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Vol. 62, Issue 2, 406-414, August 2002
Instituto de Investigaciones Bioquímicas, Universidad Nacíonal del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas, Bahía Blanca, Argentina
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The nicotinic acetylcholine receptor (nAChR) is a pentamer of
homologous subunits with composition 
2
in adult
muscle. Each subunit contains four transmembrane domains (M1-M4).
Position 8' of the M3 domain is phenylalanine in all heteromeric subunits, whereas it is a hydrophobic nonaromatic residue in non-
subunits. Given this peculiar conservation pattern, we studied its
contribution to muscle nAChR activation by combining mutagenesis with
single-channel kinetic analysis. Construction of nAChRs carrying
different numbers of phenylalanine residues at 8' reveals that the mean
open time decreases as a function of the number of phenylalanine
residues. Thus, all subunits contribute through this position
independently and additively to the channel closing rate. The
impairment of channel opening increases when the number of
phenylalanine residues at 8' increases from two (wild-type nAChR) to
five. The gating equilibrium constant of the latter mutant nAChR is
13-fold lower than that of the wild-type nAChR. The replacement of
F8', L8', L8', and V8' by a series of hydrophobic amino
acids reveals that the structural bases of the observed kinetic effects
are nonequivalent among subunits. In the subunit, hydrophobic amino acids at 8' lead to prolonged channel lifetimes, whereas they lead
either to normal kinetics ( and subunits) or impaired channel
gating ( subunit) in the non- subunits. The overall results
indicate that 8' positions of the M3 domains of all subunits contribute
to channel gating.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
nicotinic acetylcholine receptor (nAChR) is a pentamer of homologous
subunits. The primordial nAChR pentamer presumably contained only one
type of subunit, exemplified by the 7 homopentamer found in the
brain. However, evolution led to subunit diversity, resulting in a wide
spectrum of structurally and functionally different nAChRs (Le Novere
and Changeux, 1995
). nAChRs at the motor synapse are heteropentamers
with a subunit composition of 2 in
adult muscle. All subunits are characterized by the presence of a
pair of adjacent cysteines in the N-terminal domain. Non- subunits
(lacking the pair of cysteines) have derived from subunits (Ortells
and Lunt, 1995). Sequence comparison of subunits reveals several
candidate residues in transmembrane domains differentially conserved
between the two types of subunits (heteromeric and non-
subunits). In addition, many of these residues are also conserved
between non- subunits and homomeric subunits. This peculiar
pattern of conservation led us to believe it could represent structures
important for proper function. We therefore examined contributions to
channel activation of residues in the M3 transmembrane domain of the
heteromeric muscle nAChR, which are conserved among homomeric and
non- subunits but differ in the heteromeric subunits.
Each nAChR subunit contains an amino-terminal extracellular domain of
approximately 210 amino acids, four transmembrane domains (M1-M4) and a
short extracellular carboxy-terminal tail. The M2 domain of each
subunit contributes to the cation-selective channel, and agonist
binding triggers its twisting to allow ion flow (Unwin, 1995). The
locations, secondary structures, and functional roles of the M1, M3,
and M4 transmembrane domains are not as well understood. The pattern of
incorporation of the hydrophobic probe
3-trifluoromethyl-3-m-[125I]-iodophenyldiazirine
(TID) in Torpedo californica nAChR (Blanton and Cohen,
1994), NMR (Lugovskoy et al., 1998) and Fourier transform infrared
spectroscopy studies (Baenziger and Methot, 1995; Methot et al., 2001)
support a transmembrane organization of M3 in an -helix with some
contact with the lipid bilayer, whereas cryoelectron microscopic
studies suggest -sheet structures for M1, M3, and M4 (Unwin, 1993).
A few lines of experimental evidence indicate that M3 is a key
component of the nAChR channel gating apparatus: 1) by using 7/3
chimeric subunits, Campos-Caro et al. (1997) demonstrated that the M3
domain influences the gating of neuronal nAChRs; 2) a mutation at
position 9' of M3 of the subunit has been found in a patient
suffering from a congenital myasthenic syndrome (Wang et al., 1999).
Kinetic analysis of engineered mutant nAChRs revealed that this
position is essential for the opening and closing of the nAChR (Wang et
al., 1999); and 3) tryptophan substitutions at lipid-exposed residues
of the 
M3 transmembrane domain of T. californica nAChR
increased the macroscopic currents of the resulting nAChRs (Cruz-Martin
et al., 2001).
Position 8' of the M3 domain is phenylalanine in all heteromeric subunits but is a hydrophobic nonaromatic residue in homomer-forming subunits and non- subunits. Given this particular conservation pattern, we study its contribution to channel gating by combining site-directed mutagenesis and single-channel recordings. Our studies reveal that all subunits contribute independently and additively to the
closing step through this position. In addition, the
concentration-dependence of receptor activation is shifted toward
higher agonist concentrations as a function of the number of
phenylalanine residues at position 8' of M3.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of Mutant Subunits.
Mouse cDNAs were subcloned
into the cytomegalovirus-based expression vector pRBG4 (Sine, 1993).
Mutant subunits were constructed using the QuikChange site-directed
mutagenesis kit (Strategene, Inc., La Jolla, CA). Restriction mapping
and DNA sequencing confirmed all constructs.
Expression of nAChR.
HEK293 cells were transfected with ,
, , and cDNA subunits (wild-type or mutants) using calcium
phosphate precipitation at a subunit ratio of 2:1:1:1 for
///, respectively, essentially as described previously
(Bouzat et al., 1994, 1998). All subunits contained a valine at
position 433 of M4 according to the GenBank database (Salamone et al.,
1999). For transfections, cells at 40 to 50% confluence were incubated
for 8 to 12 h at 37°C with the calcium phosphate precipitate
containing the cDNAs in DMEM plus 10% fetal bovine serum. Cells were
used for single-channel measurements 1 or 2 days after transfection.
Patch-Clamp Recordings.
Recordings were obtained in the
cell-attached configuration (Hamill et al., 1981) at a membrane
potential of 
70 mV and at 20°C. The bath and pipette solutions
contained 142 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2,
1.7 mM MgCl2 and 10 mM HEPES, pH 7.4. Patch pipettes were pulled from 7052 capillary tubes (Garner Glass, Claremont, CA) and coated with Sylgard (Dow Corning, Midland, MI).
Pipette resistance ranged from 5 to 7 M
. Acetylcholine (ACh) at
final concentrations of 1 to 1000 µM or choline at 100 µM or 20 mM
was added to the pipette solution.
70 mV and analyzed at a bandwidth of 5 kHz to avoid detection of some
blockages that could be resolved at 10 kHz. In this manner, channel
kinetics can be reduced to those of the closed-to-open reaction
(Grosman and Auerbach, 2000; Bouzat et al., 2002).
Open- and closed-time histograms were plotted using a logarithmic
abscissa and a square root ordinate (Sigworth and Sine, 1987) and
fitted to the sum of exponential functions by maximum likelihood using
the program TACFit (Bruxton Corporation). Open probability within
clusters (Popen) was determined experimentally at
each ACh concentration by calculating the mean fraction of time that
the channel is open within a cluster.
Kinetic Analysis.
Kinetic analysis was performed as
described previously (Wang et al., 1997; Bouzat et al., 2000, 2002).
The analysis was restricted to clusters of channel openings, each
reflecting the activity of a single nAChR. Clusters of openings
corresponding to a single channel were identified as a series of
closely spaced events preceded and followed by closed intervals longer
than a critical duration (
crit). This duration
was taken as the point of intersection of the predominant closed
component and the succeeding one in the closed-time histogram. The
predominant closed duration component became shorter with the increase
of agonist concentration. Consequently, we assume that this component
reflects the set of transitions between unliganded closed and
diliganded open states. To minimize errors in assigning cluster
boundaries, we analyzed only recordings from patches with low channel
activity in which both components are clearly differentiated from one
another. Because each cluster contains one more opening than closing,
to avoid biasing in favor of openings, only clusters containing more
than 10 openings were considered for further analysis. In addition,
clusters showing double openings were rejected.
; Bouzat et
al., 2000, 2002). Typically, the distributions contained an
approximately Gaussian dominant component and minor contributions of
clusters with different properties. Clusters showing open time, closed
time, and open probability values within 2 S.D. of the mean of the
major component were selected. Typically, more than 80% of the
clusters were selected and only the non-Gaussian appendages were
omitted (Bouzat et al., 2002). Mean values obtained from distributions
of open probability, mean open channel duration, and mean closed
channel duration of clusters do not change significantly after the
selection procedure (Bouzat et al., 2002). In addition, comparison of
closed- and open-time histograms before and after selection indicates
that the selected clusters are representative of the predominant
population of nAChR in the patch (Bouzat et al., 2000).
The resulting open and closed intervals from single patches at several
ACh concentrations were analyzed according to kinetic schemes using the
program MIL (QuB suite, State University of New York, Buffalo).
Briefly, the program allows simultaneous fitting of recordings at
different agonist concentrations and estimates the rate constants using
a maximum likelihood method that corrects for missed events (Qin et
al., 1996). The dead time was typically 30 µs. Probability density
functions of open and closed durations were calculated from the fitted
rate constants and instrumentation dead time and superimposed on the
experimental dwell-time histogram as described by Qin et al. (1996).
Calculated rates were accepted only if the resulting probability
density functions correctly fit the experimental open- and
closed-duration histograms.
For wild-type and some mutant nAChRs activated by Ach, the opening rate
of the diliganded nAChR, 2 in Scheme 1, was
constrained to its previously determined value (Sine et al., 1995; Wang
et al., 1997; Salamone et al., 1999) because brief closings caused by
gating and channel blocking become indistinguishable at high ACh
concentrations (Wang et al., 1997; Salamone et al., 1999; Bouzat et
al., 2000). When 2 was allowed to vary freely,
MIL failed to converge to a well-defined set of rate constants and approached a value of about 100,000/s (Salamone et al., 1999; Bouzat et
al., 2000). Also, association and dissociation rate constants were
assumed to be equal at both binding sites (Akk and Auerbach, 1996; Wang
et al., 1997; Salamone et al., 1999). Rate constants are shown with
S.D.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Additivity of the Effects of Mutations at Position 8' of M3.
Position 8' of the M3 domain is phenylalanine (F) in all heteromeric
subunits but is a hydrophobic nonaromatic residue in homomer-forming subunits and non- subunits (Fig.
1). Replacement of F8' in 1 by its
homologous in the 7 subunit, isoleucine, increases the mean open
time of the resulting nAChRs (Fig. 2). On
the contrary, the reverse mutations at , and subunits decrease the mean open time. At 1 µM ACh, open-time distributions of
wild-type nAChRs show a major component of about 900 µs with a
relative amplitude larger than 0.8 in all recordings (Table 1). nAChRs containing the mutant F8'I
subunits form channels characterized by increased mean open times
whereas open durations of nAChR channels containing the mutant L8'F,
V8'F, or L8'F subunits are significantly briefer than those of
wild-type (Table 1).
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F8' I nAChR is also
observed when channels are activated by choline instead of acetylcholine. Mean open times of channels recorded in the presence of
100 µM choline were 190 ± 10 µs and 540 ± 30 µs for
wild-type and F8' I nAChRs, respectively.
Given that the introduction of phenylalanine residues to 8' of non-
subunits decreases the measured duration of apparent openings and the
removal of phenylalanine from the subunit produces the opposite
effect, we investigated the relationship between the number of F
residues at 8' and the channel mean open time. We combined wild-type
and mutant subunits to construct single nAChRs containing a variable
number of F residues, ranging from 0 (F8'I) to 5. As shown in Fig.
3, the mean open time decreases systematically with the increase in the number of F residues at 8'.
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subunit seem to be slightly briefer, indicating
a not very significant asymmetry in the contribution of the different subunits to the duration of the open state (Fig. 4 and Table 1).
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).
For both wild-type and mutant nAChRs, histograms of closed intervals
show a predominant component that becomes progressively briefer with
increasing ACh concentration. This component reflects the set of
transitions between unliganded closed and diliganded open states. Table
2 shows the mean durations of the
predominant closed component for wild-type and mutant nAChRs activated
by 30 µM ACh. As shown in Table 2, this time increases with the
number of phenylalanine residues present at position 8'. Statistically,
the duration of this component is significantly longer in the F = 5 nAChR than in the control (F = 2) (p < 0.005, Student's t test). Prolonged closed times could be caused
by changes in rate constants underlying either ACh binding or channel
gating steps.
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Kinetic Changes Caused by the Presence of Phenylalanine Residues at Position 8' of M3. To identify unequivocally the kinetic steps affected by the presence of phenylalanine residues at position 8', and to quantify the kinetic changes, we fitted kinetic schemes to the open- and closed-dwell time histograms of mutant nAChRs.
nAChR channels were activated by a range of desensitizing concentrations of ACh (10 to 300 µM) to produce clear clusters of events corresponding to a single channel (Sakmann et al., 1980). Because each cluster of channel openings reflects the activity of a
single nAChR, closed and open times of the selected clusters were used
for the kinetic analysis.
For wild-type nAChRs, we used the classical activation scheme shown in
Scheme 1, where two agonists (A) bind to
the receptor (R) in the resting state with association rates
k+1 and k+2, and dissociate
with rates k
1 and k2.
Receptors occupied by one agonist open with rate
1 and close with rate
1, and nAChRs occupied by two agonist
molecules open with rate 2 and close with rate
2. At high agonist concentrations (higher than
100 µM ACh), channel blockade is evident; thus, the blocked state, A2B, is included in the scheme. Omitting blockade
from Scheme 1 does not affect significantly the calculated rate
constants (Salamone et al., 1999; data not shown). Because some of the
blockages at 300 µM ACh are resolved under the present conditions,
the incorporation of the channel blockade to the model improves the
fitting of the histograms corresponding to high agonist concentrations
(data not shown).
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). We simultaneously analyzed
recordings obtained at multiple ACh concentrations (10-300 µM) with
the aim of representing all the states in Scheme 1 in the analysis.
Rate constant estimates obtained for wild-type nAChR (F = 2 in
Table 3) agree with those previously
reported for mouse nAChR (Akk and Auerbach, 1996; Wang et al., 1997;
Salamone et al., 1999; Bouzat et al., 2000).
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F8'I subunit,
lacking F residues at 8' (F = 0), shows that the closing rate decreases in this mutant nAChR (Table 3). This result agrees with the
observation that in this mutant nAChR the duration of the open state
increases. As described above for wild-type, 2 had to be constrained to its known value to allow the fitting. As a
consequence, if the opening rate changed, it was not possible to detect
it. In this respect, when the fixed value of 2
was systematically reduced from 50,000 s1 to
30,000 s1, the best description based on
likelihood was obtained with the highest value of
2. The fit using 2 = 50,000 s1 was e2,
e5, and e15 times more
likely than with 2 equal to 45,000, 40,000, and 30,000 s1, respectively.
To determine whether the rate of channel opening increased in nAChRs
lacking F residues at 8', we used saturating concentrations of choline,
an agonist with a very slow opening rate (Grosman and Auerbach, 2000).
Recordings of F8'I nAChRs activated by 20 mM choline appear, as for
wild-type, in clusters of channel events showing a 50% reduction in
channel amplitude due to open-channel block (2.8 ± 0.3 pA instead
of about 5.5 pA for channels activated by ACh or choline at
concentrations lower than 100 µM at 70 mV (see Grosman and
Auerbach, 2000; Bouzat et al., 2002; Fig.
6). For both wild-type and mutant
choline-activated nAChRs, open-time histograms are well described by a
single component and closed-time histograms show a main component that
corresponds to closings within clusters (Fig. 6). Given that 20 mM
choline is a saturating agonist concentration (Grosman and Auerbach,
2000), we fitted dwell times from the selected clusters to a kinetic
scheme containing only one open and one closed state. Under these
conditions, the estimates for opening and closing rate constants are:
wild-type: 90 ± 20 s1 and 1732 ± 214 s1 for and , respectively (mean ± S.D. of seven different patches); F8'I, 60 ± 20 s1 and 893 ± 230 s1 for and , respectively (mean ± S.D. of four different recordings). Thus, the resulting rate constant
estimates indicate that F8'I does not affect significantly the rate
of channel opening (p > 0.1, Student's t
test) and confirm the decrease in the rate of channel closing
(p < 0.01).
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F8' I nAChRs decrease about 50% in the presence of 20 mM choline, we estimate KB
(kb/k+b) to be
approximately 20 mM for both nAChRs in agreement with Grosman and
Auerbach (2000). Considering the classical blocking scheme (C 
O OB), the mean duration of apparent openings at 20 mM choline would be
two-fold longer than that in the absence of blockade (Grosman and
Auerbach, 2000). Accordingly, the mean duration of apparent openings of channels activated by 20 mM choline are approximately two-fold longer
than that of channels activated by 100 µM choline for both wild-type
and F8' I nAChRs. Therefore, the closing rate-constant estimates for
choline may be underestimated by a factor of ~2 as reported
previously (Grosman and Auerbach, 2000).
Changes in Free Energy.
The channel gating equilibrium
constant for ACh-activated receptors, 
, calculated as
2/2, decreases as a
function of the number of F residues at position 8' of M3. This
constant decreases from 33 in the wild-type nAChR (F = 2) to 2.4 in the F = 5 mutant nAChR. This decrease corresponds to a free
energy change of 1.5 kcal/mol. nAChRs containing an intermediate number
of F residues between two and five show intermediate values of .
Calculated values of for nAChRs containing three and four F
residues are: 8 for F = 3 (mutant subunit); 10 and 6.9 for
F = 4 (mutant and subunits and mutant and subunits, respectively). In contrast, increases approximately
two-fold in the F = 0 mutant nAChR containing the F8'I mutant
subunit ( = 75).
Probability of Channel Opening in 8' M3 Mutants.
To determine
the overall consequences for receptor activation of mutations at 8', we
determined the open probability as a function of ACh concentration. For
wild-type nAChRs, the open probability increases with increasing ACh
concentration, showing an EC50 of about 40 µM.
The curve shifts progressively to the right as the number of F residues
increases. The profile for the F = 5 mutant nAChR shift to higher
ACh concentrations, increasing the EC50 to 110 µM and lowering the maximum Popen to
approximately 0.6 at 1 mM ACh (Fig. 7).
The EC50 and maximum open probability values for
mutant nAChRs are 57 µM and 0.88 for F = 3 (mutant subunit),
100 µM and 1.0 for F = 4 (mutant and subunits), and 100 µM and 0.92 for F = 4 (mutant and subunits). On the contrary, the profile for nAChRs lacking F residues at 8' (F = 0)
is slightly displaced to the left (EC50 = 32 µM
and maximum Popen 0.98).
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Structural Bases of Kinetic Effects of Mutations at Position 8' of
M3.
Because the mutations L8'F, L8'F, and V8'F impair
channel gating and the F8'I produces the opposite effect, we further investigated whether these kinetic changes are specific to these mutations or if they respond to a more general hydrophobic nonaromatic versus aromatic change. We engineered a series of hydrophobic side
chains at 8', recorded single-channel currents elicited by 30 µM ACh,
and analyzed the kinetic properties of the nAChR clusters. We chose a
concentration of 30 µM because it is close to the
EC50 for the adult muscle nAChR and therefore it
is sensitive to changes in activation parameters. For each cluster
within a recording, we calculated the Popen, mean
open duration, and mean closed duration; plotted their distributions;
and determined the mean values. Figure 8
shows the mean values of these parameters for the different mutant
subunits.
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subunit, substitution of F8' by the hydrophobic amino acids
alanine, leucine, and valine increases the mean open duration by about
2- (F8'A and F8'L) and 2.5-fold (F8'V). Thus, hydrophobic
amino acids similarly increase the duration of the open state. nAChRs
containing the F8'L subunit are almost identical to those containing
F8'I (Fig. 8), indicating that channel gating is not influenced by
the stereochemistry of the side chain. The mutations F8'A and
F8'V produce, in addition to the increased open time, an increase in
the duration of the closed component associated to the set of
transitions between unliganded closed and diliganded open states, such
increase leading to a slight decrease in the
Popen with respect to that observed with L and I
(Fig. 8). There seems not to be a straightforward explanation for the
increase in the closed time. In terms of hydrophobicity, alanine is
less hydrophobic than isoleucine whereas valine is similar to
isoleucine (Kyte and Doolittle, 1982) and the helical propensity in a
nonpolar environment of valine is very similar to that of leucine (Liu
and Deber, 1998). Alanine and valine have smaller volumes than
isoleucine and leucine. However, although valine has an intermediate
size between alanine and leucine, it produces a more significant
increase in the duration of the closed time than alanine does.
Preserving the aromatic group as in the F8'Y mutation leads to
wild-type kinetics (Fig. 8). However, some recordings of this mutant
nAChR revealed some clusters of prolonged openings, accounting for up
to 30% of the total clusters. The mean values of
Popen, mean open duration and closed duration of
these clusters for three different recordings were: 0.69 ± 0.02, 2.80 ± 0.30 ms, and 1.19 ± 0.10 ms, respectively.
Heterogeneous kinetics have been reported before in wild-type nAChRs
(Naranjo and Brehm, 1993) and the number and range of kinetic modes
have been shown to increase in mutant nAChRs (Milone et al., 1998; Wang
et al., 2000). We thoroughly examined clusters and detected switches
from one mode to the other in the same cluster. For example, for one
recording containing 272 clusters, 55% of the clusters corresponded to
wild-type nAChRs, 31% showed prolonged openings, and 14% showed
switches from wild-type to prolonged channels during the course of a
single cluster.
All substitutions of L8' similarly impair channel gating. As can be
seen in Fig. 8, kinetic changes are observed with changes in
hydrophobicity, aromaticity, size, or stereochemistry of the side
chain. Thus, it seems likely that the subunit strictly requires
leucine to ensure appropriate gating. Replacement of L8' by other
hydrophobic nonaromatic amino acids in the subunit does not affect
kinetics. Therefore, this subunit seems to be permissive to different
hydrophobic amino acids at 8' but not to hydrophobic aromatic amino
acids. After the mutations tested in the subunit, it seems that
this subunit behaves similarly to the subunit given that kinetic
changes occur in the V8'F although they do not occur in the V8'A.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The nAChR is the neurotransmitter-gated ion channel responsible
for the rapid propagation of electrical signals at the neuromuscular junction. Although this nAChR is by far the best characterized of the
ligand-gated ion channels, the detailed structural mechanisms underlying the rapid depolarization are not yet fully understood. The
four transmembrane domains are likely to be involved in the conformational changes associated with gating. According to few lines
of experimental evidence showing the contribution of M3 to channel
gating [Campos-Caro et al. (1997), Wang et al. (1999), Cruz-Martin et
al. (2001)], the present study demonstrates that the M3 segments of
all muscle subunits influence both opening and closing steps and
reveals the mechanistic and structural bases underlying the
contribution to function of position 8'.
Each subunit of the nAChR provides special structural features that,
when combined in the whole nAChR, have a functional significance. However, how these specific features were conserved through evolution remains unknown. Position 8' of the M3 domain is phenylalanine in all
heteromeric subunits, whereas it is a hydrophobic nonaromatic residue in non- subunits and homomer-forming subunits.
Therefore, the present work shows that this conservation pattern marks
residues that contribute to channel gating. However, why the residues
were conserved through evolution in such a way remains intriguing given that the structure-function relationships are not conserved among the
different subunits.
The present kinetic analysis indicates that mutations at position 8' of
all subunits affect mainly the gating of the nAChR. The diliganded
gating equilibrium constant (), calculated as 2/2, decreases
13-fold in the F = 5 nAChR with respect to that of wild-type. Such
a decrease corresponds to a change in free energy of the gating
equilibrium of 1.5 kcal/mol.
A relationship between the number of phenylalanine residues and the
duration of the open state is clearly observed (see Fig. 4). Increasing
the number of phenylalanine residues at 8' of M3 leads to progressively
larger decreases in the duration of the open state. Although the
mutation in the subunit seems to have a slightly increased effect
with respect to mutations at other subunits, the change in the mean
open time depends mainly on the final number of phenylalanine residues.
Thus, the additive contributions of all subunits to the closing rate
indicate that the M3 domains of , , , and subunits
contribute independently to channel closing through position 8'.
Residues at homologous positions in all subunits do not necessarily
share a common functional role and, if they do, their functional
contributions may or may not be additive. For example, symmetrical and
independent contributions to channel gating have been described before
for position 9' of the M2 segment (Filatov and White, 1995; Labarca et
al., 1995). Residues at position 12' of M2 contribute additively but
asymmetrically to channel gating (Grosman and Auerbach, 2000). In
contrast, some residues in M1 (Wang et al., 1997), M3 (Wang et al.,
1999), and M4 (Bouzat et al., 2002) show subunit-specific contributions
to channel gating.
The presence of five phenylalanine residues at 8' of M3 (F = 5)
significantly impairs channel opening. After the relationship observed
between the number of F residues and the closing rate, we expected that
the nAChR lacking F residues at 8' (F = 0) showed an increased
opening rate. However, we were unable to detect such an increase.
Because the opening rate constant of wild-type nAChRs (2 in Scheme 1) is at the upper limit of
reliable estimation, even a modest increase makes this parameter too
fast to be resolved (Grosman and Auerbach, 2000). Therefore, we
determine the opening rate of F = 0 nAChR channels activated by
saturating concentrations of a slowly opening, low-efficacious agonist
as choline. At a saturating concentration of choline, the kinetics of
the channel can be reduced to that of the closed-to-open reaction
(Grosman and Auerbach, 2000; Bouzat et al., 2002). Because the opening rate is slow in the choline-activated nAChRs, this rate constant can be
well measured; thus, an increase in such constant can be easily
detected. The calculated opening rate constant for F = 0 mutant
nAChR is similar to that of wild-type nAChR. Thus, in contrast to what
is observed for the closing rate, the opening rate is not affected by
reducing the number of phenylalanine residues with respect to that of
wild-type nAChRs (F = 2). However, increasing this number from two
to five progressively impairs the opening step in the gating pathway.
Mutagenesis studies reveal that the structural bases of the
contribution to gating of position 8' are different for the different subunits. Hydrophobic nonaromatic amino acids replacing F8' increase the duration of apparent openings. The subunit strictly requires leucine at 8' for appropriate gating: both briefer openings and prolonged intracluster closings are observed either with hydrophobic nonaromatic or aromatic amino acids. In contrast, the and subunits are permissive to different hydrophobic amino acids at 8' of
M3.
The kinetic effects of mutations at 9' position of M3 have been
described in detail by Wang et al. (1999). The gating equilibrium constant decreases 14-fold in nAChRs carrying the mutant V9'I subunit, about 3-fold in nAChRs carrying V9'I or A9'V subunits, 1.7-fold in the V9'I nAChRs, and 60-fold in nAChRs containing the
five mutant subunits. Thus, compared with the 13-fold decrease in the
gating equilibrium in the F = 5 nAChR here described, position 9'
has more profound effects on gating than position 8'. The structural bases of the functional contributions also differ between positions 8'
and 9'. Mutagenesis studies showed that both volume and stereochemistry at the side chain of residue 9' contribute to channel gating (Wang
et al., 1999). In contrast, at the 8' position kinetic changes depend
on the presence of an aromatic or hydrophobic nonaromatic amino acid in
the , , and probably subunits. However, the relationship
between gating and physicochemical properties of the residues at 8' is
complex for the subunit.
Abnormal activation of nAChR has been shown to underlie congenital
myasthenic syndromes (Engel et al., 1998). Moreover, mutation in the
residue at a position neighboring the one studied herein has been found
to be the cause of the attenuated postsynaptic response observed in the
patient (Wang et al., 1999). Consequently, if mutations naturally
occurred at the 8'position of M3 of any subunit, they could lead to a
congenital myasthenic syndrome, albeit one less severe than the one
reported by Wang et al. (1999).
The residues at 8' of M3 are located on the first third of M3. The
pattern of TID labeling of the M3 defines a strip of -helix in
contact with lipids (Blanton and Cohen, 1994). F284 of T. californica, which corresponds to position 8', has been labeled by
TID. In contrast, positions 8' of , , and subunits have not
been labeled by TID; they are thus presumably not exposed to the
lipids. Assuming that the disposition of residues in M3 is conserved
between mouse and T. californica, our results suggest that
this position influences gating independently of the packing orientation of the residues with respect to the lipid bilayer. It is
therefore possible that the presence of aromatic residues at positions
8' affects gating by changing their interactions with other residues of
the same or other subunits. In conclusion, our results contribute to
assigning a functional role to the M3 segment of all muscle nAChR
subunits as a component of the channel gating apparatus.
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Footnotes |
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Received January 7, 2002; Accepted April 30, 2002
This work was supported by grants from Ministerio de Salud de la Nación, Universidad Nacional del Sur, Agencia Nacional de Promoción Científica y Tecnológica Third World Academy of Sciences (to C.B.); and Fogarty International Research Collaboration grant 1R03-TW01185-01 [to Dr. Steven M. Sine (Mayo Foundation, Rochester, MN) and C.B.].
M.J.D.R and D.R. contributed equally to this work.
Address correspondence to: Dr. Cecilia Bouzat, Instituto de Investigaciones Bioquímicas, Camino La Carrindanga Km 7, B8000FWB Bahía Blanca, Argentina. E-mail: inbouzat{at}criba.edu.ar
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Abbreviations |
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nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; M3, third transmembrane domain; HEK, human embryonic kidney; Popen, channel open probability.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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subunit decreases the rate of agonist dissociation.
J Gen Physiol
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wt
,
F = 0, 
, F = 2, wild-type; 
, F = 3, 
, F = 4, 
, F = 4, 
, F = 5, 