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Vol. 61, Issue 2, 463-472, February 2002
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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To further define the surface of the Torpedo californica
nicotinic acetylcholine receptor (nAChR) contributing to the agonist binding site structure, we used the substituted Cys accessibility method to identify novel residues and determined the "footprint" of
residues protected from modification by the reversible competitive antagonist d-tubocurarine (dTC). nAChRs
containing single Cys substitutions within regions of the 
- or
-subunit primary structure known to contribute to the agonist
binding site were expressed in Xenopus laevis oocytes.
Cys substitutions in binding site segments A (Tyr-93 and Asn-94),
C (Tyr-198), and D (Glu-57) had been shown previously to be
accessible for modification. We now introduced cysteines from
Asp-195 to Ile-201 and from Ala-106 to Asp-113 and
identified positions accessible for modification in segments C
(Asp-195, Thr-196, Pro-197, Asp-200, and Ile-201) and E (Asn-107 and Leu-109). dTC protected against alkylation in
segments D (Glu-57) and E (Leu-109) but not in segment A
(Tyr-93 and Asn-94). In segment C, dTC protection experiments
revealed a pattern in which every other residue (196, 198, and
200, but not 197 or 201) was protected from alkylation. This
pattern of protection provides evidence that bound dTC is near amino
acids in segments C, D, and E but not in segment A, and identifies a 
-strand surface within segment C contributing to the binding site.
These results are discussed in terms of a homology model, based on the
molluscan acetylcholine binding protein crystal structure, of the
T. californica nAChR agonist binding site.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
binding sites for agonists and competitive antagonists in the nicotinic
acetylcholine receptor (nAChR) are within the extracellular domain at
the - and -
subunit interfaces. Affinity labeling and
mutagenesis studies have provided extensive evidence for a model of the
agonist site structure with contributing amino acids from three
distinct regions of the -subunits (referred to as binding site
segments A, B, and C) and from at least three regions of the (or
)-subunit (segments D, E, and F) (reviewed in Prince and Sine, 1998
;
Arias, 2000; Corringer et al., 2000). Most features of the model are
present in the binding site identified within the recently solved
structure of a molluscan, glial-derived soluble ACh binding protein
(AChBP), a homopentameric structural and functional homolog of the
N-terminal ligand binding domain of a nAChR -subunit (Brejc et al.,
2001; Smit et al., 2001).
The substituted Cys accessibility method (Karlin and Akabas, 1998) has
provided an alternative approach for characterizing structural features
of the nAChR and other ion channels. An observed irreversible change in
the functional properties of the channel, after exposure to a
water-soluble sulfhydryl reagent, suggests that the substituted Cys is
exposed at the water accessible protein surface. This technique has
been used to identify the state-dependent accessibility of amino acids
contributing to the ion conduction pathway of the nAChR (Akabas et al.,
1994; Akabas and Karlin, 1995; Zhang and Karlin, 1997, 1998). In
studies of the structure of the agonist binding site, which contains a
disulfide bond between Cys-192/193 in segment C, most Cys
substitutions are well tolerated within 184-198 and are accessible
for modification (McLaughlin et al., 1995; Spura et al., 1999; Spura et
al., 2000). Using Cys mutagenesis of Torpedo californica
nAChR, we previously tested the accessibility of positions identified
by affinity labeling and mutagenesis in segments A (Tyr-93), B
(Trp-149), C (Tyr-190 and Tyr-198), and D (Trp-55 and
Glu-57) as well as surrounding amino acids in segments A (90-96)
and D (52-58) and found that Tyr-93, Asn-94, Tyr-198, and
Glu-57 were accessible (Sullivan and Cohen, 2000). That study also
helped to define the structural requirements for ligand orientation
compatible with nAChR activation, as
[2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which
attaches thiocholine, acted as an irreversible antagonist at positions
Y93C and E57C but as a covalent agonist at Y198C. Furthermore,
a structural analog with the tethering arm shortened by one methylene
group (0.7 Å) acted as an irreversible antagonist at Y198C and at
all other accessible positions.
In this report, we extended these studies by identifying additional
accessible residues in segment C (195-201) as well as segment E
(106-113), which includes residues identified by photoaffinity labeling with the antagonists
[3H]4-benzoylbenzoylcholine (Leu-109; Wang
et al., 2000) and [3H]d-tubocurarine
(dTC) (Tyr-111; Chiara et al., 1999). With these mutant nAChRs, we
also tested whether MTSET or its analogs could act as irreversible
agonists when tethered at positions other than Y198C. In addition,
we used the panel of nAChR binding site mutants containing accessible
cysteines to identify positions that could be protected from alkylation
when the agonist binding site was occupied by dTC. Based upon
photoaffinity labeling, [3H]dTC binds to the
agonist site at the - interface near amino acids in segments C
(Tyr-190, Cys-192, and Tyr-198), D (Trp-55), and E
(Tyr-111 and Tyr-117; Chiara and Cohen, 1997; Chiara et al.,
1999), and dTC can protect against MTSET reaction at Y198C (Sullivan
and Cohen, 2000). The results we now report, which indicate protection
of alkylation of positions in segments C, D, and E but not in segment
A, are consistent with the results of photoaffinity labeling. The
pattern of protected residues within segment C indicates that this
region is organized as a -strand and identifies a surface projecting
toward the ACh binding site consistent with the structure of the
binding site in the AChBP (Brejc et al., 2001). However, the lack of
protection of Y93C is surprising in terms of that structure. Our
experimental results are compared with a homology model of the T. californica agonist binding site based on the crystal structure of
the AChBP and are generally consistent with this structure. However,
the accessibility of some residues suggests differences between the
model and the structure of the T. californica nAChR binding
site in the absence of agonist (our experimental conditions).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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cDNA Mutagenesis.
Mutants were constructed by "overlap
extension" PCR using T. californica nAChR subunit plasmids
(, , and in pMXT and in pSP64) and reagents as described
previously (Sullivan and Cohen, 2000). The mutations of amino acids
195-201 of the mature -subunit were generated using primers which
gave a PCR product of ~1000 base pairs that could be subcloned
using the unique BsiWI restriction site and the
BbvII site near the 3' end of the -subunit coding region.
Mutations of amino acids 106 to 113 in the mature -subunit were made
by generating a PCR product of ~1.13 kilobases that was subcloned
using the unique HindIII site in the vector and the
StuI site in the -subunit coding region. Each PCR mix
contained 0.5 µM primers, 50 ng of template DNA, and 0.4 mM dNTPs in
the reaction buffer supplied with the enzyme. PCR reactions were for 24 cycles with a three-step protocol (1.5 min at 95°C, 45°C, and 72°C).
Electrophysiology.
T. californica nAChR
subunit-specific cRNAs were transcribed in vitro and Xenopus
laevis oocytes were injected as described previously (Sullivan and
Cohen, 2000). Isolated, follicle-free oocytes were injected with 0.5 to
10 ng of subunit-specific RNAs in a molar ratio of 2///,
and currents elicited by ACh were measured 48 to 72 h after
injection by two-electrode voltage clamp. Under our experimental
conditions, for oocytes injected with 0.5 ng of wild-type nAChR subunit
cRNAs, maximal current responses for ACh were typically 1 to 2 µA.
For the Cys substitutions between 195 and 201 in segment C, the
maximal current responses for ACh were similar to wild-type for the
mutant nAChRs containing Cys at 195, 196, 197, or 199. As
described previously, the Y198C nAChRs showed maximal current levels
~1% of wild-type, and for the D200C and I201C mutant nAChRs,
the maximal currents were also 1 to 5% of wild-type. For Y198C, as
judged by binding of 125I--bungarotoxin to
intact oocytes, surface nAChR levels were ~50% of wild-type. Within
segment E (106-113), the maximal ACh current responses were similar
to wild-type for each substitution except for Y111C, which had
maximal responses ~2% of wild-type and surface nAChR levels <10%
of wild-type. Surface receptor expression levels of other mutants were
not quantified. Salts, atropine, ACh, and dTC were from Sigma (St.
Louis, MO). MTSET,
[3-(trimethylammonium)-propyl]-methanethiosulfonate (MTSPT),
2-aminoethylmethanethiosulfonate (MTSEA), and
4-(N-maleimido)benzyltrimethylammonium (MBTA) were from
Toronto Research Chemicals (North York, Ontario, Canada), and
Biotin-PEO-maleimide (Fig. 1) was from
Pierce (Rockford, IL). Sulfhydryl-modifying reagents were prepared as
millimolar stock solutions in recording solution and stored on ice
during use, with fresh solutions prepared approximately every 2 h.
For nAChR activation, ACh dose response curves were fit to the
equation: I / Imax = [1 + (Kapp /
[ACh])nH]
1,
where I and Imax are the currents at a given
concentration of ACh and the maximal current, respectively.
Kapp is the apparent activation
constant for ACh and nH is the Hill
coefficient. pCLAMP (Axon Instruments, Foster City, CA) and SigmaPlot
(SPSS Inc., Chicago, IL) software were used for data analysis.
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Rate Constants of nAChR Modification.
For the
sulfhydryl-reactive reagents producing irreversible inhibition of ACh
responses, the time course of the reaction with a substituted Cys
mutant in the absence of ACh was determined by recording the initial
response to ACh and then the response to ACh after repeated
applications of modifying reagent for 5-s intervals. Each application
of reagent was followed by a ~1-min wash, three ACh test applications
(5 s each), and a 1-min wash. ACh was generally applied at a
concentration equal to Kapp.
ACh-induced currents after treatment were plotted as a function of
cumulative modification time (t) and fit by a single exponential
function, It = I
+ (Io 
I) exp(t / 
)
where It is the current at a given time,
I is the amount of current remaining after the
reaction is complete, and Io is the initial
current level. 1 / is the pseudo-first-order rate constant and the
second-order rate constant, k, is (1 / ) / x, where
x is the concentration of modifying reagent.
dTC Protection Assay.
Responses to ACh at a concentration
near Kapp were measured before and
after coapplication of dTC and ACh to show that 10 µM dTC was
sufficient to reversibly block >95% of the ACh response and that the
Cys substitution itself had not interfered with the receptors' ability
to bind dTC. This initial part of the assay was also necessary to
determine that the effects of dTC were reversible. It was often
necessary to wash the oocyte for several minutes after dTC application
for full recovery of the ACh response. To measure the degree of
protection by dTC, ACh test pulses were measured before and after 10 µM dTC was coapplied with a concentration of MTSET known to cause 50 to 80% inhibition (based on rate constants). The same concentration of
MTSET was then applied in the absence of dTC, again using ACh test
pulses to measure the extent of inhibition. The degree of protection
was then determined by comparing the ratio of the extent of
modification in the absence of dTC to the extent of modification in the
presence of dTC: % protection = [1 (% InhibitiondTC/MTSET / % InhibitionMTSET)] × 100.
Homology Modeling of the T. californica
nAChR.
Molecular modeling of the extracellular domain of the
T. californica nAChR based upon the recently published
structure of the AChBP (Brejc et al., 2001) was done using Insight II
(Version 98; MSI, San Diego, CA) on a Silicon Graphics
O2 workstation. The sequences for the four
T. californica nAChR subunits (NCB accession numbers:
ACRYA1, ACRYB1, ACRYG1, and ACRYD1) were obtained from the National
Center for Biotechnology Information and the coordinates for the
structure of the AChBP (PDB number 1I9B) were obtained from the
Research Collaboratory for Structural Bioinformatics (http://www.rcsb.org). The nAChR subunit sequence alignment presented by Brejc et al. (2001) was used, and the AChBP structure was examined to ensure that insertions and deletions occurred within exposed flexible segments. The Insight II Homology module placed the nAChR sequences into the AChBP structure and the Insight II Discovery module
energy minimized the resulting structural model. Compared with the
primary structure of the AChBP, the nAChR -subunit contains insertions in two regions within the ACh binding site. Although segment
C in all neuronal nAChR -subunits aligns well with the AChBP, the
T. californica and skeletal muscle -subunits contain a
single residue insertion between Cys-193 and Tyr-198. We
designated Asp-195 as the inserted residue to preserve the
10-strand beginning at Thr-196, with Thr-196, Tyr-198, and
Asp-200 projecting in the direction of the binding site, a structure
consistent with experimental results described in this report. The
other -subunit insertion in the vicinity of the binding site was
Ala-96 in segment A. Both insertions were well tolerated upon minimization.
-, -, and -subunits, respectively, in the
segment region between the 8 and 9 -strands. Lacking
additional guidelines, we placed these insertions (164-172, 164-170, and 166-176) in external loops to preserve the
structure of the adjacent regions that were homologous to the AChBP
sequence. In our model, Ser-161, which has been identified as a dTC
selectivity determinant for mouse nAChR (Sine, 1993), occurs just after
the 8-strand and is positioned beyond Lys-34, ~15 Å from the
center of the aromatic binding pocket. Similarly, Glu-183, which was identified as a determinant of agonist
Kapp (Czajkowski et al., 1993), occurs
at the NH2 terminus of the 9-strand, which
would lie near the plane of the membrane in a nAChR, 26 Å from the
agonist binding site. Asp-177 is positioned outside of
Tyr-190/Trp-55, closer to the binding pocket than Asp-174, an
important determinant of agonist Kapp
(Martin et al., 1996; Martin and Karlin, 1997). Because segment F
residues seem more distantly related to the core structure of the
binding site and amino acids in this region have not been examined in
this study, the predicted positions of these residues are not included
in our model of the T. californica nAChR ACh binding site.
In the AChBP structure, a HEPES molecule was identified within the
aromatic pocket of the ACh binding site. We placed ACh molecules into
the equivalent positions of our nAChR model (at the - and -
interfaces) using the Insight II Docking module, and the structure
containing the ACh molecules was energy minimized. A single orientation
for each ACh molecule was favored (docking energy of 30 kcal), which
placed the ACh nitrogen within the aromatic pocket of the ligand
binding site consisting of Tyr-93, Trp-149, Tyr-190,
Tyr-198, and Trp-55/Trp-57.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Functional Properties of nAChRs with Cys Substitutions in Binding
Site Segments C and E.
nAChR functional properties were assessed
by measuring ACh-elicited currents using two-electrode voltage clamp.
Each of the substitutions within 195-201 and 106-113,
when expressed with other wild-type subunits, resulted in functional
nAChRs (Table 1). For wild-type nAChR,
the Kapp for ACh was 30 ± 8 µM. For the segment C mutant nAChRs, there was a significant
rightward shift of Kapp only for the
Cys substitution at Y198C. The mutant nAChRs with substituted Cys
adjacent to Tyr-198 at either Pro-197 or Leu-199 were
characterized by leftward shifts of
Kapp, whereas for the other
substitutions studied in this segment, the
Kapp values were shifted <2-fold
compared with wild-type. The Cys substitutions in segment E
(106-113) were also well tolerated. The largest shifts of
Kapp were seen for the L109C
(Kapp = 100 µM) and Y111C (Kapp = 14 µM) mutants, whereas for
substitutions at each of the other positions,
Kapp for ACh was within a factor of 2 of wild-type.
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Modification of Substituted Cysteines within Binding Site Segment
C.
For oocytes expressing wild-type or mutant nAChRs, the response
to ACh was measured at a concentration close to
Kapp. Oocytes were then exposed to
MTSET (200 µM), MTSEA (1 mM), or maleimide-PEO-biotin (1 mM) (Fig. 1)
for 5 s in the absence of agonist; after a wash of 1 to 2 min, the
ACh response was remeasured. Representative current traces are shown
for wild-type and segment C mutant nAChRs treated with MTSET (Fig.
2A), and summary data for each of the mutants are presented for the effects of MTSET, MTSEA, and
maleimide-PEO-biotin (Fig. 2B). We were particularly interested in
determining whether thiocholine tethered at positions other than
Y198C in segment C would result in covalent activation. However,
treatment with MTSET for 5 s resulted in irreversible inhibition
of the ACh response by >75% for the T196C, P197C, and D200C
mutant nAChRs, and a smaller inhibition of the I201C mutant that
increased with longer reaction times (see later). The ~10%
inhibition of the D195C or L199C mutants was less than that seen
for wild-type nAChR and was not indicative of modification of the
substituted Cys. Treatment with MTSEA also resulted in irreversible
inhibition of ACh current responses for those mutants sensitive to
MTSET. Modification with maleimide-PEO-biotin (1 mM, 5 s)
significantly inhibited the ACh responses for the same mutants and, in
addition, it inhibited irreversibly the D195C nAChR mutant.
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Tyr-198 (data not shown). MTSPT confirmed the
accessibility of residues T196C, P197C, and D200C by
inhibiting subsequent ACh responses, but it only activated the Y198C
mutant, as described previously (Sullivan and Cohen, 2000).
Modification of Substituted Cysteines within Binding Site Segment
E.
Cys substitutions at 106 to 113 were similarly tested for
their sensitivity to MTSET and MTSEA (Fig.
3). Summary data show that a 5-sec
exposure to MTSET (200 µM) or MTSEA (1 mM) inhibited ACh responses
for the N107C mutant by ~30 and 75%, respectively, whereas both
compounds inhibited the L109C response by ~90%. Effects at the
other positions tested were not sufficiently different from wild-type
to make any conclusions about their accessibility. Exposure of these
mutants to maleimide-PEO-biotin did not yield any additional
information about residue accessibility (data not shown).
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Reaction Rate Constants.
The rates of reaction were measured
to determine the effect of full modification at a particular residue in
response to a specific reagent and to give us the kinetic information
necessary to design protocols for subsequent dTC protection
experiments. For the Cys mutants inhibited irreversibly by MTSET or
MTSEA, the rates of reaction with mutant nAChRs were determined by
measuring the response to ACh after increasing reaction times and for
various reagent concentrations (Fig. 4).
For oocytes expressing Y93C, Y198C, and E57C nAChRs, ACh
responses were inhibited by >90% after full modification (Sullivan
and Cohen, 2000). In contrast, even after complete modification, the
ACh current response was not fully inhibited for a number of the
segment C and E Cys mutants characterized here. For T196C and
I201C mutant nAChRs, ACh current responses were maximally inhibited
30 to 50% by MTSEA, whereas P197C and D200C responses were
inhibited by 75% and 95%, respectively (Fig. 4A). For substitutions
in segment E, MTSEA treatment inhibited N107C responses by 80%,
whereas it inhibited L109C responses by >90% (Fig. 4B), similar to
the level of inhibition seen for the E57C mutant. MTSET treatment of
T196C nAChRs resulted in maximal inhibition of 80% (Fig. 4C),
compared with the maximal inhibition of 30% seen after reaction with
MTSEA. MTSET also more fully inhibited responses at P197C than
MTSEA. MTSET treatment inhibited to the same extent as MTSEA at
D200C (>90%) and I201C (50%). For substitutions in the
-subunit, MTSET fully inhibited the E57C and L109C nAChR,
whereas for the N107C nAChR, maximal inhibition was only 50% (Fig.
4D).
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) to
identify Cys-192/193 as amino acids of the agonist binding site in
the T. californica nAChR (after disulfide reduction). Reaction with MBTA at T196C, P197C, D200C, and L109C
resulted in irreversible inhibition of the ACh responses to the same
extent as seen after modification with MTSET (Fig. 4, E and F).
I201C and N107C (Fig. 4, E and F) nAChRs, although sensitive to
MTSEA and MTSET, were not inhibited after exposure to MBTA, suggesting that MBTA modification may be more orientation-dependent.
The apparent bimolecular reaction rate constants for MTSEA,
MTSET, or MBTA modification of the mutant nAChRs (Table
2) were determined from the rates of
reaction, which increased with increasing reagent concentration. While
the reaction rate constants at Y93C, Y198C, and E57C differed
by as much as 500-fold, at each position, the rate constants for MTSEA
and MTSET differed by <2-fold, and the rate constants for MBTA were
never larger than for MTSET. For the other positions tested within
segment C, the rate constants for MTSET (or MTSEA) varied by
~100-fold without any characteristic periodicity, and the rate
constants for MBTA varied by as much as 3000-fold at adjacent amino
acids (T196C, P197C). At T196C, MTSET (k ~ 2 × 105 M1
s1) reacted ~1000-fold faster than MTSEA,
whereas at the other positions examined, the rate constants for the two
compounds differed by <2-fold. Whereas the rate constant for MBTA
reaction with Y198C (k ~ 5 × 103
M1 s1) was only 2% of
that for MTSET, for T196C the rate constant for MBTA (k ~ 1 × 106 M1
s1) was 5-fold higher than that for MTSET and
similar to the rate constant for MBTA modification of Cys-192/193 in
reduced, native T. californica nAChR (k ~ 3 × 106 M1
s1) (Stauffer and Karlin, 1994). At D200C
the rate constant for MBTA (k ~ 5 × 104 M1
s1) was 50-fold higher than for MTSET. Within
segment E, the rate constant for MTSET reaction at L109C (k ~ 2 × 104 M1
s1) was 40-fold greater than at N107C, and
for L109C or E57C nAChRs the rate constants for MTSEA, MTSET and
MBTA were essentially the same.
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Effects of Alkylation on ACh Dose Response.
Analysis of the
kinetics of MTSET modification of the mutant nAChRs revealed that after
full modification, ACh responses were inhibited by 
90% at some
positions (Y93C, P197C, D200C, E57C, L109C) but not at
others (T196C, I201C). For the latter positions, it was clear
that the tethered thiocholine modified the ACh response but did not
prevent ACh binding. Additional experiments were carried out to
determine whether, at the other positions, tethered thiocholine acted
as an irreversible antagonist or alternately as a modifier of ACh
binding and/or gating. We characterized ACh dose response curves before
and after modification to determine whether the inhibition seen at a
fixed concentration of ACh resulted from reduction only of the maximal
response or also from a modification of
Kapp. If modification resulted in a
nAChR no longer capable of being activated by ACh, then any ACh induced
currents could result only from remaining unmodified nAChRs, and the
response after exposure to MTSET would be characterized by a decreased maximal current without change of
Kapp. For example, modification of
Y93C or E57C nAChRs by MTSET or MTSEA resulted in reductions of
maximum current without change of Kapp
(Sullivan and Cohen, 2000). If after modification, ACh was still able
to gate the ion channel, but with either the binding or gating altered,
then the response could be characterized by a shift of
Kapp.
D195C and
L199C), the shifts of the ACh dose response curves after MTSET
treatment (~20% reduction of maximal response,
Kapp shift <1.5-fold) were similar to
the effects of MTSET on wild-type nAChR (Fig.
5, A and B; data not shown). For two
positions with substantial responses after full modification (T196C
and I201C), Kapp values were
shifted by <2-fold (Fig. 5, A and C). The P197C nAChR was inhibited
by >90% when tested at 30 µM ACh (Fig. 4C). This inhibition resulted from a 10-fold increase in
Kapp with less than a 20% reduction
of the maximal response (Fig. 5B). After full modification of the
D200C mutant, the maximal response was reduced by only 80% and the
Kapp value increased 2-fold (Fig. 5C).
Thiocholine tethered at T196C, P197C, D200C, or I201C
altered either ACh binding or gating, but did not act as a covalent
antagonist that prevented the binding of ACh.
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-subunit, modification of either E57C or
L109C can cause >90% inhibition of the ACh response when tested at
a concentration near Kapp (Fig. 4D).
For the E57C mutant, limited modification of nAChRs by exposure to
100 µM MTSET for 5 s resulted in a 70% reduction of maximal
response with no shift of Kapp (Fig.
5D). Treatment of the L109C mutant with 200 µM MTSET for 5 s,
which was sufficient for maximal modification (Fig. 4D), resulted in a
reduction of the maximum response by only 70% accompanied by a 3- to
4-fold increase in Kapp (Fig. 5D).
Thus, with thiocholine tethered at L109C, ACh was still able to bind and gate the ion channel.
dTC Protection Experiments.
From our previous work and the
experiments presented here, we identified accessible residues in ACh
binding site segments A (Y93C and N94C), C (D195C, T196C,
P197C, Y198C, D200C, and I201C), D (E57C), and E
(N107C and L109C). However, the fact that modification of the
substituted cysteines led to altered ACh responses does not establish
that these positions actually contribute to the structure of the ACh
binding site. The altered responses could result from an allosteric
modification of the structure of the binding site or from a
perturbation of the conformational transition necessary for channel
gating. If MTSET is within the agonist binding site when it reacts with
a substituted Cys, then that reaction should be inhibited by the
presence of a reversible agonist or antagonist that is bound in
proximity to that position.
Y93C. Y93C nAChRs were inhibited by ~90%
when MTSET was applied in the presence of dTC, and the remaining ACh
response was inhibited by a further treatment by MTSET alone.
Similarly, dTC did not prevent reaction of MTSET with N94C (not
shown). In contrast, for E57C in segment D, L109C in segment E,
and for some of the positions in segment C, dTC did protect against
modification by MTSET. dTC provided 90% protection of the E57C and
L109C nAChRs. For example, for E57C, the ACh response was
inhibited by only 7% after the oocyte was perfused with MTSET in the
presence of dTC and then inhibited by 75% after perfusion with MTSET
alone.
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T196C, MTSET was
used at 1 µM for that position. Consistent with lower rate constants
for modification of the other positions (Table 2), MTSET was tested at
10 µM for P197C, at 100 µM for D200C, and at 1 mM for
I201C. dTC protected against MTSET modification at T196C and
D200C but not at P197C and I201C. For the T196C receptor,
dTC protected by ~80% in the experiment shown in Fig. 6; in two
additional experiments, dTC protected T196C by ~60%. For the
D200C receptor in the experiment shown (Fig. 6) and in an additional
experiment, dTC protected by 80 to 90%. dTC did not protect the
P197C nAChR from modification: there was ~40% inhibition of the
ACh response after treatment with MTSET in the presence or absence of
dTC. Similarily, dTC did not protect the I201C nAChR from
modification. Exposure to 1 mM MTSET for 5 s in the presence of
dTC caused ~20% inhibition of the subsequent I201C ACh response,
whereas application of 1 mM MTSET alone caused only a further 10%
inhibition of the ACh response. Because there was more inhibition after
exposure to MTSET in the presence of dTC than in its absence, we
conclude that dTC afforded no protection at I201C. Thus, within
segment C, dTC binding protected against alkylation at T196C,
Y198C (previously shown), and D200C, but not at P197C or
I201C.
A Homology Model of the T. californica nAChR Binding
Site.
The studies described above were carried out before the
publication of the structure of the molluscan AChBP (Brejc et al., 2001). To facilitate discussion of our results, we developed a model of
the T. californica nAChR - binding site based upon the
structure of the AChBP (Fig. 7). The
amino acids identified by affinity labeling and mutagenesis as
contributors to the ACh binding site of the nAChR are located at each
subunit interface, with amino acids of segments A, B, and C contributed
from one subunit and amino acids from segments D, E, and F from the
other. Secondary structure elements are identified by the ribbon
representation and key binding site side chains depicted in ball and
stick representation. After energy minimization of the AChBP model
backbone containing the primary sequences of the extracellular regions
of the T. californica nAChR subunits, no significant
movement was noted for the structures containing the amino acids of
binding site segments A-E. As discussed in "Materials and Methods",
the size of the insertions in the segment F region of the - or
-subunit prohibits any confident prediction of the position of those
insertions, and we do not depict amino acids from this region in our
model of the binding site. With that caveat, the most prominent
structure changes between the ACh binding site of the AChBP and the
model of the T. californica nAChR binding site were due to
single amino acid substitutions, primarily within segment E.
|
Tyr-93, Trp-149, Tyr-190, Tyr-198, and Trp-55. Tyr-93 and Trp-149 are positioned in segments immediately after -strands (AChBP 4 and 7, respectively). Tyr-190 and Tyr-198 are on the same side of antiparallel -strands (AChBP 9 and 10) with the turn formed by the Cys-192/193 disulfide, which also contributes to the top of the binding pocket. The side chains of the amino acids
identified from -subunit segments E (Leu-109, Tyr-111, Tyr-117, and Leu-119) and D (Trp-55, Glu-57) are on a
common surface of three adjacent -strands (AChBP 5', 6, 2)
which form a sheet extending to 1 that includes Lys-34, an
affinity determinant for agonists (Prince and Sine, 1996) and for
-conotoxin M1, a peptide antagonist (Sine et al., 1995; Bren
and Sine, 2000). As predicted from the results of affinity labeling and
mutagenesis (Chiara et al., 1999; LeNovere et al., 1999), the segment E
amino acids are on antiparallel strands with a three-amino-acid
turn centered on Asp-113. Whereas Trp-55 contributes to the base of the pocket, the side chains of Leu-109, Tyr-111, and
Tyr-117 form part of the entrance to the pocket. Leu-119, a
position identified by Cys mutagenesis of mouse nAChR as important for -bungarotoxin binding (Sine, 1997; Osaka et al., 2000), is the side
chain from segment E that projects closest to the aromatic side chains
forming the binding pocket, and the turn at Asp-113 is most distant
(~20 Å) from the aromatic pocket. While the positions of segment F
agonist/antagonist affinity determinants are not included in Fig. 7,
their distances from the center of the aromatic binding pocket are
described in Materials and Methods.
The subunit primary structure of the AChBP is most closely related to
the extracellular domain of nAChR -subunits; as a homopentamer, it
is more similar in structure to the 7 homopentameric neuronal nAChR
than to the muscle-type nAChR. It has yet to be determined whether the
AChBP undergoes conformational changes analogous to those seen with the
nAChR. Because the AChBP binds ACh with reasonably high affinity
(K = 4 µM) (Smit et al., 2001), it is plausible that its binding
site structure differs from that of the nAChR in the resting (closed
channel) state, which has low affinity for agonist, and is more similar
to that of the nAChR in either the desensitized or open channel states,
which bind ACh with high affinity.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we have determined the accessibility for
modification of individually substituted cysteines within segments C
(195-201) and E (106-113) and have further assessed their accessibility in the presence of the competitive antagonist dTC. We
previously identified Y93C, N94C, Y198C, and E57C as
accessible for modification (Sullivan and Cohen, 2000). Similar to the
results obtained for segments A and D, within segment E, only two
positions (N107C, L109C) were clearly identified by our assay as
accessible for modification. Within segment C, all of the positions
tested demonstrated accessibility for modification, except for
L199C. The pattern of protection by dTC identifies residues within
the binding site likely to be in close proximity to bound dTC, and the
selective protection of residues within segment C identifies the
surface of a -strand projecting into the dTC/agonist binding site.
With the availability of a model of the T. californica nAChR extracellular domain, based upon the AChBP structure, it becomes possible to evaluate our results to identify consistencies and differences between the model and the structure of the nAChR binding site in the absence of agonist.
Modification of Substituted Cysteines within Segments C and E.
Our studies in segment C complement previous studies in which
functional embryonic mouse Cys mutant nAChRs (183-197) were expressed on the cell surface and accessible for modification by a
thiol-specific biotin, with the exception of Y190C (Spura et al.,
2000). We found that ACh responses were readily quantified for each
mutant T. californica nAChR containing Cys substitutions within 195-201, and all residues within 195-201, with the
exception of L199C, were accessible for modification. For most of
these positions, introduction of either a primary amine (after MTSEA reaction) or quaternary amine (after MTSET or MBTA reaction) caused an
altered response to ACh rather than an irreversible inhibition of
binding. For the Y93C, E57C, and Y198C receptors, earlier results indicated that covalent modification prevented the binding of
ACh. Clearly, direct radioligand binding studies are required to
determine the equilibrium constants for ACh binding to the modified Cys
mutant nAChRs.
106-113 of segment E, ACh
responses were modified after MTSEA or MTSET reaction with N107C and
L109C. Reaction of the Y111C nAChR with any of the reagents had
no effect on ACh responses. This was surprising, because Tyr-111 is
photolabeled by [3H]dTC and is a dTC affinity
determinant (Chiara et al., 1999). However, substitution of Tyr-111
by arginine had no effect on ACh equilibrium binding affinity or on the
concentration dependence of channel activation, and it is quite likely
that MTSET (or MBTA) may have reacted with Y111C without altering
the ACh response.
dTC Protection and Binding Site Structure.
Because our
functional assay identified modification of substituted cysteines in
segments A (Y93C, N94C), C (196-201, except L199C), D
(E57C), and E (N107C, L109C), we wanted to determine how dTC
binding altered the accessibility of cysteines for modification. If
bound dTC sterically occluded access of MTSET to a binding site Cys,
the rate of reaction would be reduced in proportion to dTC occupancy.
Within segment C, dTC protected substituted cysteines from alkylation
at 196, 198, and 200 but it did not protect 197 or 201.
This dTC protection pattern is readily explained if this portion of
segment C were organized as a -strand with the side chains of
196, 198, and 200 on a common surface projecting toward the
dTC/ACh binding site, as is seen in the structure of the molluscan
AChBP (Brejc et al., 2001) and in the nAChR binding site model (Fig. 7, blue).
Y198C, E57C, and L109C from
modification but not Y93C or N94C is consistent with the results of [3H]dTC photolabeling, where there was no
detectable incorporation of [3H]dTC into
Tyr-93 but there was photoincorporation into Tyr-198, Trp-55,
and Tyr-111. In addition, substitutions at Tyr-93 did not alter
dTC affinity (Sine et al., 1994). However, within the AChBP and
T. californica nAChR homology model binding sites, Tyr-93 forms one of the walls deep within the binding pocket along with the
side chains from Tyr-190, Tyr-198, Trp-149, and Trp-55. Based upon that structure, we would expect dTC to protect Y93C from
modification (but not N94C, located beyond the pocket). There are
potential explanations why dTC did not protect Y93C from
modification. First, in the structure of the binding site based upon
the AChBP, inspection of the water accessible surface (Connolly
surface, not shown) indicates that there is also significant accessibility to the tyrosyl side chain from outside the pocket that
may provide alternative access for modification of Y93C as well as
N94C by MTSET. Alternatively, the orientation of segment C in the
nAChR binding site may be displaced from that in the model, because
Cys-192/193 in the model would actually prevent dTC access to
Tyr-190 or Trp-55 within the pocket. dTC also binds to the AChBP
with high affinity (Smit et al., 2001), and it was noted that for dTC
to have access to the aromatic pocket, the binding site would have to
open up, perhaps by a movement of the -hairpin 9-10 that
contains segment C residues (Brejc et al., 2001).
Although our results demonstrate clearly that D200C is accessible
for modification, Asp-200 in our model and the equivalent amino acid
in the AChBP are actually in an interior position with very little
surface accessibility. The rate constant for modification of D200C
by MTSET is only 1% of that of T196C or Y198C; for modification
by MBTA, however, kD200C is 10-fold higher than
kY198C. The rate constant for modification of
D200C by MBTA is 50-fold higher than kD200C
for MTSET or MTSEA. It is possible that the structure of the AChBP
predicts accurately the accessibility of Asp-200 in the nAChR and
that the accessibility we see for D200C results from a structural
perturbation caused by the substitution. However, for D200C the
Kapp for ACh is similar to wild-type, and we think it is more likely that the reactivity observed for D200C is evidence that the accessibility of Asp-200 is quite different than predicted from the AChBP structure and may vary in a
ligand-dependent manner. Our results suggest that, in the absence of
agonist, the aromatic pocket of the nAChR binding site has a more
relaxed structure such that D200C is accessible. This would happen
if, for example, the antiparallel -strands containing the segment C
amino acids are further from the other elements of the binding pocket.
Previous studies have provided evidence for ligand-dependent changes in
the structure of the binding site. The Cys-192/193 disulfide in
native T. californica nAChR was readily reduced by
dithiothreitol in the absence of ligands or in the presence of dTC or
other antagonists, but in the presence of Ach or other full agonists,
the rate constant for reduction was reduced 100-fold (Damle and Karlin,
1980). Photolabeling patterns for
[3H]p-(N,N-dimethyl)aminobenzenediazonium
in the resting and desensitized states of the nAChR provided evidence
that in the two conformations the positions of Tyr-93 and Trp-149
must differ relative to the segment C residues (Galzi et al., 1991). In
addition, there is evidence that Asp-200 is involved in an agonist
dependent change in structure. Analysis of the functional consequences
of the D200N substitution in mouse nAChRs indicates that the
substitution can affect the rate constant for channel opening (Akk et
al., 1996) as well as the equilibrium between the resting and
desensitized states (Osaka et al., 1998).
Agonist Activation.
Early studies with native nAChRs
established that bromoacetylcholine acted as a covalent agonist
after reduction of the Cys-192/193 disulfide (Damle and Karlin,
1978; Chabala and Lester, 1986). We established previously that
covalent modification of Y198C with MTSET, MTSPT, or
bromoacetylcholine resulted in the addition of a covalent agonist
(Sullivan and Cohen, 2000). Reaction with either MTSET or MTSPT at
other segment C residues tested resulted only in inhibition of ACh
responses. Thus, interactions of the tethered trimethylammonio at a
distance of ~7 Å from the Cys-SH of Y198C can result in receptor
activation, and this interaction is not accessible from other
substituted cysteines within 195-201. This conclusion assumes that
the naturally occurring disulfide bond at Cys192-193 formed in the
Y198C mutant and that there was not aberrant disulfide bond
formation leaving Cys192 or Cys193 as free sulfhydryls. On
studies with mouse nAChR Y198C, the maximal current response was
potentiated 2-fold after treatment with 1 mM dithiothreitol (McLaughlin
et al., 1995), raising the possibility of aberrant disulfide bond
formation that was relieved in a reducing environment. Serine
substitution at Y198 causes a shift of ~300-fold in agonist
binding affinity, similar to that seen with Cys substitution (Sine et
al., 1994); therefore, the ~100-fold increase in Y198C Kapp for ACh that we observed is not
itself an indicator of aberrant disulfide bond formation. Under our
experimental conditions, both wild-type and Y198C T. californica nAChRs showed a decrease (~50%) in current
response, tested at half-maximal ACh concentrations, after treatment
with 1 mM dithiothreitol (data not shown), thus providing no evidence
for unnatural disulfide bond formation in the Y198C T. californica mutant.
P194,
T196, L119). It will also be important to map the binding site
surface protected by agonists, as well as by competitive antagonists
smaller than dTC, and to determine the changes in accessibility and
protection patterns when the binding site structure is altered
allosterically by desensitizing noncompetitive antagonists.
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Footnotes |
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Received August 10, 2001; Accepted November 6, 2001
This research was supported in part by United States Public Health Service grant NS19522 (J.B.C.) and by a Muscular Dystrophy Association Research Development grant (D.S.).
Dr. Jonathan B. Cohen, Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115. E-mail: jonathan_cohen{at}hms.harvard.edu
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Abbreviations |
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nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; AChBP, acetylcholine binding protein of Lymnaea stagnalis; MTSET, ([2-(trimethylammonium)-ethyl]-methanethiosulfonate; dTC, d-tubocurarine; MTSEA, 2-aminoethylmethanethiosulfonate; MTSPT, [3-(trimethylammonium)propyl]-methanethiosulfonate; MBTA, 4-(N-maleimido)benzyltrimethylammonium.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-subunit.
Biochemistry
34:
12496-12500[CrossRef][Medline].-neurotoxin binding sites in acetylcholine receptor from Torpedo californica.
Biochemistry
17:
2039-2045[CrossRef][Medline].-subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine receptor binding site.
J Biol Chem
259:
11662-11665-conotoxin M1 selectivity.
Neuron
15:
205-211[CrossRef][Medline].This article has been cited by other articles:
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), 1 mM MTSEA (
), or 2 mM maleimide-PEO-biotin
(
). Bars represent the mean change in current ± S.D. from
experiments on at least three oocytes. Percentage change in current was
calculated as: [(Iafter MX / Ibefore 
)
current inhibition was determined as in Fig. 
.
