Radioligand binding studies have demonstrated that, under equilib-rium conditions, the nAChR carries two high affinity Keywords acetylcholine; loop D; mutagenesis; nicotinic receptor; ooc
Trang 1The contribution of residues aArg55 and cGlu93
Ankur Kapur, Martin Davies, William F Dryden and Susan M.J Dunn
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada
The muscle-type nicotinic acetylcholine receptor
(nAChR) is the prototype of the Cys-loop ligand-gated
ion channel (LGIC) super-family that includes the
neuronal nicotinic, c-aminobutyric acid (GABA) type
A, 5-hydrotryptamine type 3 (5-HT3) and glycine
receptors This is largely a consequence of the
abun-dance of this receptor in Torpedo electric organ, which
facilitated its early purification and characterization
The Torpedo nAChR is a pentameric transmembrane
protein complex in which four structurally related subunits (a, b, c, d) in a stoichiometry of 2 : 1 : 1 : 1 assemble to form a central cation-selective ion channel [1,2] The a and b subunits of the Torpedo receptor referred to in this report correspond to the a1 and b1 subunits in the nomenclature recommended by the International Union of Pharmacology [3] Radioligand binding studies have demonstrated that, under equilib-rium conditions, the nAChR carries two high affinity
Keywords
acetylcholine; loop D; mutagenesis; nicotinic
receptor; oocytes
Correspondence
S.M.J Dunn, Department of Pharmacology,
University of Alberta, Edmonton, Alberta,
T6G 2H7 Canada
Fax: +780 4924325
Tel: +780 4923414
E-mail: susan.dunn@ualberta.ca
(Received 22 October 2005, revised 13
December 2005, accepted 23 December
2005)
doi:10.1111/j.1742-4658.2006.05121.x
The Torpedo nicotinic acetylcholine receptor is a heteropentamer (a2bcd) in which structurally homologous subunits assemble to form a central ion pore Viewed from the synaptic cleft, the likely arrangement of these sub-units is a–c–a–d–b lying in an anticlockwise orientation High affinity bind-ing sites for agonists and competitive antagonists have been localized to the a–c and a–d subunit interfaces We investigated the involvement of amino acids lying at an adjacent interface (c–a) in receptor properties Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used
to investigate the consequences of mutating aArg55 and cGlu93, residues that are conserved in most species of the peripheral nicotinic receptors Based on homology modeling, these residues are predicted to lie in close proximity to one another and it has been suggested that they may form a salt bridge in the receptor’s three-dimensional structure (Sine et al 2002 J Biol Chem 277, 29 210–29 223) Although substitution of aR55 by phenyl-alanine or tryptophan resulted in approximately a six-fold increase in the
EC50 value for acetylcholine activation, the charge reversal mutation (aR55E) had no significant effect In contrast, the replacement of cE93 by
an arginine conferred an eight-fold increase in the potency for acetyl-choline-induced receptor activation In the receptor carrying the double mutations, aR55E-cE93R or aR55F-cE93R, the potency for acetylcholine activation was partially restored to that of the wild-type The results sug-gest that, although individually these residues influence receptor activation, direct interactions between them are unlikely to play a major role in the stabilization of different conformational states of the receptor
Abbreviations
5-HT 3A receptor, serotonin type 3 A receptor; a-BgTx, alpha-bungarotoxin; ACh, acetylcholine; AChBP, acetylcholine binding protein; dTC, d-tubocurarine; GABA, c-aminobutyric acid; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; PTMA,
phenyltrimethylammonium; WT, wild-type.
Trang 2binding sites for agonists and competitive antagonists
[4,5] It is now generally agreed that these sites lie at
the interfaces between the a–c and the a–d subunits
[6] Labeling and mutational studies have identified
several key amino acids lying in discrete
noncontigu-ous ‘loops’ of the a-subunits (designated as loops
A–C, the ‘primary component’), together with amino
acids in the neighboring c and d subunits (lying in
loops D–F, the ‘secondary component’) that
partici-pate in forming these binding pockets [7–9]
Although none of the ligand-gated ion channel
fam-ily to which the nAChR belongs has been crystallized,
the published structure of a related protein [10], the
acetylcholine binding protein (AChBP), lends credence
to current ideas of high affinity binding site location
The AChBP, which is secreted by the glial cells of the
snail, Lymnaea stagnalis, is a truncated homologue
of the extracellular amino terminal domains of the
nAChR[see 10] Inspection of its structure has
rein-forced earlier predictions that the residues involved in
forming the binding sites occur at subunit–subunit
interfaces and that the stretches of amino acids that
have been implicated in binding are arranged in
loop-like structures
The structural homology of all subunits in the LGIC
family suggests that each of the five subunit–subunit
interfaces contributes to ligand binding and⁄ or the
conformational changes that are involved in the
trans-duction mechanism(s) that link agonist binding to
channel opening This is particularly true of the
homo-pentameric receptors, e.g the a7 neuronal nAChR and
the 5-HT3A homomeric receptor In these receptors,
there are five identical interfaces that presumably play
equivalent roles in ligand recognition and receptor
function In the heteromeric receptors, the roles of all
five subunit interfaces are less clear Due to structural
homology, all subunits carry all putative binding loops
(A–F), suggesting that each interface has the potential
to form a binding site, albeit with a distinct affinity
arising from the nonequivalence of the intersubunit
contacts within the pentamer Alternatively, these
homologous loops at each interface may contribute to
receptor assembly and⁄ or play a role in the
conforma-tional changes that result in channel activation or
receptor desensitization
In the case of the Torpedo nAChR, the importance
of the a-subunit in ligand binding has long been
recog-nized [11-15], but the involvement of non a-subunit
residues has become clear only more recently The first
direct evidence for the contribution of the c and d
sub-units to ligand recognition came from photoaffinity
labeling studies using [3H]nicotine and [3
H]d-tubocura-rine (dTC), which identified residues cW55 and the
homologous dW57 (lying in what is now referred to as the loop D domain) as specific sites of ligand incorpor-ation [5,15-17] In the present study, we have investi-gated the effects of mutations of the equivalent residue (aR55) lying in loop D of the a-subunit, i.e at the opposite side of the subunit from residues (in loops A– C) that have previously been implicated in agonist binding (Fig 1) Within the LGIC family, this residue
in the peripheral nAChR is unique; whereas almost all subunits in the family have an aromatic residue at this position, a positively charged arginine residue is con-served in all peripheral a-subunits (see Fig 1) Previ-ous comparative modeling studies have revealed that E93 of the c-subunit (lying in putative binding loop A) may lie in close proximity to aR55, leading to the pro-posal that an ionic interaction between these two resi-dues may stabilize receptor conformation [18,19] This
53
A
B
A
h
A B C
F
A B C
D
D
D
E F
-+ +
-+
-α
α
β
δ
γ
Fig 1 Loop D of the LGIC family (A) Amino acid sequence align-ments of residues lying in loop D of the a1, c and d subunits from Torpedo californica (T Ca) nAChR, human (H) a1 nAChR subunit, b2 of rat GABAAreceptor, rat 5-HT3Asubunit and AChBP Number-ing shown is for the Torpedo nAChR a1 subunit The positively charged R55 residue is unique to the peripheral nAChR a1 subunit since other members of the LGIC family have an aromatic amino acid in this position (B) Schematic representation of the subunit arrangement of the Torpedo nAChR showing the ‘six binding loop’ model of high affinity ligand binding sites Also represented is loop
D of the a-subunit (not previously implicated in ligand binding), which lies at the b-a and c–a subunit interfaces.
Trang 3residue is also conserved in peripheral nAChR c (and
e) subunits We therefore also investigated the effects
of a charge reversal mutation of this residue (cE93R)
both alone and in combination with the aR55
muta-tions Our results demonstrate that mutations of these
residues, which lie at an interface (c–a) that has not
previously been implicated in receptor function, can
have significant effects on ligand binding and⁄ or
chan-nel gating However, we conclude that a direct
inter-action between aR55 and cE93 is unlikely to make a
major contribution to nAChR properties
Results
Functional effects of aR55 mutations
The functional responses of wild-type (WT) or mutant
receptors expressed in Xenopus oocytes were studied
using two-electrode voltage clamp techniques Figure 2
shows the concentration-effect curves for
ACh-medi-ated responses The WT nAChR receptor has an EC50
value for ACh-induced activation of 24 lm with an
estimated Hill coefficient of 1.6 The substitution of
aArg55 with glutamic acid (aR55E) or lysine (aR55K)
resulted in a statistically insignificant shift in the EC50
values for ACh activation to 29 and 47 lm,
respect-ively, and had no significant effect on the cooperativity
of receptor activation In contrast, the aR55F and
aR55W mutations caused a five- to six-fold shift in the
EC50 for ACh activation to 112 and 151 lm,
respect-ively In addition, the Hill coefficients for Ach-induced
activation for these mutant receptors were significantly reduced in comparison with the WT nAChR (Table 1) The effects of phenyltrimethylammonium (PTMA)
on activation of WT and mutant receptors were also investigated PTMA is a poor partial agonist of the
WT nAChR and it elicits a maximum current of only 1.5 ± 0.1% of the ACh response (data not shown) The WT receptor was activated by PTMA with an
EC50 of 57 lm and a Hill coefficient of 2.1 ± 0.2 (Fig 3A, Table 2) In contrast, PTMA failed to acti-vate the aR55F and aR55W mutant receptors, even at concentrations up to 10 mm Instead, PTMA acted as
a competitive antagonist of these mutant receptors (see Fig 3A) Co-application of PTMA and ACh to the aR55F (Fig 3A) and aR55W (data not shown) recep-tors resulted in a concentration-dependent inhibition
of ACh-evoked currents with apparent KI values of
103 and 88 lm, respectively Thus PTMA-induced channel activation (in WT nAChR) and inhibition (in mutant receptors) occurs over a similar concentration range suggesting that, although the mutations affected the apparent efficacy of this ligand, they had little effect on its affinity
Effects of d-tubocurarine on aR55 mutant nAChRs
We further examined the ability of the competitive ant-agonist, dTC to inhibit ACh-evoked currents in WT and mutant receptors (Fig 3B) For WT nAChR,
Fig 2 ACh activation of wild-type (WT) and aR55 mutant
recep-tors Concentration-effect curves obtained from oocytes expressing
WT (n), aR55E (h), aR55K (n), aR55W (e) and aR55F (s) nAChR.
Data are normalized to Imaxfor each individual point The data
rep-resent the mean ± SEM from at least three oocytes The data
obtained from curve-fitting are summarized in Table 1.
Table 1 Concentration-effect data for ACh activation of wild-type (WT) and mutant receptors expressed in Xenopus oocytes Data represent the mean ± SEM Values for log EC 50 and Hill coefficient (nH) were determined from concentration-effect curves using GRAPH-PAD PRISM software Log EC50and Hill coefficients from individual curves were averaged to generate final mean estimates The val-ues in parentheses are the number of oocytes used for each recep-tor type Statistical analysis was performed by comparing the log
EC 50 and n H of the mutant receptors to the WT nAChR ( a p<0.001, b
p<0.05) using one-way analysis of variance ( ANOVA ) followed by Dunnett’s post-test to determine the level of significance c p<0.001 compared with the aR55F receptor.
Receptor
Log EC 50 ± SEM
( M )
EC 50 (l M ) nH± SEM
EC50 mutant ⁄
EC50WT
aR55E ) 4.54 ± 0.12 (3) 28.6 1.2 ± 0.1 1.2 aR55K ) 4.33 ± 0.08 (3) 47.2 1.2 ± 0.1 1.9 aR55F ) 3.95 ± 0.06 (4) a 112 0.8 ± 0.02 a 4.6 aR55W ) 3.82 ± 0.18 (4) a
151 0.8 ± 0.1a 6.2 cE93R ) 5.52 ± 0.10 (3) a 3.03 1.4 ± 0.02 0.12 cE93R-aR55E ) 4.94 ± 0.07 (7) b,c 11.5 1.1 ± 0.07 0.47 cE93R-aR55F ) 4.89 ± 0.20 (3) c
12.9 1.3 ± 0.1 0.53
Trang 4preperfusion with dTC produced a concentration dependent inhibition of ACh-evoked currents charac-terized by an apparent KI of42 nm dTC also inhib-ited Ach-evoked currents in the receptors carrying the aR55F and aR55W mutations with apparent KIvalues
of 52 and 34 nm, respectively (see Table 2) These results suggest that mutations at position 55 of the a-subunit do not affect either the binding affinity for dTC or its ability to competitively inhibit ACh-evoked currents In these experiments, although dTC alone did not elicit detectable whole cell currents, we observed that low concentrations of dTC (1–3 nm) potentiated ACh- evoked currents (by up to 25%) in both WT and mutant receptors (Fig 3B)
Expression levels and maximum amplitude
of WT and mutant nAChR Fig 4 compares the density of binding sites for 125 I-labelled a-BgTx for the WT and mutant receptors with the maximum ACh-evoked current Injection of 50 ng
of WT subunit cRNAs resulted in a robust expression
of 125I-labelled a-BgTx binding sites (approximately 3.1 fmolÆoocyte)1) All of the aR55 mutations were well tolerated and, after their coexpression with WT b-, c- and d- subunits, their expression levels (in terms
of125I-labelled a-BgTx binding sites) were in the same range as the WT nAChR Although the expression levels of the receptors carrying the R55K and R55E mutations were statistically higher than that of the
WT, the normalized currents (nAÆfmol)1) were com-parable In contrast, after normalization to the density
of toxin binding sites, the peak currents mediated by
A
B
Fig 3 The effects of PTMA and dTC on WT and aR55 mutant
receptors (A) Data show concentration-effect curves for the
activa-tion of WT nAChR(n) and inhibiactiva-tion of the ACh-induced response of
aR55F (s) mutant receptors (Table 2, see text for details) (B)
Inhi-bition of ACh-evoked currents by dTC acting on the WT (n) and
R55F (s) mutant receptors (see Table 2) For each receptor, the
ACh concentration used to induce responses was equivalent to its
EC 50 value for activation of that subtype Similar data show a lack
of significant effect of dTC on the aR55W and R55E mutant
recep-tors (data not shown).
Table 2 Effects of PTMA and dTC on WT and mutant receptors.
Data were analyzed as described in the legend to Fig 3 K I values
were determined as described in Experimental procedures Each
experiment was repeated in 3–4 oocytes for each receptor
sub-type No significant differences were observed between the WT
and mutant receptors.
Receptor
log IC 50 ± SEM K I (l M ) log IC 50 ± SEM K I (n M )
WT ) 4.24 ± 0.06 a 57.0 a ) 7.07 ± 0.11 42.5
a Data obtained from PTMA-induced channel activation (Hill
coeffi-cient ¼ 2.1 ± 0.2) All other data are from the effects of the ligand
on ACh-induced currents.
WT αR55K αR55E αR55F αR55W
0 2 4 6 8 10
fmol
0 2 4 6 8 10
Fig 4 Surface nAChR expression of WT and mutant receptors in Xenopus oocytes Maximum ACh-evoked currents (Imax) were determined using concentrations determined from concentration-effect curves (as shown in Fig 2) Surface receptor levels were determined in the same oocytes by measuring 125 I-labelled a-BgTx binding as described in Experimental procedures The data repre-sent the mean ± SEM of 3–11 determinations from individual oocytes and are presented in Table 3.
Trang 5the aR55F and aR55W mutants were reduced by
approximately three- to five-fold, respectively,
com-pared with the WT receptor (Table 3; see Discussion)
Overall, these results suggest that mutation of aR55,
which is predicted to lie at the a–c and a–b interfaces,
does not play a major role in receptor assembly or
sur-face expression
Influence of aR55F and aR55W mutant receptors
on the binding of acetylcholine
The binding properties of ACh were investigated in
intact Xenopus oocytes expressing WT and mutant
nAChR The affinity of the mutant receptors for ACh
was characterized by its inhibition of the initial rate
of 125I-labelled a-BgTx binding to Torpedo nAChR
expressed on the surface of oocytes (Fig 5) ACh
inhibited the initial rate of125I-labelled a-BgTx binding
to the WT nAChR in a concentration-dependent
man-ner with an IC50of 544 nm (nH¼ 0.8) The IC50 (nH)
of the aR55F and aR55W mutants was estimated to
be 454 nm (1.0) and 313 nm (0.9), respectively, and did
not differ from that of the WT nAChR Thus, despite
the reduction in the potency of ACh in mediating
functional responses in the mutant receptors, the
equi-librium (high affinity) binding of ACh appears to be
unaltered
Effects of cE93R mutation on the sensitivity of
agonist and antagonist
As noted in the Introduction, it has been suggested
that the proximity of aR55 and c93E may be
con-ducive to an ion-pairing interaction (see Fig 6)
Surprisingly, the cE89R mutation resulted in an
approximately eight-fold increase in the apparent
potency of ACh-induced activation As shown in
Fig 7A (see Table 1), the EC50for this mutant recep-tor was reduced to 3 lm (Hill coefficient of 1.4) from the value of 24 lm measured in the WT In contrast, the apparent affinity for the competitive antagonist, dTC (as determined by inhibition of ACh-evoked cur-rents in oocytes), for the cE93R mutant receptor was unaltered as compared with the oocytes expressing WT nAChR (KI 55 and 42 nm, respectively, see Fig 7B, Table 2) This figure also illustrates that, in contrast to the WT receptor (see above), the potentiating effects
of low concentrations of dTC were abolished by the cE93R mutation
Effects of double mutations of aR55 and cE93
In order to investigate whether the aR55F and cE93R mutations have an additive effect, we studied receptors carrying the double mutations, aR55E-cE93R and aR55F-cE93R These double mutant receptors had
EC50 values for ACh-induced activation of11.5 and 12.9 lm (Fig 7A, Table 1), which approach those of the WT receptors The Hill coefficients for these dou-ble mutants were not significantly different from the
WT receptor
Discussion The conformational changes that result in activation
of the nAChR channel are poorly understood, but are thought to involve an agonist-induced rotation of the
Table 3 Surface expression and normalized ACh-evoked maximum
currents in WT and mutant receptors expressed in Xenopus
oocytes All oocytes were injected with 50 ng of total cRNA
enco-ding WT or mutant subunit nAChR The value in parentheses is the
number of oocytes used for each receptor type.
Surface
binding
(fmolÆoocyte)1
± SEM )
I max (nA ± SEM )
Normalized peak current (nAÆfmol)1)
% peak current (mutant I max ⁄
WT Imax)
aR55E 9.2 ± 0.7 (3) 8680 ± 501 934.4 86.4
aR55K 8.0 ± 1.5 (5) 3652 ± 654 455.1 42.1
aR55F 3.8 ± 0.7 (6) 1445 ± 340 384.6 35.6
aR55W 5.9 ± 1.3 (7) 1154 ± 210 197.0 18.2
-10 -9 -8 -7 -6 -5 -4 0
20 40 60 80 100 120
log [ACh] (M)
Fig 5 ACh binding to Torpedo nAChR expressed on the surface of intact Xenopus oocytes ACh inhibited the initial rate of 125 I-labelled a-BgTx binding in a concentration dependent manner in WT (n) and aR55F receptors (s) The data represent the mean ± SEM of 2–3 determinations performed in duplicate giving log IC50± SEM values
of )6.26 ± 0.14 and )6.34 ± 0.06, respectively (IC 50 s of 544 and
454 n M , respectively) Similar experiments with the R55W mutant gave a log IC50value of )6.50 ± 0.20 (IC 50 of 313 n M ) There were
no significant differences between any of these receptor subtypes.
Trang 6subunits that is eventually communicated to the ion
channel pore [20,21] It is widely accepted that the
nAChR carries two high affinity binding sites located
at the interfaces between a–c and a–d subunits [7], but
the involvement of other interfaces in receptor function
is relatively unexplored In the present study, we have
therefore investigated the role of specific residues lying
at an adjacent interface, i.e residues aR55 (loop D)
and cE93 (loop A) which, in the muscle counterpart,
have been proposed to interact and to possibly play a
critical functional role in receptor properties [19]
Amino acid sequence alignments of loop D (see
Fig 1A) reveal that the peripheral nAChR a-subunits
carry a unique amino acid at position 55, i.e an
argin-ine residue rather that an aromatic amino acid that is
conserved in most other subunits of the Cys-loop
LGIC family There is considerable evidence to suggest
that, in a number of subunits, the residue in the
equiv-alent position plays an important role(s) in modulating
agonist⁄ antagonist sensitivity Mutations of cW55 and
dW57 in the Torpedo nAChR have been shown to
affect the affinity for dTC and ACh [17,22] while the
W54 of the neuronal nicotinic a7 receptor has been
shown to contribute to the binding of agonists [23] In
the GABAA receptor, the F64L mutation of the a1
subunit had a dramatic effect on GABA sensitivity
[24] and mutations of the F77 residue of the c2 subunit
significantly affected ligand affinity for the
benzodi-azepine binding site [25] In addition, the GABAA
receptor b2Y62 residue has been shown to be an
important determinant of high affinity agonist binding
[26] In the 5-HT3A receptor, the homologous W89
residue has been reported to contribute to both dTC
and granisetron binding [27] Thus, the conserved
aro-matic residue in this position of most LGIC subunits
appears to play an important role and the unusual occurrence of a positively charged amino acid in the peripheral nAChR a-subunit first prompted the present investigation
The present results demonstrate that R55, which is conserved in the peripheral a-subunits, is not essential for subunit assembly, as mutations in this position did not have a detrimental effect on the expression of 125 I-labelled a-BgTx binding sites on the oocyte surface Not surprisingly, the conservative substitution, aR55K had no significant effect on the concentration depend-ence of ACh-induced activation More surprisingly, the charge reversal mutation, aR55E, also had no signifi-cant effect on receptor activation properties These results are strong evidence that a positively charged residue in this position of the peripheral nAChR a-subunit is neither obligatory for agonist recognition nor does it play a major role in the transduction mech-anism that couples agonist binding to channel activa-tion
The aR55F and aR55W mutations resulted in a modest but significant decrease in the sensitivity to ACh (by approximately five- and six-fold, respectively)
In these mutant receptors, PTMA failed to induce
a measurable response However, since PTMA is such
a poor agonist on the WT nAChR, the lack of a response could be attributable to either a greatly reduced sensitivity of the receptor towards the ligand
or to a further reduction of conductance to undetecta-ble levels The lack of any response to PTMA was exploited to differentiate between these possibilities [28], and the results reveal that the effects of the muta-tions are on PTMA efficacy rather than affinity The apparent KI for PTMA-inhibition of ACh-responses mediated by the aR55F and aR55W mutant receptors
N O N
N N
O N
O O
5.7 Å
D I N N E L V I Human ε
D V N N E L V I Human γ
D V N N E L V V
Torpedoγ
D N N N Q L V I
Torpedoδ
D A N N Y L V L
Torpedoβ
D A N N Y L V L
Torpedoα
97 96 95 94 93 92 91 90 Loop A
Fig 6 Representation of the extracellular
domains of the c–a subunits based on the
crystal structure of AChBP The positions of
cE93 and aR55 at the subunit interface are
indicated and modeling suggests that these
residues are located approximately 6 A˚
apart Also shown are amino acid sequence
alignments of residues in loop A of
repre-sentative nAChR subunits.
Trang 7were similar to its EC50value for activation of the WT
receptor (Fig 3A, Table 2) suggestive of an unaltered
affinity for its binding site(s)
A reduced sensitivity of the aR55F and aR55W
mutant receptors to ACh-activation was accompanied
by a reduced maximum current response When the
measured peak currents were normalized to cell-surface
expression (nAÆfmol)1) the current responses were
sub-stantially lower than displayed by the WT receptor
This may reflect a reduction in single channel
conduct-ance of the mutant receptors, a decreased efficacy of
ACh-mediated currents or the possibility that some of
the expressed receptors are nonfunctional Distinction
between these possibilities requires further analysis at the single channel level We also observed a significant reduction in the Hill slope of the activation curves in the mutant receptors While the interpretation of chan-ges in Hill coefficients is controversial, the simplest explanation is that these mutations reduce the level of cooperativity between different agonist binding sites [29]
Our present findings are consistent with previous reports that mutations of the homologous residue (W54) in the a7 nAChR W54 resulted in a reduction of ACh potency without a disruption of a-BgTx binding [23] The present results point to a role of aR55 in the transduction mechanism rather than in direct agonist binding However, there is some evidence in the litera-ture that this region may also contribute to binding site formation A synthetic peptide equivalent to a55–74 of TorpedonAChR was shown to be able to bind a-BgTx but this binding was inhibited by an R55G substitution
in the synthetic peptide [30] However, since a synthetic peptide is unlikely to have a similar conformation as the equivalent domain in the native receptor, it is diffi-cult to correlate the two sets of results
The apparent affinity of the competitive antagonist, dTC, was measured by its ability to inhibit ACh-induced currents The potency for dTC-ACh-induced inhi-bition in WT and mutant receptors was similar suggesting that the mutations had not altered dTC affinity When the binding of ACh was measured by its ability to inhibit the initial rate of 125I-labelled a-BgTx binding to individual oocytes, its apparent affinity was also unaltered from that of the WT recep-tor As these experiments are designed to measure high affinity binding sites that exist under equilibrium con-ditions, the results suggest that, under these circum-stances, agonist binding has been unaltered Thus the major effect of the aR55F and aR55W mutations appears to be on the potency for ACh-induced func-tional responses, i.e on the transition(s) between rest-ing and activated states of the receptor
In the case of the cE93R mutation, the ability of low concentrations of dTC to potentiate ACh-induced currents was apparently lost Steinbach and Chen [31] previously reported that dTC can act as a weak agon-ist of the fetal muscle nAChR and suggested that, at low dTC concentrations, the simultaneous binding of one agonist molecule and one dTC molecule might eli-cit channel opening The above data on the WT recep-tor are consistent with such a mechanism The most parsimonious explanation of the loss of the potentiat-ing effect in the mutant receptor is that the mutation results in the loss of the ability of dTC to act as such
a ‘coagonist’
0
20
40
60
80
100
120
A
B
log [ACh] (M)
0
20
40
60
80
100
120
log [dTC] (M)
Fig 7 Effects of the cE93R mutation (A) Concentration-effect
curves for ACh activation of WT (n), cE93R (,) and the double
mutants, cE93R-aR55E (.) and cE93R-aR55F (e) nAChR The data
represent the mean ± SEM from at least three oocytes and are
nor-malized to the I max for each oocyte Data are summarized in
Table 1 (B) Concentration dependent inhibition of ACh evoked
cur-rents by dTC in oocytes expressing the WT (n) and cE93R (,)
mutant receptors The ACh concentration used in the experiments
corresponded to their EC50 concentrations determined for each
receptor Each curve was generated from at least three oocytes.
The apparent K I of dTC on oocytes expressing the cE93R receptors
(55 n M ) is not significantly different from the WT nAChR (42 n M ).
Trang 8Although the c–a interface has not previously been
implicated in ligand binding or receptor function,
structural models of the adult human nAChR based
on AChBP have suggested putative interactions at this
interface i.e a salt bridge between eE93 (a loop A
resi-due) and aR55 (a loop D residue [18,19]) Our results
obtained with the cE93R substitution clearly
demon-strate that this is a ‘gain-of-function’ mutation that
results in an approximately eight-fold decrease in the
EC50 for ACh activation One possible explanation is
that this mutation facilitates the rotational movements
at intersubunit contact points that have been suggested
to occur during channel activation [20,21,32] The
receptor carrying the double charge-reversal mutation
(cE93R-aR55E) was activated by ACh with an EC50
that approached that of the WT receptor, although the
EC50 values (Table 1) remained statistically different
Taken together, these results suggest that, in the WT
receptor, an interaction between aR55 and cE93 is
unlikely to stabilize either the resting conformation (as
the mutation aR55E had little effect on activation) or
the activated state (as mutation cE93R increased ACh
potency) However, these residues lying at the c–a
interface do appear to play a role in receptor
activa-tion and⁄ or the signal transduction mechanism
In summary, we have identified a residue, R55 in
loop D of the extracellular ligand binding domain of
a-subunit that modulates ACh sensitivity and that lies
at some distance from the ‘classical’ high affinity
bind-ing sites for ACh This residue has not previously been
implicated in nAChR function However, our data
complement earlier work to suggest that loop D
resi-dues occurring in nonbinding domains may play
important roles in receptor function[see 26] In
addi-tion, we show that E93 of the Torpedo nAChR
c-sub-unit has a significant effect on agonist-induced
activation, as substitution by the positively charged
arginine increased ACh potency by approximately
eight-fold Although our data do not support a critical
role for a direct interaction between aR55 and cE93,
they demonstrate that residues lying at interfaces
adja-cent to those that have been implicated in agonist
binding influence receptor function
Experimental procedures
Materials
ACh, a-BgTx and dTC were obtained from Sigma-RBA
(Natick, MA, USA).125I-labelled a-BgTx (2000 CiÆmmol)1)
was from Amersham Life Science (Arlington Heights, IL,
USA) Restriction enzymes and cRNA transcript
prepar-ation materials were purchased from Invitrogen
(Burling-ton, ON, Canada), Promega (Madison, WI, USA) or from New England Biolabs (NEB, Pickering, ON, Canada) Pfu Turbo DNA polymerase for mutagenesis experiments was from Stratagene (La Jolla, CA, USA) All other chemicals were obtained from Sigma or other standard sources The a-, b- (in the SP64 plasmid) and d-subunit (in the SP65 plasmid) cDNA clones of the Torpedo nAChR were gener-ous gifts from H A Lester (California Institute of Technology, Pasadena, CA, USA) The c-subunit cDNA (in the SP64-based plasmid, pMXT) was a gift from J B Cohen (Harvard Medical School, Boston, MA, USA)
In vitro transcription and site-directed mutagenesis
The plasmid cDNAs were linearized by digestion with either EcoRI (for the a-subunit), FspI (for the b-subunit)
or XbaI (for the c- and d-subunits) In vitro cRNA tran-scription was performed using the methods described by Goldin and Sumikawa [33] Briefly, the linearized cDNA templates (5 lg) were transcribed in vitro using SP6 RNA polymerase (Promega) in the presence of ribonucleotide triphosphate (NTP mix, Invitrogen) and RNA capping analogue (NEB) The RNA transcripts were extracted using 25 : 24 : 1 (v⁄ v) phenol–chloroform–isoamyl alcohol Finally, the RNA pellets were resuspended in diethylpyro-carbonate-treated water at a concentration of 1 lgÆlL)1 The a-subunit mutants (R55F, R55W, R55K and R55E) were constructed using Stratagene’s QuikChange site-direc-ted mutagenesis protocol Synthetic oligonucleotide muta-genic primers were typically 23–34 base pairs long (with 10–15 base pairs lying on either side of the mismatch region) A similar approach was undertaken to engineer the cE93R mutation Restriction endonuclease digestion and DNA sequencing subsequently verified the presence of the mutation
Expression in Xenopus oocytes and electrophysiology
Isolated, follicle-free oocytes were microinjected with 50 ng
of total subunit cRNAs in a ratio of 2a : 1b : 1 c : 1d Oocytes were maintained in ND96 buffer (96 mm NaCl,
2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm Hepes,
pH 7.6) supplemented with 50 lgÆmL)1gentamicin at 14C for at least 48 h prior to recording Currents elicited by bath application of agonist were measured with a Gene-Clamp 500 amplifier (Axon Instruments, Foster City, CA, USA) using standard two-electrode voltage clamp tech-niques at a holding potential of )60 mV Electrodes were filled with 3 m KCl and those with resistances of 0.5–3.0
MW were used The recording chamber was perfused con-tinuously (at a flow rate of 5 mLÆmin)1) with low Ca2+ ND96 buffer, in which the CaCl2 concentration was
Trang 9reduced to 0.1 mm in order to slow the rate of receptor
desensitization [34] Atropine (1 lm) was included in the
perfusion buffer to block endogenous muscarinic receptors
present in the oocytes [35] Agonist-evoked responses were
measured by applying the drug via the perfusion system for
15Ờ20 s followed by a 15-min wash-out period to ensure
full recovery from desensitization For measuring the effects
of antagonists, oocytes were preperfused with various
con-centrations of antagonist in low Ca2+ ND96 buffer for
2 min, before initiating the response by application of
solu-tion containing ACh (at a concentrasolu-tion eliciting 50% of
the maximum response, EC50) and including the same
con-centration of antagonist as used in the preperfusion
Binding of125I-labelled a-BgTx to intact oocytes
Binding assays were performed on individual oocytes that
had previously been used in the electrophysiological
experi-ments To measure the density of nAChR binding sites
expressed on the oocyte surface (fmol), oocytes were
incu-bated with 5 nm 125I-labelled a-BgTx in a final volume of
100 lL of low Ca2+ ND96 buffer containing 5 mgẳmL)1
bovine serum albumin for 2 h at room temperature [36,37]
Excess unbound toxin was removed by washing the oocytes
three times with 1 mL of ice-cold low Ca2+ND96 buffer
This procedure was performed by manually transferring the
oocyte from one solution to another using a broad
micropi-pette tip to pick up the oocyte with minimal transfer of the
original solution Non-specific binding was estimated by
carrying out parallel studies of125I-labelled a-BgTx binding
to uninjected oocytes Non-specific binding determined in
oocytes expressing WT or mutant receptor in the presence
of excess cold ACh was comparable to that estimated using
uninjected oocytes (data not shown) Bound 125I-labelled
a-BgTx was measured by c-counting (Gamma8000,
Beck-man) Using these data, the maximum currents (Imax)
meas-ured for WT and mutant receptors were normalized to the
concentration of binding sites in terms of nAẳfmol)1 For
competition curves, oocytes were incubated for 40 min with
various concentrations of ACh in a 96-well plate prior to
the addition of 2.5 nm 125I-labelled a-BgTx After 40 min,
125I-labelled a-BgTx binding was stopped by the addition
of 1 lm unlabeled a-BgTx In the absence of the competing
ligand, ACh,125I-labelled a-BgTx binding was30% of the
available a-BgTx-binding sites (data not shown)
Non-speci-fic binding was determined in the presence of 100 mm ACh
Data and statistical analysis
Competition and concentration-effect curves for both
elec-trophysiological and radioligand binding experiments were
analyzed by nonlinear regression techniques using
graph-pad prism3.0 software (GraphPad, San Diego, CA, USA)
Data from individual oocytes were normalized to the Imax
value obtained for that oocyte
For receptor activation, concentration-effect curves for agonist activation were analyzed using the following equa-tion:
IỬ ImaxơLn=đEC50ợ ơLỡn where I is the measured agonist-evoked current, [L] is the agonist concentration, EC50 is the agonist concentration that evokes half the maximal current (Imax) and n is the Hill coefficient In each experiment, the current (I) is nor-malized to the Imaxand the normalized data are presented
as percentage response
The IC50 was determined from competitionỜinhibition curves by fitting to the following equation:
f Ử 100=ơ1 ợ đơX=ơIC50ỡn where f is the fractional (%) response remaining in the presence of inhibitor at concentration [X], IC50is the inhib-itor concentration that reduced the amplitude of ACh-evoked current by 50% and n is the Hill coefficient ACh inhibition of initial rate of125I-labelled a-BgTx binding was also fit by the above equation
The KI(apparent) value was calculated using the Cheng-Prusoff equation [38]:
KIỬ IC50=ơ1 ợ ơL=EC50 where [L] is the ACh concentration used in the experiment and EC50 is the ACh concentration that evokes half the maximal current
Statistical analysis was performed using one-way analysis
of variance (anova, http://www.physics.csbsju.edu/stats/ anova.html) followed by DunnettỖs post-test to determine the level of significance
Acknowledgements This work was supported by the Canadian Institutes
of Health Research We are especially grateful to Isabelle Paulsen for expert technical assistance
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