
in calcium-free ND96, and delivered to cells via computer-controlled perfusion
systems. The holding potential was 260mV unless otherwise specified. EC50
at least three different batches. Further details are available in Supplementary
NMR. The NMR sample was prepared containing 1mM peptide, 200mM
sodium dodecyl-d25 sulphate, 150mM sodium chloride, 20mM sodium phos-
phate, 1mM EDTA, 20mM 3,3,3-trimethylsilylpropionate and 10% D2O at
pH6.0 to a final volume of 550ml in a 5-mm Ultra-Imperial grade NMR tube
(Wilmad). All experiments were recorded at both 298K and 303K on a Bruker
DRX500 spectrometer equipped with a z-shielded gradient triple resonance
probe using standard procedures. Two dimensional nuclear Overhauser and
points and acquisition times of 26 and 102ms in the indirectly and directly
acquired dimensions, respectively. Dataprocessingandanalysiswere carriedout
Received 24 March; accepted 8 August 2005.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements S.C.R.L. is a Wellcome Trust Senior Research Fellow in
Basic Biomedical Science. The work at Caltech was supported by the National
Institutes of Health. We thank K. L. Price and A. J. Thompson for assistance with
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare no competing
financial interests. Correspondence and requests for materials should be
addressed to D.A.D. (email@example.com).
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