From the * Receptor Biology Laboratory, We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine
receptor (AChR) The acetylcholine receptor (AChR)1 from vertebrate
skeletal muscle is a pentamer of homologous subunits
with compositions To understand the essential function of the AChR,
investigators have worked to establish structure-function relationships for the various structural domains.
Thus it is clear that agonist binds to two sites in the extracellular domain, that the M2 domains from each
subunit form the ion permeation pathway, and that
binding triggers twisting of the M2 domains from the
center to the perimeter of the channel to cause opening (Unwin, 1995 Table I.
Comparison of Sequences of M1 Domains
Muscle Research Laboratory, Department of
Neurology, Mayo Foundation, Rochester, Minnesota 55905; and § Department of Biophysical Sciences, State University of New York at
Buffalo, Buffalo, New York 14214
ABSTRACT
subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously
showed that receptors containing
N217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that
the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation
from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for
N217K. The apparent affinity of
ACh binding, measured by competition against the rate of 125I-
-bungarotoxin binding, is also enhanced 20-fold
by
N217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the
,
, or
subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The
results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation
that stabilizes agonist bound to the resting state of the AChR.
INTRODUCTION
2
in fetal or
2
in adult muscle (Mishina et al., 1986
). Each subunit contains an
amino-terminal extracellular domain of approximately 210 residues followed by four candidate transmembrane
domains (M1-M4). The five subunits are packed pseudosymmetrically to form a cylindrical structure containing two acetylcholine binding sites coupled to a central
ion channel. The binding sites are thought to lie approximately 30 Å above the plane of the membrane
(Unwin, 1993
; Valenzuela et al. 1994
), formed by extracellular domain portions of
,
, or
subunit pairs
(Blount and Merlie, 1989
, 1991
; Sine, 1993
), whereas
the transmembrane domains are thought to be encompassed within the membrane where the M2 domain forms the cation-selective channel (Unwin, 1993
, 1995
). The
essential function of the AChR is to transduce a local
perturbation caused by binding of agonist into movement of the remote M2 domains that opens the channel.
). On the other hand, the contribution of the M1 domain is not as well understood as that
of the M2 domain. In particular, the secondary structure of M1, its disposition relative to the membrane or
the binding sites, and its contribution to AChR function are not established. M1 is unique among transmembrane domains in that it is poised in the linear sequence between binding site residues in the extracellular domain and the M2 channel lining. It is readily
identified as a 26 residue hydrophobic segment flanked
by the palindromic sequences PLYF . . . FYLP (Table I).
Whether M1 is a
sheet or an
helix is not known, but
in either configuration its length is more than adequate to span the membrane. The presence of a conserved proline (P221) in the middle of M1 suggests a
discontinuous structure (Suchnya et al., 1993
), perhaps
dividing it into two types of structural and functional
domains. Labeling studies with a hydrophobic reagent
revealed accessible residues in the middle of M1 (C222, L223, F227, and L228), perhaps accessible through the
lipid bilayer (Blanton and Cohen, 1994
), whereas these
same residues (C222 and L223) mutated to cysteine are
not labeled by hydrophilic sulfhydryl reagents. On the
other hand, several residues in the amino-terminal third of M1 (between P211 and P221) are accessible to
hydrophilic sulfhydryl reagents when mutated to cysteine, and the pattern of accessibility suggests an unordered structure of this segment (Akabas and Karlin,
1995
). Thus, approximately the carboxyl-terminal two-thirds of M1 may be folded in the membrane while the
amino-terminal third may extend above it.
Recent insights into structure-function relationships
have come from mutations in human AChR that cause
congenital myasthenic syndromes (CMS). In the cases
described to date, the mutant residue is conserved across
species and in some cases across all members of the superfamily, and pathogenicity can be traced to a change in a specific step in receptor activation (Ohno et al.,
1995; Sine et al., 1995
; Ohno et al., 1996
). We recently
described a mutation in the synaptic third of the M1
domain of the
subunit (
N217K) that causes a CMS
(Engel et al., 1996
).
N217 is conserved across all species and subtypes of
subunits and is present in the
equivalent position in the
,
, and
subunits (Table
I). When expressed in 293 HEK cells, receptors containing
N217K activate in prolonged episodes strikingly similar to those observed at the CMS end plates.
Here we use single channel recording to examine the
kinetics of activation of AChRs containing
N217K. We
show that the prolonged activation episodes are due
primarily to slowing of the rate of ACh dissociation from
the binding site. The results reconfirm the importance
of agonist binding affinity in governing the duration of
the synaptic response and show that the M1 domain contributes to binding site affinity.
Construction of Mutant AChR cDNAs and Expression in 293 HEK Cells
Mouse AChR subunit cDNAs were generously provided by Drs.
John Merlie, Norman Davidson (,
, and
subunits; referenced
in Sine, 1993
), and Paul Gardner (
subunit, Gardner, 1990
) and
were subcloned into the CMV-based expression vector pRBG4
(Lee et al. 1991
) for expression in 293 HEK cells. Mutations were
constructed either by bridging restriction sites with synthetic
double-stranded oligonucleotides or by overlap PCR. For the
N217K,
N217Q, and
N217E mutations a 31-bp oligonucleotide bridged from a HincII to a SapI site. For
N217K, a 43-bp
oligonucleotide bridged from a HincII to a BbsI site. For
N217K,
a 130-bp fragment harboring the mutation was constructed by
overlap PCR and ligated between MspI and NheI sites. For
N217K, a 230-bp fragment harboring the mutation was constructed by overlap PCR and ligated between Bst1107I and KpnI
sites. The presence of each mutation and the absence of unwanted mutations was confirmed by dideoxy sequencing. Human embryonic kidney fibroblast cells (293 HEK) were transfected
with mutant or wild-type AChR subunit cDNAs using calcium
phosphate precipitation as described (Bouzat et al., 1994
).
Patch-clamp Recordings from AChRs Expressed in HEK Cells
Recordings were obtained in the cell-attached configuration
(Hamill et al., 1981) at a membrane potential of
70 mV and a temperature of 22°C (Bouzat et al., 1994
). Bath and pipette solutions contained (mM): KCl 142, NaCl 5.4, CaCl2 1.8, MgCl2 1.7, HEPES 10, pH 7.4. Single channel currents were recorded using
an Axopatch 200A at a bandwidth of 50 kHz, digitized with a
PCM adapter at 94 kHz (VR-10B; Instrutech Corp., Great Neck,
NY), transferred to a Macintosh computer using the program Acquire (Instrutech Corp.), and detected by the half-amplitude
threshold criterion using the program MacTac (Instrutech Corp.)
at a final bandwidth of 9 kHz. Data acquisition typically commenced within 1 min of seal formation. Open and closed duration histograms were constructed using a logarithmic abscissa
and square root ordinate (Sigworth and Sine, 1987
). For analysis
of currents recorded at limiting low ACh concentrations, the histograms were fitted by the sum of exponentials by maximum likelihood. The resulting time constants and relative areas were used
to calculate the rate constants
2,
2, and k
2 in SCHEME I (see RESULTS).
Currents elicited by intermediate and high ACh concentrations were analyzed to obtain estimates of all the rate constants in
SCHEMES I and II (see RESULTS). Clusters of events corresponding to a single channel were identified as a series of closely spaced openings preceded and followed by closings longer than a specified duration; this duration was taken as the point of intersection of the predominant closed time component in the histogram and the succeeding closed time component. A uniform filter bandwidth of 9 kHz and a dead time of 22 µs were imposed for all recordings. For each recording, kinetic homogeneity was determined by computing the mean open duration (o) and open
probability (Popen) of each cluster and plotting their distributions
for visual inspection (Sine and Steinbach, 1987
; Auerbach and
Lingle, 1987
). Typically the distributions contained a dominant,
approximately Gaussian component, as well as minor contributions of clusters with very different properties. Clusters of events
belonging to the dominant component were systematically selected for further analysis by accepting clusters with
o and Popen
within two standard deviations of the mean of the major component. The selection process typically retained >90% of the original clusters for further analysis.
The resulting open and closed intervals, from single patches at
several ACh concentrations, were transferred to an IBM RS6000 computer, and analyzed according to either SCHEME I or II using an interval-based maximum likelihood method that incorporated corrections for missed events (Qin et al., 1996). Briefly, the method
computes the likelihood, or joint probability of obtaining the experimental series of open and closed dwell times given the kinetic scheme, and maximizes the likelihood by optimizing parameters in the scheme (Ball and Sansom, 1989
). The likelihood
was maximized using a forward-backward recursive procedure to
calculate the likelihood function and its derivatives with respect
to model parameters (Qin et al., 1997
) and an optimizer that
combined calculation of the approximate inverse Hessian matrix
of second derivatives of the likelihood function and an exact line
search with adaptive step sizes (Fletcher, 1981
). After fitting, standard errors of the rate constants were determined from the curvature of the likelihood function at its maximum; these were obtained as the diagonal elements of the approximate inverse Hessian matrix generated by the optimizer. Standard errors calculated
in this manner assume a quadratic form of the likelihood function near its maximum, and correspond to standard errors determined by the half-likelihood-interval method (Colquhoun and
Sigworth, 1995
).
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 histograms as described by Qin et al. (1996). To check the
final set of rate constants, open and closed intervals were simulated according to SCHEME I, the fitted rate constants and dead
time (Clay and DeFelice, 1983
), binned into histograms and compared with the theoretical probability density functions.
For wild-type, N217Q , and
N217E receptors, recordings included in the analysis were obtained at the following ACh concentrations (µM): 10, 20, 30, 50, 100, 200, and 300. For the
N217K
receptor, recordings for analysis were obtained at the following
ACh concentrations (µM): 0.3, 1.0, 2, 3, 5, 10, 20, 30, 100, and
300. For each ACh concentration, the number of kinetically homogeneous clusters ranged from 17 to 62, and the corresponding numbers of events ranged from 1,400 to 4,000.
ACh Binding Measurements
3 d after transfection, intact HEK cells were harvested by gentle
agitation in PBS plus 5 mM EDTA. The esterase inhibitor diisopropylphosphofluoridate (1 µM) was added to the PBS/EDTA solution, and the cells were incubated for 15 min. Cells were briefly centrifuged, resuspended in high potassium Ringer's solution (140 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2, 1.7 mM MgCl2,
25 mM HEPES, 30 mg/liter BSA, adjusted to pH 7.4 with 10-11
mM NaOH), and divided into aliquots for measurements of ACh
binding. Specified concentrations of ACh were added 30 min before addition of 125I-labeled -bungarotoxin (5 nM), which was
allowed to bind for 30 min to occupy approximately half of the
surface receptors. The binding reaction was stopped by adding
potassium Ringer's solution containing 300 µM d-tubocurarine,
followed by filtration using a cell harvester (Brandel Inc.). Radioactivity retained by the glass fiber filters (GF-B, 1 µm cutoff;
Whatman Inc., Clifton, NJ) was measured with a gamma counter.
The initial rate of 125I-
-bungarotoxin binding was determined to
yield fractional occupancy of sites by ACh (Sine and Taylor,
1979
). Competition measurements were analyzed according to
the Hill equation.
![]() |
where Y is fractional occupancy by ACh and Kov is an overall dissociation constant.
To investigate the kinetic basis of the prolonged activation episodes of the N217K AChR, we recorded single
channel currents from 293 HEK cells transfected with
either wild-type or mutant
plus complementary
,
,
and
subunit cDNAs. Currents were elicited by a range
of desensitizing concentrations of ACh, as this allows
identification of clusters of events due to a single AChR
channel (Sakmann et al., 1980
). For the
N217K AChR,
openings appear in readily recognizable clusters at concentrations as low as 0.3 µM ACh, whereas for the wild
type AChR, clustering requires concentrations of at
least 3 µM. The channel traces show that closed intervals within clusters become more brief with increasing ACh concentrations and that they are more brief at a
given concentration for the
N217K compared to the
wild-type AChR (Fig. 1). Qualitatively, the briefer closed
intervals observed with
N217K indicate a change in
the rate of one or more of the following steps governing reopening of the channel: agonist association, agonist dissociation, or opening of the doubly occupied
channel.
Table II.
Kinetic Parameters for AChRs Containing Wild-type or Mutant |
Table III. Kinetic Parameters from Measurements at Low ACh Concentrations |
To identify kinetic steps affected by N217K, we analyzed the kinetics of channel opening and closing according to the following activation scheme:
Scheme I.
where two agonists (A) bind to the receptor in the resting state (R) with association rates k+1 and k+2 and dissociate with rates k1 and k
2. Receptors occupied by
one agonist open with rate
1 and close with rate
1,
while receptors occupied by two agonists open with
rate
2 and close with rate
2. To account for channel
block by high concentrations of ACh, we included the
blocked state A2RB with the blocking and unblocking rate constants k+b and k
b. To estimate the set of rate
constants, SCHEME I was fit to the data by computing
the likelihood of the experimental series of open and
closed times given a set of trial rate constants and changing the rate constants to maximize the likelihood; the
fitting analysis included dwell times obtained for the
entire range of ACh concentrations. The advantage of
fitting recordings obtained at multiple rather than single ACh concentrations is all the states in SCHEME I are
represented over a range of concentrations. In addition, for wild-type receptors,
2 was constrained to the
value obtained from measurements at limiting low ACh
concentrations described below. This constraint was
necessary because the wild-type receptor opens very
rapidly and is blocked at ACh concentrations similar to
its intrinsic affinity (Table II), so closings due to gating
and blocking become indistinguishable at high ACh
concentrations. The simultaneous fit to all of the data,
shown as smooth curves superimposed on the open
and closed duration histograms, reasonably describes the kinetics of wild-type and
N217K AChRs (Fig. 1).
Focusing on the kinetic steps governing closed durations, we see that the rate of ACh dissociation is greatly
slowed by N217K (Table II). ACh dissociates from the
wild-type receptor at a rate similar to the rate of channel opening, predicting approximately two openings per
activation episode after brief exposure to agonist. By
contrast, ACh dissociates from the
N217K AChR 10- to 20-fold more slowly, allowing greater than ten openings per activation episode. The initial fitting analysis
allowed dissociation rate constants for the two binding
sites to be free parameters, but the fit was not significantly better than with the constraint of equal dissociation rate constants for the two sites. For both the constrained and unconstrained analyses, the two binding
sites showed essentially equivalent dissociation rate
constants for both
N217K and wild-type receptors.
Unequal dissociation rate constants have been described
for Torpedo (Sine et al., 1990
) and fetal mouse (Zhang
et al., 1995
) receptors; features of those data indicating
nonequivalent dissociation rate constants were biexponential distributions of the major concentration-dependent closings at intermediate but not high ACh concentrations. In the present study,
N217K and wild-type receptors show monoexponential distributions of the
major concentration-dependent closings over the range
of ACh concentrations examined (Fig. 1); Akk and
Auerbach (1996)
also observed equal dissociation rate
constants for adult mouse receptors. The overall data
suggest that differences in the agonist dissociation rate
constants for the two sites depend on species and on
whether the
subunit pairs with the
,
, or
subunit.
We further examined equivalence of the dissociation rate constants by expanding SCHEME I to allow independent binding of ACh to each site.
[View Larger Version of this Image (306K GIF file)]Scheme II.
SCHEME II predicts a fourth closed time component, which can be distinguished if the two binding sites are neither equivalent nor widely different in their ACh affinities. Fitting SCHEME II to the data, however, revealed equivalent dissociation rate constants for each binding site (Table II).
Association rate constants are not affected by N217K
(Table II) and are close to the diffusion limited values
reported previously from single channel kinetic analysis of Torpedo and adult mouse AChRs (Sine et al.,
1990
; Akk and Auerbach, 1996
). As observed for the agonist dissociation rate constants, association rate constants are equivalent at each binding site for both wild-type and
N217K AChRs; allowing the association rate
constants for the two sites to be free parameters gave
no better fit than when they were constrained to be
equal. Several features of the closed duration histograms indicate that
N217K slows ACh dissociation without a change in association: the long duration component of closed times is roughly equally dependent on
ACh concentration for both receptor types, but the
mean of this component is always more brief at a given
concentration for
N217K than for wild type. Thus the
overall effect of
N217K on affinity of the resting state of the receptor is a 10-fold decrease in the dissociation
constant for ACh binding.
The rate of opening of the doubly occupied AChR,
2, though very fast for both mutant and wild type, is
slowed by ~50% by
N217K (Table II). The reduced
2
leads to a longer mean duration of the doubly occupied
closed receptor, approximately (
2 + k
2)
1, which is
seen in the closed duration histograms as an increase in the time constant of the major component of brief
closings (Fig. 1). For the wild-type receptor,
2 was determined from analysis of currents obtained at limiting
low ACh concentrations, so the results of the global fit
demonstrate its consistency across a range of concentrations. For the
N217K receptor, the slower rate of
ACh dissociation, combined with the slower rate of channel opening, allows
2 to be estimated even at high ACh
concentrations. Although
2 is slowed by
N217K, opening is still rapid enough to elicit multiple reopenings
per activation episode, as the other pathway away from
the doubly occupied closed state, agonist dissociation, is slowed even more.
The life time of the doubly occupied open channel is
increased about twofold by N217K, owing to slowing of
the closing rate
2 (Table II). A second class of briefer
openings is detected at the lowest but not at the highest
ACh concentrations, and is therefore ascribed to opening
of singly occupied receptors. Owing to the predominance
of the doubly occupied open channel during synaptic
activity, the slower closing rate would double the duration of an activation episode, compounding the effect
of the increase in the number of openings per episode.
To confirm the rate constant estimates and to illustrate the overall consequences of N217K, we determined the mean open probability within clusters at
each concentration of ACh and compared it with the
dose-response relationship calculated from the kinetically determined rate constants. The calculated dose-response curves superimpose upon the Popen measurements, supporting the rate constant estimates and revealing a 20-fold decrease in the EC50 for activation of
the
N217K AChR (Fig. 2).
Structural Basis of Kinetic Effect of N217K
The preceding results establish that replacing asparagine with lysine at 217 slows the rate of agonist dissociation from the binding site. To gain insight into the
structural basis of this effect, we made the glutamine
and glutamate mutations at position 217 of the
subunit. We again recorded single channel currents over a
range of desensitizing concentrations of ACh and analyzed the kinetics using SCHEMES I and II. The analysis
reveals virtually indistinguishable sets of activation rate
constants for
N217Q and wild-type receptors, showing
that introducing a larger side chain alone is not responsible for the kinetic effect of
N217K (Table II). Introducing the negatively charged glutamate with
N217E
also produces activation rate constants similar to those
of wild type; the association rate constants are slowed with
N217E, leading to somewhat lower agonist affinity of each binding site (Table II). The dose-response
relationship calculated from the kinetic parameters reasonably describes the measured Popen values, confirming that
N217Q and
N217E cause little or no change
in activation properties of the receptor (Fig. 2). Thus introducing a negatively charged or an enlarged side
chain at position 217 of the
subunit fails to slow the
rate of agonist dissociation, suggesting that the structural basis of
N217K is introduction of a positive charge.
N217 is conserved not only across subunits of all
species, but also across the
,
, and
subunits (Table
I). To determine whether the effect of the
N217K mutation is specific to the
subunit, and is therefore localized, we mutated the equivalent asparagine in the
,
,
and
subunits. We recorded single channel currents
elicited by 30 µM ACh and determined the mean open
probability within clusters of openings. We chose a concentration of 30 µM because it is close to the EC50 for
the wild-type receptor and therefore should be sensitive
to changes in activation parameters (see Fig. 2). The
measured open probabilities for receptors containing either
N217K,
N217K, or
N217K are within the range
obtained for wild type, and are clearly lower than obtained for
N217K (Fig. 3). Thus slowing of ACh dissociation by the N217K mutation is specific to the
subunit, indicating a local rather than a global perturbation.
Kinetic Parameters Obtained at Low Concentrations of ACh
We also estimated the parameters 2,
2, and k
2 from
currents elicited by limiting low concentrations of ACh.
Estimating these three rate constants relies on choosing a concentration of ACh low enough so reopening
after agonist dissociation is very slow. Thus we used
ACh at concentrations of 1 µM for wild type,
N217Q ,
and
N217E and 50 nM for
N217K; these concentrations elicit threshold responses as shown by the dose-response measurements (Fig. 2). The traces obtained
at low ACh concentrations show that wild-type,
N217Q ,
and
N217E receptors open one or two times per activation episode, whereas
N217K receptors open many
times per episode (Fig. 4). For all four receptor types,
closed duration histograms are described as the sum of
two exponentials, with a long duration component due
to periods between activation episodes elicited by different channels and a brief component due to transient interruptions of episodes due to a single channel. The
corresponding burst duration histograms are described
as the sum of two exponentials, with the long duration
component due to receptors with two bound agonists
and the brief component to receptors with a single
bound agonist. Qualitatively, the presence of
N217K
prolongs the activation episode by increasing the number of openings per burst.
The measured parameters obtained at low agonist
concentrations are related to steps in SCHEME I as follows. The mean duration of brief closings equals (2 + k
2)
1, and the number of brief closings per burst of
long duration openings equals
2/k
2. Omitting the
negligible contribution of brief closings in A2R, the
mean burst duration equals (1 +
2/k
2)/
2. Estimates
of
2 and k
2 obtained from these relationships are presented in Table III; they agree closely with the estimates
obtained from the global kinetic analysis (Table II).
The estimate of
2 obtained from low ACh concentrations was used as a fixed parameter in the global fitting
analysis to constrain the range of parameters for receptors with low affinity and rapid rates of opening (Fig. 1
and Table II); this consistency across a wide range of
ACh concentrations supports the accuracy of
2 for
these receptors. For the
N217K AChR, essentially identical estimates of
2 were obtained independently from
the low concentration and global analyses. Thus the parameters estimated at low ACh concentrations confirm
that
N217K primarily slows the rate of agonist dissociation and that the kinetic effect is due to the positively
charged lysine.
Measurements of Equilibrium Binding of ACh
We further examined the consequences of N217K by
measuring equilibrium binding of ACh by competition
against the initial rate of 125I-
-bungarotoxin binding
(Sine and Taylor, 1979
). The wild-type AChR binds ACh
with micromolar apparent affinity, whereas the
N217K
AChR binds 20-fold more tightly (Fig. 5 A). Receptors
containing either
N217Q or
N217E bind with affinities similar to wild type, as observed for their gating kinetics, confirming that the effect of
N217K is due to
the presence of the positively charged lysine. Similarly,
substitution of
N217K,
N217K, or
N217K for the
corresponding wild-type subunit does not affect the apparent affinity for ACh, again confirming that the consequences of the N217K mutation are specific to the
subunit (Fig. 5 B).
Binding of ACh at equilibrium is determined by contributions of the resting, open channel, and desensitized
states of the receptor, each of which binds agonist with
different affinity (for review see Changeux, 1990). While
the increased affinity of the resting state contributes to the
increase in equilibrium binding affinity of the
N217K
receptor, changes in ACh affinity for the desensitized
state or the allosteric constant governing the distribution of resting and desensitized states may also contribute. Our preliminary experiments indicate that
N217K
does not affect the affinity of the desensitized state for
ACh. Assuming a wild-type value of 40 nM for the affinity of the desensitized state (Sine et al., 1995
), we computed the allosteric constant for desensitization using
our measured parameters for activation (Table II). The
results indicate a marked increase in the allosteric constant for desensitization from a wild-type value of 4 × 10
4 to 0.5 for
N217K.
We previously showed that N217K causes a slow channel congenital myasthenic syndrome by prolonging the
elementary activation episode elicited by ACh (Engel et
al., 1996
). Here we trace the kinetic defect to slowing of
the rate of ACh dissociation from the binding site; prolonged occupancy by ACh allows the channel to open
repeatedly before it can dissociate. Rate constants for channel opening and closing are also slowed but to much
smaller extents. The kinetic fingerprint of
N217K is strikingly similar to that of our previously described SCCMS
mutation
G153S, which is near residues in the extracellular domain that contribute to the binding site (Sine
et al., 1995
). The present results are surprising because
N217K is in the M1 transmembrane domain, whereas its primary effect is at the binding site, some 30 Å above
the plane of the membrane (Unwin, 1993
; Valenzuela
et al. 1994
). The kinetic effect of
N217K results from
introduction of the positively charged lysine side chain
and is not observed when lysine is introduced into the
corresponding positions of the
,
, or
subunits. Thus slowing of agonist dissociation is not due to a global
perturbation but rather to a local perturbation of the
linkage between the M1 domain of the
subunit and
the binding site. The results have implications for structure-function relationships of AChR and for how agonist binding affinity affects the time course of the synaptic response.
Present understanding of the topology of the M1 domain points to an allosteric rather than a direct effect
of N217K in slowing agonist dissociation. The available data indicate that approximately the carboxyl-terminal two-thirds of M1 may be folded in the membrane while the amino-terminal third may extend above it.
Thus
N217 appears to lie just outside the membrane,
where it is accessible to hydrophilic sulfhydryl reagents
when mutated to cysteine (Akabas and Karlin, 1995
). Exposure of residue 217 to aqueous solution would render a lysine side chain at this position positively charged at physiological pH.
N217 is also four residues amino-terminal to the conserved P221, which borders a stretch
of four residues accessible to a hydrophobic labeling
agent (Blanton and Cohen, 1994
). If P221 is the most
amino-terminal residue of M1 embedded in the membrane, the intervening four residues are not long enough to extend N217 30 Å to the binding site. Thus
N217
likely comprises part of the inner wall of extracellular
vestibule and contributes to the linkage between the
channel gating apparatus and the binding site.
Our findings show that the perturbation caused by
N217K propagates to the binding pocket to enhance
the fit of ACh for the resting state of the receptor. Although it is clear that the binding site and channel gate
are functionally coupled to produce rapid and efficient
gating, the results presented here are the first to show
spread of a perturbation in a transmembrane domain to the binding sites in the extracellular domain. The results
suggest the presence of a structure that physically links
the binding site and channel gating apparatus. Because
the perturbation is due to the positively charged lysine
side chain, in the wild-type receptor
N217 may serve as
a hydrogen bond acceptor for a positively charged donor.
The findings reconfirm the importance of ACh binding affinity in governing the time course of the synaptic
response (Sine et al., 1995). Magleby and Stevens (1972)
showed that the decay of the end plate current is governed by properties intrinsic to the post synaptic AChR.
The time constant for decay approximately equals the
mean channel open time multiplied by the number of openings per activation episode, or (1/
2)(1 +
2/k
2). At the normal synapse,
2 is very fast to provide fast onset
of the response, but k
2 is similarly fast to rapidly terminate the response ( Jackson, 1989
). By contrast, synapses
harboring the
N217K receptor show a prolonged end
plate current primarily because the number of openings
per activation episode is increased due to slowing of k
2.
Original version received 30 December 1996 and accepted version received 24 March 1997.
Address correspondence to Steven M. Sine, Ph.D., Department of Physiology and Biophysics, Mayo Foundation, 200 First Street, S.W., Rochester, MN 55905. Fax: 507-284-9420.
1 Abbreviations used in this paper: AChR, acetylcholine receptor; CMS, congenital myasthenic syndromes.This work was supported by NIH grants to S.M. Sine (NS31744), A. Auerbach (NS23513), and A.G. Engel (NS6277).