Correspondence to: Anthony Auerbach, 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:auerbach{at}buffalo.edu.
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Abstract |
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We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the subunit (
S269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the
S269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an
92-fold increased gating equilibrium constant, which is consistent with an
10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening
28-fold, slows channel closing
3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh,
S269I acetylcholine receptors open at a rate of
1.4 x 106 s-1 and close at a rate of
760 s-1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of
140 s-1. Ile mutations at positions flanking
S269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the
subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the
S269I mutation, Ile mutations at equivalent positions of the ß,
, and
subunits do not affect apparent open-channel lifetimes. However, in ß and
, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2M3L of the
subunit moves before the corresponding linkers of the ß and
subunits.
Key Words: nicotinic receptors, allosteric proteins, single-channel kinetics, transition state
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INTRODUCTION |
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The muscle acetylcholine receptor channel (AChR)1 is a ligand-activated ion channel that mediates neuromuscular transmission in vertebrates (reviewed in 2ß
), each having four putative membrane-spanning domains (M1, M2, M3, and M4; reviewed in
The second transmembrane segment (M2) from each subunit lines the narrow region of the ion-permeation pathway and is critical for both ion permeation (7 AChRs (
3,
7, and ß4 subunit-containing AChRs (
1 homomers (
1 homomers (
The effect of naturally occurring mutations in the M2M3L also suggests a role in channel gating. The slow-channel congenital myasthenic syndrome (SCCMS) mutation S269I prolongs the AChR's apparent open time (
1 subunit of the glycine receptor, shorten the apparent open time (
To characterize the M2M3L of the muscle AChR, we engineered a set of mutations in this region of the different subunits of adult mouse receptors and studied their effects on the kinetics of activation of single AChRs. Our results indicate that M2M3L mutations affect activation by selectively altering channel gating. Furthermore, the results suggest that during channel opening the M2M3L of the subunit moves before the corresponding linkers of the ß and
subunits.
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METHODS |
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Mutagenesis and Expression
Mouse cDNA clones were obtained as described by -subunit clone contained an incidental M4 mutation (V433A) that has no functional consequences (
24 h after addition of the calcium-phosphate precipitate, and electrophysiological recordings started
24 h later.
Patch-Clamp Recordings and Analysis
Recordings were performed in the cell-attached patch configuration (22°C. The pipette solution contained (mM): 142 KCl, 5.4 NaCl, 1.8 CaCl2, 1.7 MgCl2, 10 HEPES/KOH, pH 7.4. In most experiments using ACh, the bath solution was the same as the pipette solution. In all other experiments, the bath solution was Dulbecco's phosphate-buffered saline (mM): 137 NaCl, 0.9 CaCl2, 2.7 KCl, 1.5 KH2PO4, 0.5 MgCl2, and 8.1 Na2HPO4, pH 7.3. Single-channel currents were amplified using a patch-clamp amplifier (PC-505; Warner Instrument Corp. or 200B; Axon Instruments, Inc.) and were digitized at 100 kHz.
To separate the more rapid binding and gating steps from the slower desensitization steps, clusters of single-channel activity were identified and used to estimate open probability (Popen), time constants, and gating rate constants. Clusters were defined as a series of openings separated by closures shorter than a critical time, c (
c was determined as described by
c was the longest time interval that defined clusters still best fitted with a C
O kinetic model, as described by
4 kHz or a hidden-Markov-modelbased algorithm (program SKM) at a bandwidth of
18 kHz. Only clusters whose overall mean open time was within ±2 SD of the mean of the corresponding patch were retained for further analysis (typically >98% of the original data; programs LPROC or SELECT).
Equilibrium concentrationresponse curves were analyzed in the framework of Scheme 1:
where C and O denote the closed and open channel, respectively, A denotes the agonist, OA2B represents the open channel blocked by the agonist itself, and CA2* is a shut state whose mean lifetime is independent of the agonist concentration (1 ms and is therefore distinct from the long-lived desensitized state(s) that gives rise to the longer-lived closed intervals between clusters. Popen values were estimated from the idealized current traces as the fraction of time the channel is open within a cluster. Plots of Popen vs. agonist concentration were fitted with Equation 1 to estimate the microscopic agonist-dissociation equilibrium constant (Kd = k-/k+) and the diliganded-gating equilibrium constant (
2 = ß2/
2):
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(1) |
where c = KB/(A + KB) and KB is the dissociation equilibrium constant for channel block (k-b/k+b; 2 mM for ACh and 20 mM for choline), and Kg is the equilibrium constant between OA2 and CA2* (k+g/k-g; 0.05 for ACh, as in Salamone et al. (1999), and ignored in the presence of choline). Equation 1 corresponds to the classical sequential model (
The opening rate constants of the various constructs in the presence of ACh were estimated by fitting plots of ß' vs. agonist concentration (A) with the following empirical equation (Hill equation, Equation 2):
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(2) |
where ß' (the "effective" opening rate) is defined as the reciprocal of the slowest component of the closed-time distribution, ß2 is the opening rate constant, A50 is the agonist concentration at which ß' is half maximal (i.e., ß2/2), and n is the Hill coefficient. ß' at each ACh concentration was estimated from the fit of log-binned (O kinetic scheme, an effective bandwidth of
18 kHz, and a dead time of
25 µs, as described by
The distribution of open times in the presence of low concentrations of ACh was analyzed in the context of an MWC-like kinetic model (Scheme II;
Where k-/k+ (= Kd) and j-/j+ (= Jd) are the microscopic agonist-dissociation equilibrium constants from the closed and open channels, respectively, ß0/0 (=
0), ß1/
1 (=
1), and ß2/
2 (=
2) are the gating equilibrium constants of un-, mono-, and diliganded receptors, respectively, k+D/k-D is the desensitization equilibrium constant, and k+g/k-g is the equilibrium constant between OA2 and CA2*. Because this model was used to interpret data recorded at low concentrations of ACh, blockade by the agonist itself was ignored. According to Scheme 2, to satisfy detailed balance,
1 =
0Kd/Jd and
2 =
0(Kd/Jd)2.
Single-channel analysis programs of the QuB suite are available at www.qub.buffalo.edu.
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RESULTS |
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The SCCMS Mutation S5I
The M2M3 linkers of the different subunits of muscle, neuronal, and Torpedo electroplaque AChRs are all 15 amino acids long and contain several conserved residues, including two Pro (Table 1). The homology between aligned residues is largely lost, however, when the anion-selective members of the superfamily of nicotinoid receptors (GABA and glycine receptors) are included in the comparison, although the first Pro is still present.
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The naturally occurring mutation S269I (position 5 in our numbering system;
S5I) causes a congenital myasthenic syndrome (
S5I AChRs in the presence of 0.2 µM ACh are shown in Fig 1. The open-interval duration histogram (-70 mV; 4 kHz) was best fitted with two exponential components, the mean of the slowest being
4.5 ms. In the wild type, under otherwise identical experimental conditions, the mean of the slowest (and predominant) component was
0.75 ms (Table 2).
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At an effective bandwidth of 4 kHz, the minimum detectable time using the "half-amplitude" idealization criterion is
45 µs (
1% of the brief sojourns in the diliganded closed state (
10 µs on average) are detected. As a consequence, apparent open intervals often include two or more consecutive openings and, therefore, are not true estimates of the reciprocal of the diliganded closing rate constant. Taking the sequential model (Scheme 1) as an approximation of the channel's kinetic behavior, and neglecting the (short) time spent in the closed diliganded state (CA2), the mean duration of the slowest component of these apparent openings is given by:
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(3) |
where (1 + ß2/2k-) is the mean number of openings per burst. In the experiments at low concentrations of ACh, the probability that sojourns in shut states other than CA2 were shorter than the time resolution of 45 µs (and thus that were included within an apparent opening) is negligible. Likewise, the probability that a sojourn in CA2 was longer than 45 µs (and thus that terminates an apparent opening) is vanishingly small. In conclusion, an "apparent opening," defined as a series of openings separated by closures shorter than 45 µs, is likely to reflect a sojourn in the combined set of states (CA2 + OA2).
From Equation 3, it can be seen that the prolonged open-time constant of S5I AChRs may arise from one or a combination of the following: an increased opening rate constant (ß2), a decreased closing rate constant (
2), or a decreased agonist-dissociation rate constant (2k-). To distinguish between these possibilities, we studied the effect of the
S5I mutation on gating by using choline as the agonist. Choline slows channel gating so open and closed intervals can be fully resolved (
S5I AChRs recorded in saturating 20-mM choline. The opening rate constant (ß2) was 7,098 ± 1,227 s-1, and the closing rate constant (
2) was 1,487 ± 313 s-1. These are, respectively, 27.7x faster and 3.33x slower than those of wild-type AChRs activated by choline (
S5I mutation increases the gating equilibrium constant
92-fold, mostly due to an increase in ß2.
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Next, we sought to determine whether the S5I mutation affects binding affinity. Single-channel currents elicited over a range of choline concentrations were recorded, and intracluster Popen values were estimated and compared with that of ACh (Fig 2). The lower efficacy (i.e., smaller
2) of choline allowed us to fit the Popen-vs.-concentration data with Equation 1, yielding
2 = ß2/
2 = 2.5 ± 0.9 and Kd = k-/k+ = 3.3 ± 1.4 mM. This value of
2 is consistent with the rate constants obtained by single-channel kinetic modeling of currents elicited by 20-mM choline with a KB of 20 mM and assuming a sequential model for channel block. The Kd for wild-type AChRs activated by choline is not known with certainty because the extremely low efficacy of this agonist makes this doseresponse curve difficult to measure. However, the wild-type Kd is in the millimolar range (
S5I mutation does not substantially alter the affinity of the closed-channel transmitter binding site for choline, but rather predominantly affects channel gating.
The gating (2) and agonist dissociation (Kd) equilibrium constants of ACh-activated receptors could not be directly estimated from the doseresponse data (by fitting Equation 1) because of the high ACh
2 value. However, the Popen-vs.-concentration relationship of the
S5I mutant activated by ACh revealed an EC50 of
4.0 µM, which is
10x lower than that of the wild type determined under identical conditions (
>>1),EC50
.Therefore, the
10-fold decrease in ACh EC50 can be accounted for by an
100-fold increase in
2, which is similar to the 92-fold increase in
2 measured in the presence of choline. These results indicate that
2 is increased to the same extent in the presence of ACh or choline. An increase in
2 can arise from an increase in the unliganded gating equilibrium constant,
0 (ß0/
0) and/or an increase in the closed/open agonist-affinity ratio, Kd/Jd (Scheme 2). The agonist-insensitive effect of the
S5I mutation on
2 suggests that this mutation increases the unliganded gating equilibrium constant with little, if any, effect on the affinity ratio, which is expected to be sensitive to the nature of the agonist. Several SCCMS mutations, including
S5I, have been shown to increase unliganded gating, thus turning choline, a normally inert molecule, into a significant agonist (
Although gating of S5I receptors in the presence of ACh is exceedingly fast for the rate constants to be measured directly, the closing and opening rate constants can be predicted based on the results in the presence of choline. This is because the structure of the AChR at the transition state of the gating reaction is likely to be the same regardless of the particular ligand bound (
S5I receptors open
27.7x faster, and close
3.33x slower, than the wild type; i.e., ß2
1.4 106 s-1 and
2
750 s-1.
Even though the predicted value of ß2 cannot be confirmed experimentally from the duration of shut intervals, the effect that such a fast opening rate constant would have on the apparent open-time distribution can be calculated. In particular, we focused on the time constant of the slowest component of the distribution (o), which, for Scheme 1, is approximately given by Equation 3 at any concentration of agonist. According to this equation,
o for the
S5I mutant should be 48 ms, which is more than 10x longer than the observed
o of
4.5 ms.
However, as ß2 increases and 2 decreases, the sequential-model simplification (Scheme 1 and Equation 3) becomes inappropriate because the probability that a burst of openings terminates from the open state (i.e., by agonist dissociation from the open state followed by closing or by directly entering a desensitized state) is no longer negligible. Accordingly, an MWC-like kinetic model (
o values. Considering Scheme 2, it can be shown that
o (the slowest component of the open-time distribution) is an increasing function of agonist concentration whose value at infinite agonist concentration is given by Equation 3, and at zero agonist concentration (our conditions, approximately) by Equation 4:
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(4) |
where is the sum of all the rate constants leading away from OA2 other than
2. These are the dissociation rate constant from the diliganded open channel (2j-), the rate constant leading to the long-lived desensitized state (k+D), and the rate constant leading to CA2* (k+g).
Using the wild-type value of 40,000 s-1 for 2k- ( value of 200 s-1 accounts for all three experimental observations in the presence of ACh: (a) a
o value of 0.75 ms for the wild type; (b) a
o value of 4.5 ms for the mutant at low ACh concentration, and (c) an
10-fold smaller EC50 for the mutant. We were unable to find other combinations of ß2,
2, 2k-, and
values that could account simultaneously for these three findings.
Mutational Analysis of Position 5
The S5I mutation increases both the hydrophobicity and size of the side chain. To further characterize the structurefunction relationships of this position, we engineered a series of polar and hydrophobic side chains at
5 (Fig 1 and Table 2). Using ACh as the agonist, hydrophobic substitutions (Phe, Cys) increased
o nearly 10-fold, while polar substitutions (Asp, Gln, and Lys) were without effect. Ala, which has the highest mutational probability with Ser, also yielded wild-type-like
o values.
Some of these S5 mutants were also studied using choline as the agonist (Fig 3 A). The
S5C mutation had essentially the same effect as the
S5I mutation; that is, it produced a large increase in the opening rate constant (26-fold) and a modest decrease in the closing rate constant (3.7-fold). The
S5Q mutant was similar to the wild type, with essentially no change in the opening (
1.3-fold increase) or closing (
1.2-fold increase) rate constants. For both mutations,
o values were calculated using Equation 4 with 2k- = 40,000 s-1 and
= 200 s-1, assuming that only ß2 and
2 change upon mutation. For
S5C and
S5Q receptors, the calculated values were
4.5 and 0.77 ms, respectively, in reasonable agreement with the experimental values of 7.5 and 0.6 ms (Table 2).
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The similarity of the effects of S/Q and C/I residues suggests that the volume of the 5 sidechain (
89 and
144 Å3 for S and Q, and
109 and
167 Å3 for C and I, respectively;
5 increases the AChR gating equilibrium constant.
Mutational Analysis of the -subunit M2M3L Domain
To further explore the role of the M2M3L in gating, we made additional mutations at different positions of the subunit (Fig 4 and Table 2). Both T
A and T
I mutations at position
3 shortened
o of ACh-gated currents by a factor of
4.5 (0.160.17 vs. 0.75 ms in the wild type). For these mutations, kinetic modeling of single-channel clusters elicited by ACh was possible because the opening rate constant was slower than the wild type's and, therefore, the use of choline was not needed. ß2 was reduced from
50,000 s-1 in the wild type to 2,127 s-1 and 1,730 s-1 in the Ala and Ile mutants (Fig 5), whereas
2 increased from 2,500 s-1 in the wild type to
6,000 s-1 in both mutants. Thus, the consequences of these
T3 mutations are nearly opposite to those of the
S5I mutation; i.e., ß2 decreases
26-fold and
2 increases
2.4-fold. The net effect of these mutations is to decrease
2 by
62-fold. Returning to Equation 4, the shorter
o in
T3A and
T3I AChRs mainly reflects a reduced number of openings per burst (because of a much smaller ß2). Like mutations at position
5, mutations of
3 residues also had a larger effect on channel opening than on channel closing.
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The doseresponse properties of T3I and
T3A activated by ACh are shown in Fig 5. The Kd values of these two mutants for ACh were similar to that of the wild type, while the
2 values were smaller. This result is consistent with the suggestion that the nearby mutation
S5I does not alter the Kd. Together, these results suggest that the M2M3L of the
subunit is not an important determinant of agonist affinity.
We next studied the effect of S A and S
I mutations of the
4 residue. With ACh as the agonist,
S4A AChRs had a
o of
1.2 ms, which is not very different from that of the wild type, while the
S4I mutant had a
o of
0.36 ms (Fig 4 and Table 2). Further kinetic and doseresponse analyses of the
S4I mutant in the presence of ACh are shown in Fig 5. ß2
1,500 s-1, which is >30x slower than the wild-type's value. In this regard,
S4I resembles the
T3I mutant. Unlike the
3 mutant, however,
2
2,777 s-1, which is similar to that of the wild type. Again, mutations at the
4 position mostly affect the channel opening rate constant. Doseresponse analysis of
S4I indicates that this mutation decreases the Kd, but less than approximately threefold (Fig 5).
In summary, positions 3, 4, and 5 of the M2M3L of the subunit were probed using mutagenesis. Mutations in this region mainly affect channel gating with little effect on agonist binding. Hydrophobic substitutions at position 5 increase, and at positions 3 and 4 decrease, the channel opening rate constant.
The M2M3L Domain in Other Subunits
In the ß and subunits, an S
I mutation in position 4 produced channels having prolonged
o values. With ACh as the agonist,
o was
9.1 ms for ßS4I and 6.6 ms for
S4I AChRs, respectively (Fig 6). Again, we quantified the effects of the mutations on the opening and closing rate constants using choline as the agonist (Fig 7). For ßS4I, ß2 was
3.9-fold faster and
2 was
6.1-fold slower than the wild type (
2 increased
23.5-fold). The effect of
S4I was less pronounced, as ß2 was only
2.8-fold faster and
2 was
2.2-fold slower than in the wild type (
2 increased approximately sixfold). Application of Equation 4 assuming that only
0 changes upon mutation and using a
value of 200 s-1 yields
o values of 3.7 and 2.2 ms for the ßS4I and
S4I mutant receptors, respectively.
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The S4I mutation did not affect channel gating. We scanned nearby residues in
by mutation to Ile, and none yielded AChRs having prolonged
o values (Fig 6 and Table 2).
Change in the Length of the M2M3L
To explore the effect of changes in length, we engineered -subunit mutants having longer or shorter M2M3L regions. Decreasing the linker size by one residue shortened
o (
0.28 ms), while adding one residue lengthened it (
1.46 ms) (Fig 4 and Table 2). The briefer
o of the deletion mutant suggests that ß2 is slower and
2 is
1.5-fold faster when the M2M3L is shorter. The
o of the addition mutant suggests that ß2 is faster, and/or
2 is slower, when the M2M3L is longer.
Mapping the Gating Transition State at the M2M3L
To probe the closed-like-vs.-open-like character of the M2M3L at the gating transition state, we examined the correlation between rate and equilibrium constants of the gating reaction for the side-chain series at positions 3 (S, A, and I),
4 (S and I) and
5 (S, Q, C, and I). A linear correlation ("linear free-energy relationship;"
, is a measure of how "open" the probed region is when the transition state is reached, with
= 0 being fully closed and
= 1 being fully open.
For the 3 position,
= 0.789 ± 0.010 in the presence of ACh (the correlation coefficient between log ß and log
is 0.985). For the
4 position,
= 0.971 in the presence of ACh as well (two-point relation). For the
5 position,
= 0.694 ± 0.028 in the presence of choline (Fig 3 B; the correlation coefficient between log ß and log
is 0.988). These results suggest that the conformation of the region around
S3-5 at the transition state of the gating allosteric transition is
70% open-like (
30% closed-like). It is important to note that
values are likely to be insensitive to the nature of the particular ligand used as the agonist (
values estimated in the presence of choline are good estimates of the corresponding values in the presence of ACh.
values of the ß4 and
4 positions were calculated from two-point relations. With choline as the agonist, these values were 0.431 in ß and 0.568 in
. The
value corresponding to the
subunit, however, could not be measured because none of the tested mutations in the M2M3L of this subunit caused a significant change in the gating equilibrium constant.
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DISCUSSION |
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S5I Is a Typical SCCMS Mutant
Like many other SCCMS mutants (S5I AChRs have a prolonged apparent open time (Table 2), an increased sensitivity to the metabolite choline (Fig 3 A and
S5I mutation and, therefore, that the increased gating in the presence of ACh can be accounted for by a more favorable isomerization step (an increased unliganded gating equilibrium constant). Although the term "slow channel" that describes the SCCMS phenotype refers to the slow decay of the end-plate current, the predominant underlying mechanism of the disease is the accelerated channel opening:
S5I AChRs are hyperactive channels that open too fast. Along similar lines, "fast-channel" congenital myasthenic syndromes (e.g.,
P121L;
Mechanistic Basis of the SCCMS Phenotype of S5I Receptors
It is difficult to analyze the kinetic and doseresponse properties of AChRs that open extremely rapidly, such as S5I. The brevity of sojourns in the closed diliganded state results in many missed closures, and this distortion of the data makes it impossible to estimate binding and gating rate constants directly from the dwell-time series. Moreover, in these cases, the gating equilibrium constant is very large, hence the estimates of gating (
2) and agonist-dissociation (Kd) equilibrium constants obtained by fitting Popen-vs.-concentration curves are ill-determined (i.e., have large coefficients of variation).
Another consequence of a very fast opening rate constant is that the EC50 value may no longer depend on the agonist affinity for the closed state of the receptor. According to Scheme 2, when >>1,(
), where Jd is the microscopic dissociation equilibrium constant from the open state.
To overcome the limitations imposed by using ACh as the agonist, we analyzed the single-channel behavior of the S5I mutant in the presence of choline, a low-efficacy agonist that opens the channel relatively slowly. In the presence of choline, we found that the mutation affects gating (
28-fold increase in ß2,
3.3-fold decrease in
2) while having little effect on agonist affinity for the closed state. The latter was also suggested by the analysis of Popen-vs.-ACh concentration data of nearby mutations that decrease
2 (
T3I,
T3A, and
S4I). Because the mutation increases the gating equilibrium constant to the same extent regardless of whether ACh or choline is bound to the receptor, we also suggest that the agonist affinity for the open channel is largely unaffected. These results clearly indicate that the predominant effect of the
S5I mutation is to favor the channel's isomerization step.
The observed prolongation of o in the presence of ACh by the
S5I mutation was similar to the value predicted using Equation 4, with
equal to 200 s-1. Considering that open channels desensitize at a rate <10 s-1 (
50 s-1 (
140 s-1; that is,
70 s-1 from each binding site if they were equivalent. This value is
300x slower than the dissociation rate constant from the closed conformation, and (only) about three times faster than the dissociation rate constant from the desensitized state (
Previous estimates of the agonist-dissociation rate constant from diliganded open AChRs consisted of calculations in the context of cyclic kinetic schemes, assuming microscopic reversibility and identical association rate constants of the agonist to the closed and open conformations. For ACh, values of 0.2 s-1 for adult-type (2ß
;
2ß
;
Applying a similar approach, we can calculate the dissociation rate constant from diliganded open channels using published values of the agonist-dissociation rate constant from diliganded closed channels and the equilibrium constants of mono- and diliganded gating. For ACh bound to adult-type AChRs, values include 3.7 s-1 (from data in
The reason for the disparity of these estimates is not clear, but is most likely related to inaccuracies in the identification of monoliganded openings (
Taken together, our results suggest that S5I receptors can open as fast as at
1.4 x 106 s-1. Experiments with other very-fast-opening mutants, as well as confirmation of the
value estimated here, are needed to explore the issue of a speed limit for AChR gating. An upper limit of 108 s-1 has been proposed for the T
R conformational change of hemoglobin (
Mutational Analysis of the M2M3L Domain
The results indicate that the hydrophobic amino acids Phe, Cys, and Ile at the 5 position increase the diliganded gating equilibrium constant and produce the SCCMS phenotype. The less hydrophobic residues Asp, Lys, Gln, Ala, and Ser result in wild-typelike gating. As noted before, the large variation in the size of these side chains indicates that residue volume is not a significant factor with regard to gating. It is also interesting that the
5 mutants have either a wild-type or an SCCMS-like phenotype, in spite of the variety of residues tested. Taken alone, this pattern would suggest that the local environment of
S5 changes from polar to nonpolar upon channel opening.
It is remarkable that similar modifications to adjacent residues in the M2M3L have such diverse effects on gating. As opposed to the effect of S I and S
A mutations at position
5, similar substitutions at positions
3 (T
I and T
A) or
4 (S
I) impair gating (i.e., decrease
2), making the interpretation of the structurefunction results less straightforward. This suggests a marked dependence of the effect of mutations on the position in the primary sequence of the M2M3L. This pattern is different from that of other regions of the AChR like M2, where, regardless of the position, mutations have either little effect or favor (rather than impair) gating (e.g.,
The effect of M2M3L mutations was different in the different subunits even though they were at positions that were homologous in terms of sequence. Even though mutations in ß and yielded AChRs with increased
2 values, characteristic of the SCCMS phenotype, position 4 was more sensitive to mutation than position 5, as opposed to the situation in the
subunit. Also, the
o values for the ß- and
-subunit mutants predicted by Equation 4, assuming that only
0 changes upon mutation and a value of 200 s-1 for
, differ from the experimentally derived
o values. More experiments are needed to determine the agonist-binding properties of the ß and
M2M3L mutants. Interestingly, mutations at positions
36 did not have any effect on channel gating whatsoever. Thus, we conclude that, in the M2M3L, homology in sequence does not coincide with homology in function. It is unlikely that this interpretation is caused by the vagaries of primary-structure alignment because the M2M3L motif is very well conserved (Table 1). That mutations in the
subunit do not affect gating is yet additional evidence for the distinct role of this subunit in the receptor's function (
Physical Mechanism
It is remarkable that the gating behavior of the -subunit M2M3L mutants was trimodal, being either that of the wild type (
S5D,
S5K, and
S5A), of the SCCMS mutant
S5I (
S5I,
S5C, and
S5F), or of the
T3 mutants (
T3A,
T3I, and
S4I). Considering that these side chains cover a rather wide range of physicochemical properties, we would have expected to observe a continuum of gating phenotypes if the local environment of the side chains themselves had been a critical determinant of the gating equilibrium constant. Instead, the modality of the results suggests that the mutations cause the M2M3L as a whole to adopt one of at least three conformations, each leading to a discrete phenotype.
M2M3L Mutations in Other Receptors
Mutations of the M2M3L domain have been studied in other ionotropic receptors. In glycine receptors, these have been shown to selectively impair gating (according to our numbering system: 1 R-3L,
1 R-3Q,
1 K2E, and
1 Y5C;
1 K2E mutant (
3 position of muscle AChRs. In GABAC receptor
1 homomers, the R2A mutation (
I mutations at positions
5, ß4, and
4 of the muscle AChR. Mutation of position 5 of the neuronal AChR subunits
3,
7, or position 6 of ß4 impair gating without affecting binding (
In summary, the M2M3L is an important domain with regard to channel gating. However, whether it acts as the crucial transduction element between the binding sites and the pore, as it has often been suggested (e.g.,
The M2M3L and the Gating Reaction Pathway
Through value analysis (
, ß, and
subunits. In the
subunit,
0.7. This suggests that, during the opening reaction, the closed
open conformational rearrangement of this domain is >70% complete at the transition state (or <30% complete when going in the open
closed direction). In the
and ß subunits, however,
was found to be
0.57 and
0.43, respectively.
values have been suggested to reflect the sequence of conformational rearrangements with the movement of regions having larger
values preceding those with smaller ones (
subunit leads the conformational change in the opening direction, being followed by
, and then by ß, at least as far as the M2M3L domains are concerned.
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Footnotes |
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1 Abbreviations used in this paper: AChR, acetylcholine receptor channel; M2, second transmembrane segment; SCCMS, slow-channel congenital myasthenic syndrome.
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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 (NS-23513) and S.M. Sine (NS-31744), the American Heart Association (New York State Affiliate) to C. Grosman, the Howard Hughes Medical Institute (Medical Student Research Training Fellow) to F.N. Salamone, and the W.M. Keck Foundation to the State University of New York at Buffalo.
Submitted: 17 April 2000
Revised: 9 June 2000
Accepted: 5 July 2000
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References |
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Auerbach, A., and Akk, G. 1998. Desensitization of mouse nicotinic acetylcholine receptor channels. J. Gen. Physiol. 112:181-197
Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1992. Short Protocols in Molecular Biology. John Wiley & Sons, New York, NY. 9.1.19.1.6.
Campos-Caro, A., Sala, S., Ballesta, J.J., Vicente-Agullo, F., Criado, M., and Sala, F. 1996. A single residue in the M2M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors. Proc. Natl. Acad. Sci. USA. 93:6118-6123
Chen, J., and Auerbach, A. 1998. A distinct contribution of the subunit to acetylcholine receptor channel activation revealed by mutations of the M2 segment. Biophys. J. 75:218-225
Cohen, B.N., Labarca, C., Czyzyk, L., Davidson, N., and Lester, H.A. 1992. Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region. J. Gen. Physiol. 99:545-572[Abstract].
Colquhoun, D., and Sakmann, B. 1985. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J. Physiol. 369:501-557[Abstract].
Colquhoun, D., and Sigworth, F.J. 1995. Fitting and statistical analysis of single-channel records. In Sakmann B., Neher E., eds. Single-Channel Recording. New York, NY, Plenum Publishing Corp, 483-585.
Corringer, P.J., Bertrand, S., Galzi, J.L., Devillers-Thiéry, A., Changeux, J.P., and Bertrand, D. 1999. Mutational analysis of the charge selectivity filter of the 7 nicotinic acetylcholine receptor. Neuron. 22:831-843[Medline].
Croxen, R., Newland, C., Beeson, D., Oosterhuis, H., Chauplannaz, G., Vincent, A., and Newsom-Davis, J. 1997. Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome. Hum. Mol. Genet. 6:767-774
Davies, P.A., Pistis, M., Hanna, M.C., Peters, J.A., Lambert, J.J., Hales, T.G., and Kirkness, E.F. 1999. The 5-HT3B subunit is a major determinant of serotonin-receptor function. Nature. 397:359-363[Medline].
del Castillo, J., and Katz, B. 1957. Interaction at endplate receptors between different choline derivatives. Proc. R. Soc. Lond. B Biol. Sci. 146:369-381[Medline].
Eaton, W.A., Henry, E.R., and Hofrichter, J. 1991. Application of linear free energy relations to protein conformational changes: the quaternary structural change of hemoglobin. Proc. Natl. Acad. Sci. USA. 88:4472-4475[Abstract].
Edelstein, S.J., Schaad, O., Henry, E., Bertrand, D., and Changeux, J.P. 1996. A kinetic mechanism for nicotinic acetylcholine receptors based on multiple allosteric transitions. Biol. Cybern. 75:361-379[Medline].
Edelstein, S.J., Schaad, O., and Changeux, J.P. 1997. Myasthenic nicotinic receptor mutant interpreted in terms of the allosteric model. Crit. Acad. Sci. (Paris). 320:953-961.
Edmonds, B., Gibb, A.J., and Colquhoun, D. 1995. Mechanisms of activation of muscle nicotinic acetylcholine receptors and the time course of endplate currents. Annu. Rev. Physiol. 57:469-493[Medline].
Fersht, A. 1999. Structure and mechanism in protein science. New York, NY, W.H. Freeman and Co, pp. 540570.
Filatov, G.N., and White, M.M. 1995. The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating. Mol. Pharmacol. 48:379-384[Abstract].
Fisher, J.L., and Macdonald, R.L. 1998. The role of an subtype M2-M3 His in regulating inhibition of GABAA receptor current by zinc and other divalent cations. J. Neurosci. 18:2944-2953
Galzi, J.L., Devillers-Thiéry, A., Hussy, N., Bertrand, S., Changeux, J.P., and Bertrand, D. 1992. Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature. 359:500-505[Medline].
Grosman, C., and Auerbach, A. 2000a. Kinetic, mechanistic, and structural aspects of unliganded gating of acetylcholine receptor channels. A single-channel study of M2 12' mutants. J. Gen. Physiol. 115:621-635
Grosman, C., and Auerbach, A. 2000b. Asymmetric and independent contribution of M2 12' residues to diliganded gating of acetylcholine receptor channels. A single-channel study with choline as the agonist. J. Gen. Physiol. 115:637-651
Grosman, C., Zhou, M., and Auerbach, A. 2000. Mapping the conformational wave of acetylcholine receptor channel gating. Nature. 403:773-776[Medline].
Hagen, S.J., Hofrichter, J., Szabo, A., and Eaton, W.A. 1996. Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding. Proc. Natl. Acad. Sci. USA. 93:11615-11617
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch clamp technique for high resolution current recordings from cells and cell-free membrane patches. Pflügers Arch. 391:85-100[Medline].
Imoto, K., Busch, C., Sakmann, B., Mishina, M., Konno, T., Nakai, J., Bujo, H., Mori, Y., Fukuda, K., and Numa, S. 1988. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 335:645-648[Medline].
Itzhaki, L.S., Otzen, D.E., and Fersht, A.R. 1995. The structure of the transition state for folding of chymotrypsin inhibitor 2 analyzed by protein engineering methods: evidence for a nucleationcondensation mechanism for protein folding. J. Mol. Biol. 254:260-288[Medline].
Jackson, M.B. 1988. Dependence of acetylcholine receptor channel kinetics on agonist concentration in cultured mouse muscle fibres. J. Physiol. 397:555-583[Abstract].
Karlin, A., and Akabas, M.H. 1995. Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins. Neuron. 15:1231-1244[Medline].
Kusama, T., Wang, J.-B., Spivak, C.E., and Uhl, G.R. 1994. Mutagenesis of the GABA 1 receptor alters agonist affinity and channel gating. Neuroreport. 5:1209-1212[Medline].
Labarca, C., Nowak, M.W., Zhang, H., Tang, L., Deshpande, P., and Lester, H.A. 1995. Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors. Nature. 376:514-516[Medline].
Leffler, J.E., and Grunwald, E. 1963. Rates and equilibria of organic reactions. New York, NY, John Wiley & Sons, pp. 458 pp.
Lewis, T.M., Sivilotti, L.G., Colquhoun, D., Gardiner, R.M., Schoepfer, R., and Rees, M. 1998. Properties of human glycine receptors containing the hyperekplexia mutation 1 (K276E), expressed in Xenopus oocytes. J. Physiol. 507:25-40
Lingle, C.J., Maconochie, D., and Steinbach, J.H. 1992. Activation of skeletal muscle nicotinic acetylcholine receptors. J. Membr. Biol. 126:195-217[Medline].
Lynch, J.W., Rajendra, S., Pierce, K.D., Handford, C.A., Barry, P.H., and Schofield, P.R. 1997. Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel. EMBO (Eur. Mol. Biol. Organ.) J. 16:110-120
Magleby, K.L., and Stevens, C.F. 1972. The effect of voltage on the time course of end-plate currents. J. Physiol. 223:151-171[Medline].
Mihic, S.J., Ye, Q., Wick, M.J., Koltchine, V.V., Krasowski, M.D., Finn, S.E., Mascia, M.P., Valenzuela, C.F., Hanson, K.H., and Greenblatt, E.P. et al. 1997. Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature. 389:385-389[Medline].
Monod, J., Wyman, J., and Changeux, J.-P. 1965. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12:88-118[Medline].
Ohno, K., Hutchinson, D.O., Milone, M., Brengman, J.M., Bouzat, C., Sine, S.M., and Engel, A.G. 1995. Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the subunit. Proc. Natl. Acad. Sci. USA 92:758-762[Abstract].
Ohno, K., Wang, H.-L., Milone, M., Bren, N., Brengman, J.M., Nakano, S., Quiram, P., Pruitt, J.N., Sine, S.M., and Engel, A.G. 1996. Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor subunit. Neuron. 17:157-170[Medline].
Ortells, M.O., and Lunt, G.G. 1995. Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci. 18:121-127[Medline].
Qin, F., Auerbach, A., and Sachs, F. 1996. Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys. J. 70:264-280[Abstract].
Qin, F., Auerbach, A., and Sachs, F. 1997. Maximum likelihood estimation of aggregated Markov processes. Proc. R. Soc. Lond. B Biol. Sci. 264:375-383[Medline].
Rovira, J.C., Ballesta, J.J., Vicente-Agullo, F., Campos-Caro, A., Criado, M., Sala, F., and Sala, S. 1998. A residue in the middle of the M2M3 loop of the beta4 subunit specifically affects gating of neuronal nicotinic receptors. FEBS Lett. 433:89-92[Medline].
Rovira, J.C., Vicente-Agullo, F., Campos-Caro, A., Criado, M., Sala, F., Sala, S., and Ballesta, J.J. 1999. Gating of alpha3beta4 neuronal nicotinic receptor can be controlled by the loop M2M3 of both alpha3 and beta4 subunits. Pflügers Arch. 439:86-92.
Sachs, F., Neil, J., and Barkakati, N. 1982. The automated analysis of data from single ionic channels. Pflügers Arch. 395:331-340.
Sakmann, B., Patlak, J., and Neher, E. 1980. Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist. Nature. 286:71-73[Medline].
Salamone, F.N., Zhou, M., and Auerbach, A. 1999. A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics. J. Physiol. 516:315-330
Schoepfer, R., Conroy, W.G., Whiting, P., Gore, M., and Lindstrom, J. 1990. Brain -bungarotoxin binding protein cDNAs and Mabs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron. 5:35-48[Medline].
Sigurdson, W., and A. Auerbach. 1994. Frequent, long-lived spontaneous and mono-liganded openings of nicotinic acetylcholine receptors with M2 mutations. Neuroscience Society Annual Meeting Abstract. 20:1132.
Sigworth, F.J., and Sine, S.M. 1987. Data transformations for improved display and fitting of single channel dwell-time histograms. Biophys. J. 52:1047-1054[Abstract].
Sine, S.M. 1993. Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of residues that determine curare selectivity. Proc. Natl. Acad. Sci. USA. 90:9436-9440[Abstract].
Ternström, T., Mayor, U., Akke, M., and Oliveberg, M. 1999. From snapshot to movie: analysis of protein folding transition states taken one step further. Proc. Natl. Acad. Sci. USA. 96:14854-14859
Villarroel, A., Herlitze, S., Koenen, M., and Sakmann, B. 1991. Location of a threonine residue in the -subunit M2 transmembrane segment that determines the ion flow through the acetylcholine receptor channel. Proc. R. Soc. Lond. B Biol. Sci. 243:69-74[Medline].
Villegas, V., Martínez, J.C., Avilés, F.X., and Serrano, Luis. 1998. Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain. J. Mol. Biol. 283:1027-1036[Medline].
Wang, H.L., Auerbach, A., Bren, N., Ohno, K., Engel, A.G., and Sine, S.M. 1997. Mutation in the M1 domain of the acetylcholine receptor subunit decreases the rate of agonist dissociation. J. Gen. Physiol. 109:757-766
Zamyatnin, A.A. 1972. Protein volume in solution. Prog. Biophys. Mol. Biol. 24:109-123.
Zhou, M., Engel, A.G., and Auerbach, A. 1999. Serum choline activates mutant acetylcholine receptors that cause slow channel congenital myasthenic syndromes. Proc. Natl. Acad. Sci. USA. 96:10466-10471