Correspondence to: Claudio Grosman, Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, SUNY at Buffalo, 124 Sherman Hall, Buffalo, NY, 14214. Fax:716-829-2569 E-mail:grosman{at}buffalo.edu.
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Abstract |
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The spontaneous activity of adult mouse muscle acetylcholine receptor channels, transiently expressed in HEK-293 cells, was studied with the patch-clamp technique. To increase the frequency of unliganded openings, mutations at the 12' position of the second transmembrane segment were engineered. Our results indicate that: (a) in both wild type and mutants, a C O kinetic scheme provides a good description of spontaneous gating. In the case of some mutant constructs, however, additional states were needed to improve the fit to the data. Similar additional states were also needed in one of six patches containing wild-type acetylcholine receptor channels; (b) the
12' residue makes a more pronounced contribution to unliganded gating than the homologous residues of the
, ß, and
subunits; (c) combinations of second transmembrane segment 12' mutations in the four different subunits appear to have cumulative effects; (d) the volume of the side chain at
12' is relevant because residues larger than the wild-type Ser increase spontaneous gating; (e) the voltage dependence of the unliganded gating equilibrium constant is the same as that of diliganded gating, but the voltage dependences of the opening and closing rate constants are opposite (this indicates that the reaction pathway connecting the closed and open states of the receptor changes upon ligation); (f) engineering binding-site mutations that decrease diliganded gating (
Y93F,
Y190W, and
D200N) reduces spontaneous activity as well (this suggests that even in the absence of ligand the opening of the channel is accompanied by a conformational change at the binding sites); and (g) the diliganded gating equilibrium constant is also increased by the 12' mutations. Such increase is independent of the particular ligand used as the agonist, which suggests that these mutations affect mostly the isomerization step, having little, if any, effect on the ligand-affinity ratio.
Key Words: nicotinic receptors, allosteric proteins, site-directed mutagenesis
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INTRODUCTION |
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The acetylcholine receptor channel (AChR)1 is a synaptic receptor ion channel (
In wild-type AChRs, as in any other allosteric protein, the gating equilibrium constant of the unliganded receptor is finely tuned to ensure the appropriate response both in the presence and absence of the ligand (
There have been only a few studies of AChR spontaneous gating (, ß, and
subunits exhibit more frequent spontaneous openings than complete
2ß
receptors, but their respective mean open life times and single-channel conductances are indistinguishable (
/
-less receptors, in the presence of ACh and at the single-channel level, unequivocal (
The frequency of unliganded openings has been shown to increase as a result of several different mutations, most notably in the second transmembrane segment (M2) (, and Thr in
, ß, and
), which is near the middle of the M2 segment, for three reasons. First, it was known that a mutation at this position of the
subunit increases spontaneous activity (
12' T
I,
12' T
P,
and ß subunits do not face the channel conduction pathway (
-neurotoxins suggested that, in Torpedo,
12' is accessible from the extracellular side (
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Here, we show that the unliganded activity of M2 12' mutants largely resembles that of AChRs from embryonic cultured mouse muscle (
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METHODS |
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Mutagenesis and Expression
Mouse cDNA clones were generously provided by Dr. S.M. Sine (Department of Physiology and Biophysics, Mayo Foundation, Rochester, MN), who had obtained them from the late Dr. J. Merlie and Dr. N. Davidson (, ß, and
subunits), and Dr. P. Gardner (
subunit,
subunit had a background mutation in M4 (V433A), which does not significantly affect the gating behavior of diliganded AChRs (
T264P,
Y93F,
Y190W, and
D200N cDNAs in pRBG4 were generously provided by Dr. Sine. Mutations
T254S, ßT265S,
S268A,
S268C,
S268I,
S268N, and
S268Y were engineered using the QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene Inc.) protocol. Mutations
T254P, ßT265P,
S268P (
S268T, and
T265S were engineered by overlap PCR (
Human embryonic kidney fibroblast cells (HEK 293) were transiently transfected using calcium phosphate precipitation (:ß:
:
) was applied to the cells for ~20 h, after which the medium was changed. Electrophysiological recordings started ~30 h later.
Patch-Clamp Recordings
Recordings were performed in the cell-attached patch configuration (. Pipettes outside this range were discarded in an attempt to exert some control on the area of patched membrane. Unless otherwise stated, the potential of the patch pipette was held at +65 mV, which corresponds to an estimated membrane potential of approximately -100 mV. The membrane potential was estimated based on the amplitude of the single-channel currents and the single-channel conductance. Single-channel currents were recorded using an Axopatch 200B (Axon Instruments) at a 100-kHz bandwidth, digitized at a frequency of 94.4 kHz (VR-10B, fc = 37 kHz; Instrutech Corp.), and stored on videotape. For analysis, the recordings were transferred to a PC via a digital interface (VR111; Instrutech Corp.) at a sampling frequency of 94.4 kHz.
Some of the mutants studied exhibited high levels of spontaneous activity, but some of them did not. For the latter, it was critical to distinguish between low activity and low expression levels. Rather than relying on the expression of a reporter gene, we identified AChR-expressing cells by making seals, with 5-µM ACh in the pipette, on cells having the typical morphological alterations of cells that are expressing the transfected DNA. Only those cells displaying activity in the presence of ACh were further patched using another pipette that did not contain ACh. In some cases, series of up to six seals on the same cell, alternating the presence and the absence of the agonist, were performed. Only those series displaying activity whenever ACh was present in the pipette were considered for analysis. To prevent contamination of ACh-free solutions with ACh, the pipette electrode (i.e., the Ag/AgCl wire connected to the headstage) was extensively washed with distilled water after each time it came in contact with the agonist. Separate filling needles were used throughout.
Data Analysis
The QUB suite of programs was used (www.qub.buffalo.edu). Single-channel recordings were inspected visually (program PRE) and segmented into ~1.5-slong stretches of data. Noisy sections and those containing simultaneous openings of two or more channels were excluded. Typically, >98% of the original recording was retained for the kinetic analysis. The data were further filtered digitally (Gaussian filter; effective bandwidth: ~18 kHz) and idealized (program SKM) by using a hidden-Markov modeling procedure (recursive Viterbi algorithm) known as the "segmental k-means" method (
The list of dwell times generated with SKM was used to obtain kinetic parameters in two different ways. One method consisted of estimating the mean duration of detected closures and openings (here referred to as c and
o, respectively) by averaging the corresponding idealized dwell times. This calculation is what a maximum likelihood approach would perform when applied to a two-state, C
O model without correction for missed events. The reciprocal of
c was taken as an estimate of the opening frequency which, as long as openings are brief, is a good approximation. Because the number of channels in the patch was not known, this parameter is a measure of both the opening frequency per channel and the number of channels in the patch. The open probability was calculated as
o/(
o +
c) and, because it is not a measure of the single-channel open probability, we refer to it as "patch open probability". The second procedure (program MIL;
In the case of currents elicited by saturating concentrations of choline (20 mM) or acetylthiocholine (2 mM), clusters of single-channel openings were defined as a series of openings separated by closures shorter than a critical time (crit). For every patch, different
crit values were tested and the corresponding idealized intracluster dwell-time series were analyzed with MIL (fc = 18 kHz; dead time
30 µs). Because diliganded gating is very likely to proceed from a single closed diliganded state, the longest
crit defining clusters whose kinetics of closed durations were still best modeled by a single closed state was chosen. To discriminate between models with one or two closed states, we compared the corresponding maximum log-likelihood values. The two extra free parameters of the model with two closed states were penalized by subtracting two units from its maximum log-likelihood value (asymptotic information criterion;
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RESULTS |
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In the absence of ligand, wild-type AChRs open rarely and briefly. Fig 1 shows a continuous stretch of data illustrating the spontaneous activity of recombinant, adult muscle AChRs transiently expressed in HEK-293 cells. Some kinetic parameters are listed (see Table 2 and Table 4).
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Fig 2 illustrates the effect of the M2 12' S
T mutation at the single-channel level. Both the frequency and average duration of spontaneous openings are considerably increased by the mutation. In four of five patches, multiple closed and open components were apparent, with the longest (and least frequent) type of openings occurring in bursts. However, in all patches, short-lived isolated openings accounted for the majority of events. Estimates of the opening frequency, mean duration of detected openings (
o), and patch open probability are displayed in Table 2.
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We next tested the effect of TS single mutations in the 12' positions of the other subunits. The results in Table 2 suggest that T
S substitutions in the ß or
subunit exert little (if any) effect, while this mutation in both
subunits decreases the spontaneous activity of the wild type to undetectable levels. We then expressed combinations of mutant subunits to generate AChRs that, like the wild type, have a Ser in only one type of subunit. As expected from mutational effects that are cumulative (if not additive;
display high spontaneous activity, comparable with the sum of the effects of the single mutants. Likewise, adding the T
S mutation in both
subunits to
S
T receptors reduces unliganded activity, as it does when added to the wild type. We conclude that the residues occupying the 12' position of the different subunits make asymmetric contributions to unliganded activity, the
subunit playing the most prominent role. Also, since the mutational effects do not seem to deviate from additivity, we conclude that the five subunits are likely to make independent contributions to gating.
Estimates of opening frequency, mean duration of detected openings, and patch open probability of different 12' mutants and of the Pro mutants in the four subunits are also listed in Table 2. The very large "opening frequency" of
12' T
P receptors resulted, at least in part, from an increased number of openings per burst, as analyzed below.
A Kinetic Model
The parameters in Table 2 do not fully reflect the differences between the spontaneous activity of wild-type and mutant receptors that are clear from even a cursory examination of the recordings. Particularly, the multiplicity of open states is not represented at all in Table 2, where all open durations were averaged into a single value. We therefore sought to characterize the behavior of unliganded gating in terms of rate constants. It is worth noting that the occurrence of bursts of openings was not always observed. In the S
T mutant, these bursts were recorded in four of five patches, while in the wild type they were detected in only one of six patches, even with cells corresponding to the same transfection batch. The other constructs, however, displayed this component in either all, or none, of the patches. The kinetic analysis below was performed on single-channel records from patches that exhibited these bursts. With the exception of noisy sections and those containing simultaneous openings of two or more channels, which typically represented <2% of the original data, the recordings were analyzed in their entirety.
What follows is an example of model discrimination applied to a particular dwell-time sequence (4,226 events) obtained from a patch of a cell expressing the S
T mutant. Fig 3 shows all the kinetic models tested, and Table 3 displays the corresponding maximum likelihood values expressed as log-likelihood ratios (LLRexp), taking A as the reference (i.e., maximum log-likelihood value for model i maximum log-likelihood value for A, where model i is any model from B to J). A comparison of the LLRexp values leads to the rejection of B, D, and E, which suggests that at least two closed and three open states are needed to describe the kinetics of the data, and that the second closed state has to be connected to one of the open states (compare A and E). Sojourns in this additional closed state (C4) account for the population of brief closures present within bursts of openings. The best fit to the data always connected this short-lived closed state to the slowest open state (with the exception of the
T
P mutant), in agreement with the observation that openings within a burst are longer than isolated openings. Both A and H satisfy these conditions and LLRexp = 0. C and F were rejected because the rates leading to O5 (C) and C6 (F) were negligibly small and, therefore, sojourns in these states are very rare.
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Throughout this procedure, only large LLR values were taken into account to discriminate between models and, thus, application of penalties for excess free-parameters did not affect the decisions. The LLRexp values and rate constants corresponding to Fig 3G, Fig I, and Fig J, however, did not allow a clear decision to be made. Therefore, we simulated noise-free single-channel event lists (containing approximately the same number of events as the original data) according to each model in Fig 3. Analysis of the simulated data (Table 3) and comparison with the LLRexp values confirms that G, I, and J cannot be ruled out as appropriate kinetic schemes for spontaneous gating.
We conclude that, for the S
T mutant, A, G, H, I, and J in Fig 3 are all plausible, with A, H, I (constrained), and J being the most parsimonious. Simulating dwell-time sequences containing ~10x more intervals revealed that I (constrained) and J could have been distinguished from A and H if more data had been collected. As expected, however, A and H could not have.
The maximum log-likelihood value obtained with Fig 3 A was then compared with that obtained from fitting the experimental data to a fully connected model with the same number of closed and open states. In such a model, all connections between states (regardless of their conductances) are allowed, the corresponding maximum-likelihood value being the highest possible for the given number of closed and open states (for a related approach, see S
T patches (Nos. 24) where the three types of openings were present. A model discrimination analysis of these three other patches supports all the conclusions above for patch No. 1.
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A similar procedure was applied to all other constructs that exhibited multiple open and closed components. Fig 4 shows the dwell-time histograms and the superimposed density functions of example patches, and Table 4 shows the corresponding rate constants. The kinetics of the S
T + ßT
S and
T
P constructs, like those of the
S
T mutant, were best described by Fig 3A, Fig G, Fig H, Fig I, and Fig J. The corresponding rate-constant estimates to A are shown in Table 4. For the wild type and the mutants
S
T +
T
S,
S
A,
S
N,
S
I,
S
P, and ßT
P, Fig 3 B (with only two open states) was superior. In these seven cases, the intermediate open state was of doubtful existence, a fact that could be related to its lifetime being close to (and thus difficult to distinguish from) that of either the fastest or slowest type of openings, rather than to a genuine mechanistic difference between mutants. Finally, the mutants
S
Y and
T
P were fitted best to G. Again, we think that this difference may reflect some data heterogeneity rather than a different gating mechanism. For any given mutant, the rank order of the models was the same for all patches.
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Other than the opening rates, which in our case depended on the number of channels in the patch, the rate constants varied little between constructs. However, two noteworthy differences were present. First, S
A and wild-type AChRs have larger k30 values, which make their longest-lived openings shorter than those of the other mutants (Fig 5). Nevertheless, as k34 values are also increased, these openings still occur in bursts with a "regular" number of openings. Second,
T
P AChRs have a much larger k34, which greatly increases the number of openings per burst and reduces the duration of these openings to as little as ~150 µs (Fig 5). If, for example, Fig 3 H were used instead (where the bursting behavior is modeled by a C
C
O scheme), an increase in two rate constants, k43 and k34, would be needed to account for the same finding.
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If we assume that the multiple kinetic components arise from the activity of individual channels, then, in bursty patches, the unliganded AChR can access three different open conformations with mean lifetimes of ~100 µs (>75% of the total openings), ~500 µs, and ~5 ms. From the rate-constant estimates, it can be calculated that the longest openings are grouped in bursts of, on average (and excluding the T
P mutant), ~1.29 openings separated by brief (~150 µs) gaps. In other words, ~60% of these openings occur as isolated events, ~27% occur as pairs of openings separated by a brief gap, ~9% form triplets separated by two gaps, and so on. Fig 5 shows examples of such bursts for different constructs, including the wild type. In all the cases where multiple open components were not observed, only brief isolated openings, indistinguishable from the fast predominant component described above, were recorded.
The fact that short-lived openings dominated the unliganded activity clearly suggests that these represent the main allosteric transition of wild-type gating. This is the reason why we focused on this kinetic component to study the effects of voltage and binding-site mutations on spontaneous activity (see below). It is not clear, however, what the diliganded counterpart of the longest-lived, less-frequent type of openings is.
Voltage Dependence
It has been known for several decades that the equilibrium constant of AChR-diliganded gating is voltage dependent (S
T mutant.
The voltage dependence of the opening and closing rate constants was studied in a patch that displayed only the brief type of openings (i.e., the data were fitted best to a C O model). The z
parameters were obtained from fits of rate-versus-voltage plots to the Boltzmann equation [k = ko exp(-z
F V/RT)] (Fig 6). Their values were 0.363 ± 0.024 for the opening and 0.011 ± 0.023 for the closing rate constant. Thus, in this patch, the unliganded gating equilibrium constant increased e-fold with an ~70-mV hyperpolarization, a change that was entirely due to the increase in the opening rate constant. In diliganded AChRs,
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Effect of Binding-Site Mutations
According to the constraints imposed by a cyclic model with only binding and isomerization steps, unliganded gating must involve a conformational rearrangement in both the pore and the transmitter binding sites, just like during liganded gating. However, the increased spontaneous activity often recorded from recombinant ligand-gated ion channels has been interpreted as arising from the uncoupling of the binding sites from the gate (
To distinguish between such coupled and uncoupled mechanisms of unliganded gating, we transfected HEK-293 cells with cDNA encoding for 12' S
T, 12'
T
S, ß wild type, and either
Y93F,
Y190W, or
D200N. The combination of both 12' mutations was shown, above, to yield receptors with high spontaneous activity. The three binding-site mutations in the
subunit have been shown to decrease the diliganded gating equilibrium constant by, mainly, slowing down the channel's opening rate (
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Diliganded Gating in Channels with Increased Unliganded Activity
The results presented in the above section suggest that both unliganded and diliganded gating result from the same global conformational change, even though, as suggested by the voltage-dependence experiments, the particular pathway followed in each case may differ. This supports the application of thermodynamic cycles to model the AChR's activity, according to which:
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(1) |
where Kd and Jd are the microscopic dissociation equilibrium constants from the closed and open states respectively, and 0 and
2 are the gating equilibrium constants for the unliganded and diliganded AChR, respectively. Equation 1 predicts that an increase in
0 (caused, for example, by a pore mutation) has to be accompanied by the same increase in
2 as long as the affinity ratio (Kd/Jd) is not affected.
2 values were estimated as the ratio between the opening (ß2) and closing (
2) rate constants from clusters of single-channel currents elicited at saturating concentrations of agonist (
2 upon the 12'
S
T mutation. This is because the wild-type's
2 value is already near the upper limit of reliable estimation. However, such an increase is readily apparent in the presence of choline or acetylthiocholine (the thioester counterpart of ACh), two slowly opening, low-efficacy agonists (Fig 8). The
12' mutation increases
2 by a factor of 14.5 when the AChR is occupied by choline and by a factor of 13.1 when it is occupied by acetylthiocholine (Table 6). The increase in
2 thus appears to be independent of the particular agonist used, suggesting that the 12'
S
T mutation mainly affects
0, leaving the Kd/Jd ratio almost unchanged.
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DISCUSSION |
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Kinetic Aspects
In both wild type and M2 12' mutants, a two-state C O kinetic scheme provides a good description of spontaneous gating. In the case of some 12' mutants, however, additional states were needed to improve the fit to the data and similar extra states were needed in one of six patches containing wild-type AChRs. Multiple kinetic components during unliganded gating have also been observed for a number of slow-channel congenital myasthenic syndrome (SCCMS) mutants in M2 (
T264P,
L269F,
V249F,
There are a number of possible explanations for the multiplicity of kinetic states that we observed during unliganded gating. A trivial explanation is that trace concentrations of ACh (or some other cholinergic ligand) were present and resulted in a small number of liganded openings. We consider this to be unlikely because precautions were taken to avoid such contamination. A second possibility is that the patches contained a set of heterogeneous receptors. Under our experimental conditions, these could have arisen from alternative assembly patterns and/or different extents of post-translational modifications. However, neither of these are likely to be affected by mutations in M2 (
Structural Aspects
Our results suggest that the M2 12' position of each subunit makes an asymmetric and independent contribution to unliganded gating. We showed that this is also true during diliganded gating ( subunit that stands out. Whether this reveals differences in the dynamics of the subunits during gating or results from an asymmetric orientation of the five subunits in the pore of the pentameric complex is still unclear.
Voltage Dependence
There is a discrepancy in the literature about the voltage dependence of the unliganded gating rate constants. Although
With the 12' mutants, we were able to record higher frequencies of openings. One remaining problem, however, was that a complex kinetic scheme was generally needed to fit the data. To make the analytic procedure more reliable and the interpretation of the results more straightforward, we analyzed the voltage dependence of unliganded gating in a patch that only displayed the fast class of openings (which, even in the kinetically most complex patches, accounts for the majority of openings). We found clear evidence that the opening rate constant increases as the membrane is hyperpolarized, in agreement with the results of
Coupling between Binding Sites and Pore
The results in Fig 7 indicate that the binding-site mutations Y93F,
Y190W, and
D200N impair unliganded gating. This can be interpreted as an indication that there is a conformational rearrangement around these binding-site residues during unliganded gating, whose manifestation at the pore is the openingclosing of the channel. This suggests that spontaneous openings do not arise from the autonomous activity of the M2 region.
This interpretation relies on the assumption that binding-site mutations can only cause local perturbations that do not reach the distant gate. We cannot rule out, however, the possibility that a local perturbation at the binding sites could propagate to the "autonomous gate" and affect its behavior. Nevertheless, crystallographic data of proteins shows that structural changes are usually greater near the sites of mutation and fall off rapidly with increasing distance (
Control of the Isomerization Step by Binding-Site Residues
There are two ways a binding-site mutation can reduce 2: it can reduce the ligand-affinity difference between the open and closed states (the affinity ratio), or it can interfere with the low affinity
high affinity conformational change that accompanies channel opening (
0). Here, we showed that the decrease in
2 caused by the binding-site mutations
Y93F,
Y190W, and
D200N (
0 (Equation 1). Thus, the use of spontaneously opening mutants as the background receptors on which other mutations are engineered constitutes a useful tool to identify binding-site residues that contribute to gating in forms other than by binding the agonist with different affinities in the open versus closed state. It is very likely that these residues, in addition to making contact with the ligand (at least in the case of
Y93 and
Y190), also participate in proteinprotein interactions that change upon gating. This distinction between effects on
0 or the affinity ratio would have been difficult to make if the background receptor had been the wild-type channel.
It seems that the gating conformational change of AChRs is controlled in opposite directions by residues at the pore and the binding sites. With the exception of G153S (
Diliganded Activity
In the absence of a solid estimate of the number of channels per patch, we cannot estimate 0. As a consequence, it was impossible to determine directly whether the increase in
2 caused by the 12' mutations arises exclusively from an equal increase in
0 or there is a contribution from a change in the affinity ratio as well. To solve this issue, we took an indirect approach reasoning that it is unlikely for a mutation that alters the affinity ratio of a receptor to do so to the same extent for different ligands. According to Equation 1, it is clear that a mutation that increases
0 without affecting the affinity ratio causes the same increase in
2 for different agonists. Here we suggest that the reverse is also true: the same increase in
2 for different ligands can be taken as an (indirect) evidence that the mutation in question only affects
0. By comparing the responses of wild-type and
S
T AChRs to two agonists (choline and acetylthiocholine), we concluded that the
S
T mutation mainly affects
0.
As shown in Fig 8, we cannot directly measure the effect of the 12' S
T mutation on ACh-activated AChR gating. However, from the average increase in
2 for choline and acetylthiocholine (~13.8x), we can predict that
2 for ACh would be ~380, which is 13.8x the
2 value for the wild-type AChR (calculated to be ~27.5 at -100 mV, from data in
12' mutants are linearly correlated with the corresponding gating equilibrium free-energy changes, and that the slope of such relationship (
) is ~0.3 for choline or acetylthiocholine (
2mut/
2wt)
[and, therefore,
2mut/
2wt = (
2mut/
2wt)(
- 1)]. Thus, the diliganded opening rate constant of
12' S
T receptors should be 13.80.3
2.2 times that of wild-type AChRs, or ~110,000 s-1, and the closing rate constant should be 13.8-0.7
0.16 times that of wild-type AChRs, or ~300 s-1 (at -100 mV).
In the following paper, we make extensive use of choline as a slowly opening, low-efficacy agonist to investigate the effect of 12' pore mutations on diliganded gating.
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Footnotes |
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1 Abbreviations used in this paper: AChR, acetylcholine receptor channel; M2, second transmembrane segment.
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Acknowledgements |
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We thank Karen Lau for technical assistance.
This work was supported by grants from the National Institutes of Health to A. Auerbach and from the American Heart Association, New York State Affiliate, to C. Grosman.
Submitted: 24 September 1999
Revised: 3 March 2000
Accepted: 20 March 2000
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