© 2005 Nature Publishing Group
conformation. Binding of ligand initiates a structural change that
culminates in isomerization of Pro 8* to the cis form. This change at a
crucial pivot point reorients the M2 transmembrane helix, opening
the channel. We have crudely modelled the change that would occur
if Pro 8* isomerized from the trans to the cis conformation (Fig. 5),
treating the M2
-helix as a rigid body. Because of the hinge location
of Pro 8*, 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 loop
. Although the image
of Fig. 1 is not based on atomic-resolution data, Pro 8* in its position
at the apex of M2–M3 is in close proximity to both loops 2 and 7. It
has been proposed that loops 2 and 7 ‘lock’ the M2 helix in the closed
. Expanding upon this model, we propose that loops 2
and 7 act as a caliper to immobilize Pro 8* in a trans conformation.
Arrival of agonist causes a movement of the key binding site
tryptophan (Trp 183 in the 5-HT
receptor; Trp 149 in the nACh
. This Trp is directly linked to loop 7 by a six-amino-
acid stretch of sheet structure termed
7 (dark grey in Fig. 1). Thus,
the binding site Trp may be the actuator that, perhaps via
the clamp on M2–M3. Pro 8* 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
; in any particular context the equ ilibrium 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 as sociated with agonist
binding has occurred. There is also an issue of timescale. Estimates
of the opening rate for the 5-HT
receptor are in the 10–100 s
. Intrinsic prolyl cis–trans isomerization rates in peptides are
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
. 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
We have established a clear correlation between the conformation-
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 a nd
transporters, suggesting a key role in structural changes associated
with transmembrane signalling
. The unnatural amino acid muta-
genesis approach has allowed an exquisitely detailed probe of the
Pro 8* site, yielding an experimentally bas ed model of channel
opening involv ing 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-
tion at a non-proline site or a completely different gating mechanism
should be explored.
Mutagenesis and preparation of cRNA and oocytes. Mutant 5-HT
subunits were developed using pcDNA3.1 (Invitrogen) containing the complete
coding sequence for the 5-HT
subunit from mouse neuroblastoma N1E-115
cells as previously described
. For nonsense suppression, the proline codon at
308 was replaced by TAG as previously described
. 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 previously
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 tRNA
as detailed previously
before co-injection with mRNA, tRNA-aa was deprotec ted by photolysis.
Typically, 5 ng mRNA and 25 ng tRNA-aa were injected into stage V–VI oocytes
in a total volume of 50 nl. For control experiments, mRNA was injected in the
absence of tRNA, and with the THG73 74-mer tRNA. Experiments were
performed 18–36 h after injection.
Characterization of mutant receptors. 5-HT-induced currents were recorded
from individual oocytes using two-electrode voltage clamp with eithe r a
GeneClamp 500 ampliﬁer or an OpusXpress system (Molecular Devices Axon
Instruments). All experiments were performed at 22–25 8C. Serotonin (creati-
nine sulphate complex, Sigma) was stored as 25 mM aliquots at 280 8C, diluted
in calcium-free ND96, and delivered to cells via computer-controlled perfusion
systems. The holding potential was 260 mV unless otherwise speciﬁed. EC
data were obtained from at least six independent experiments using oocytes from
at least three different batches. Further details are available in Supplementary
NMR. The NMR sample was prepared containing 1 mM peptide, 200 mM
sodium dodecyl-d25 sulphate, 150 mM sodium chloride, 20 mM sodium phos-
phate, 1 mM EDTA, 20
M 3,3,3-trimethylsilylpropionate and 10% D
pH 6.0 to a ﬁnal volume of 550
l in a 5-mm Ultra-Imperial grade NMR tube
(Wilmad). All experiments were recorded at both 298 K and 303 K on a Bruker
DRX500 spectrometer equipped with a z-shielded gradient triple resonance
probe using standard procedures. Two dimensional nuclear Overhauser and
exchange spectroscopy (NOESY) and TOCSYexperiments, with mixing times of
200 and 72.5 ms, respectively, were collected with 256 and 1,024 pairs of complex
points and acquisition times of 26 and 102 ms in the indirectly and directly
acquired dimensions, respectively. Data processing and analysis were carried out
on a Silicon Graphics O2 workstation using the programs AZARA and CCPNmr
Received 24 March; accepted 8 August 2005.
1. Lester, H. A., Dibas, M. I., Dahan, D. S., Leite, J. F. & Dougherty, D. A. Cys-loop
receptors: new twists and turns. Trends Neurosci. 27, 329–-336 (2004).
2. Meeus, N. & Lummis, S. C. R. Proline 307 in the mouse 5–-HT
binding and function. pa2online 1, 015P (2003).
3. Lummis, S. C. R. The transmembrane domain of the 5-HT
receptor: its role in
selectivity and gating. Biochem. Soc. Trans. 32, 535–-539 (2004).
4. Bera, A. K., Chatav, M. & Akabas, M. H. GABA
receptor M2–-M3 loop
secondary structure and changes in accessibility during channel gating. J. Biol.
Chem. 277, 43002–-43010 (2002).
5. Grosman, C., Salamone, F. N., Sine, S. M. & Auerbach, A. The extracellular
linker of muscle acetylcholine receptor channels is a gating control element.
J. Gen. Phys. 116, 327–-339 (2000).
6. Lynch, J. W., Han, N. L. R., Haddrill, J., Pierce, K. D. & Schoﬁeld, P. R. The
surface accessibility of the glycine receptor M2–-M3 loop is increased in the
channel open state. J. Neurosci. 21, 2589–-2599 (2001).
7. Kash, T. L., Jenkins, A., Kelley, J. C., Trudell, J. R. & Harrison, N. L. Coupling of
agonist binding to channel gating in the GABA
receptor. Nature 421, 272–-275
8. Unwin, N. Reﬁned structure of the nicotinic acetylcholine receptor at 4 A
resolution. J. Mol. Biol. 346, 967–-989 (2005).
9. Nowak, M. W. et al. In vivo incorporation of unnatural amino acids into ion
channels in Xenopus oocyte expression system. Methods Enzymol. 293,
10. Macarthur, M. W. & Thornton, J. M. Inﬂuence of proline residues on protein
conformation. J. Mol. Biol. 218, 397–-412 (1991).
11. Dang, H., England, P. M., Farivar, S. S., Dougherty, D. A. & Lester, H. A. Probing
the role of a conserved M1 proline residue in 5-hydroxytryptamine(3) receptor
gating. Mol. Pharm. 57, 1114–-1122 (2000).
12. Jabs, A., Weiss, M. S. & Hilgenfeld, R. Non-proline cis peptide bonds in
proteins. J. Mol. Biol. 286, 291–-304 (1999).
13. Dugave, C. & Demange, L. Cis-trans isomerization of organic molecules and
biomolecules: Implications and applications. Chem. Rev. 103, 2475–-2532
14. Colquhoun, D. Binding, gating, afﬁnity and efﬁcacy: the interpretation of
structure-activity relationships for agonists and of the effects of mutating
receptors. Br. J. Pharmacol. 125, 924–-947 (1998).
15. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. Structure and gating mechanism of the
acetylcholine receptor pore. Nature 423, 949–-955 (2003).
16. Taly, A. et al. Normal mode analysis suggests a quaternary twist model for the
nicotinic receptor gating mechanism. Biophys. J. 88, 3954–-3965 (2005).
NATURE|Vol 438|10 November 2005 LETTERS