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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Mutagenesis studies have suggested that the second transmembrane segment (M2) plays a critical role during acetylcholine receptor liganded gating. An adequate description of the relationship between gating and structure of the M2 domain, however, has been hampered by the fact that many M2 mutations increase the opening rate constant to levels that, in the presence of acetylcholine, are unresolvably fast. Here, we show that the use of saturating concentrations of choline, a low-efficacy agonist, is a convenient tool to circumvent this problem. In the presence of 20 mM choline: (a) single-channel currents occur in clusters; (b) fast blockade by choline itself reduces the single-channel conductance by ~50%, yet the excess open-channel noise is only moderate; (c) the kinetics of gating are fitted best by a single-step, C O model; and (d) opening and closing rate constants are within a well resolvable range. Application of this method to a series of recombinant adult mouse muscle M2 12' mutants revealed that: (a) the five homologous M2 12' positions make independent and asymmetric contributions to diliganded gating, the
subunit being the most sensitive to mutation; (b) mutations at
12' increase the diliganded gating equilibrium constant in a manner that is consistent with the sensitivity of the transition state to mutation being ~30% like that of the open state and ~70% like that of the closed state; (c) the relationship between
12' amino acid residue volume, hydrophobicity or
-helical tendency, and the gating equilibrium constant of the corresponding mutants is not straightforward; however, (d) rate and equilibrium constants for the mutant series are linearly correlated (on loglog plots), which suggests that the conformational rearrangements upon mutation are mostly local and that the position of the transition state along the gating reaction coordinate is unaffected by these mutations.
Key Words: nicotinic receptors, allosteric proteins, Brønsted plot, kinetics, double-mutant cycles
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INTRODUCTION |
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The four transmembrane segments (M1M4)1 of the acetylcholine receptor (AChR) are likely to be involved in the conformational changes associated with gating (
An adequate description of the relationship between function and structure of the M2 segment has been hampered by the fact that many mutations increase the channel opening rate constant ("gain-of-function" mutations). The opening rate constant of wild-type AChRs, in the presence of ACh, is at the upper limit of reliable estimation (~30,000100,000 s-1;
One way of dealing with this limitation is to work with a slowly opening mutant on the background of which the mutations of interest are engineered. This approach was taken by D200N mutant, a binding-site mutant with a reduced opening rate constant, as the background AChR on which M2 mutations were introduced. Here we used an alternative method: clusters of openings were elicited with 20 mM choline, a "slowly opening," low-efficacy agonist (
We applied this method to investigate the contribution of the M2 12' residues of the four different AChR subunits (a Ser in and a Thr in
, ß, and
) to gating. By dissecting the effects of mutations on either the opening or the closing rate constant, we provide a detailed picture of the relationship between structure and gating in this region of M2. The results indicate that these positions contribute in an independent and asymmetrical manner to gating, which is most affected by mutations in the
subunit. They also suggest that there is a complex relationship between gating and the physicochemical properties of the amino acid residue in
12', and that the increase in gating equilibrium constant upon mutation is mostly due to a decrease in the closing rate constant with a smaller contribution of an increase in the opening rate constant. For
12' mutations, the logarithms of the rate and equilibrium constants of gating are linearly correlated with a slope that suggests that the local environment of this M2 position is ~30% open-like (70% closed-like) at the transition state of the gating conformational change.
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METHODS |
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Expression, Electrophysiology, and Kinetic Modeling
Mouse AChR cDNA clones, transient expression in HEK-293 cells, cell-attached patch-clamp recordings, cluster definition in the presence of saturating concentrations of agonist, and kinetic analysis were as described in the preceding paper (S268T and
T264S mutations were engineered by overlap PCR (
Error Estimates
Standard errors of the calculated (as opposed to experimentally determined) variables in Table 3 and Table 4, say Y = f (Xi), were estimated according to the following expressions:
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(1) |
and
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(2) |
where Y/
Xi is the partial derivative of Y with respect to each random variable (Xi) evaluated at the corresponding experimentally derived mean values, Var(Xi) is the variance of Xi, and n is the number of different Xi variables. Equation 1 assumes that the random variables are uncorrelated (i.e., that the covariance between any given pair of Xi is zero) and it is exact only when Y is a linear function of Xi. When Y(Xi) is a nonlinear function (our case in Table 3 and Table 4), the result is only approximate because it is the variance of only the linear portion of the Taylor power-series expansion of Y(Xi). However, the smaller the values of Var(Xi), the more accurate the approximation.
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Open-Channel Noise
The experimental excess open-channel noise was calculated by subtracting the closed-level variance from the open-level variance, and is expressed as the ratio between the standard deviation (root mean square, rms) of the noise (ex) and the single-channel current amplitude (i). Such variances were calculated directly from the digitized currents once the samples ("points") were sorted into the closed and open "classes" by the idealization procedure. Thermal (
th) and shot (
sh) excess noise were calculated by Equation 3 (
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(3) |
where kB is Boltzmann's constant, T is the absolute temperature (~295°K), is the single-channel conductance, and B is the bandwidth, and (Equation 4):
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(4) |
where q is the elementary charge (1.602 x 10-19 coulomb). The sum of these two sources of excess noise was calculated by adding their variances, and it is expressed as a standard deviation.
Coupling Energies
Coupling energies between mutations (see Table 3) were calculated as:
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(5) |
where R is the universal gas constant, and the values are the diliganded gating equilibrium constants (the ratio between the opening and closing rate constants) of the background receptor (B), the single mutants (x and y), and the double mutant (x + y). Values calculated in this way are referred to as "mean" values. The corresponding standard errors were calculated by applying Equation 1 and Equation 2 to Equation 5.
From Equation 5 it can be seen that if mutational effects were additive (i.e., if Gº = 0):
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(6) |
This expression was used to calculate the expected diliganded gating equilibrium constant values (2) of double mutants (see Table 3) or the wild type (see Table 4), given the
2 values of the other three members of the cycle.
Coefficients of variation (see Table 3) were calculated as:
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(7) |
where is the difference between the observed (
observed) and the calculated (from Equation 6) values of
2 for the double mutants.
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RESULTS |
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Choline as an Agonist
Fig 1 A shows continuous single-channel traces of 12' S
T AChRs in the presence of a saturating concentration (20 mM) of choline. At this concentration, openings occurred in clusters separated by long-lived silent periods. As with openings elicited by ACh, each cluster represents the activity of a single channel, and the silent intervals between them correspond to sojourns in desensitized states (
O reaction scheme. Fig 2 shows the mean open and closed times, on a cluster-by-cluster basis, for seven patches containing the 12'
S
T mutant.
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One consequence of working with high concentrations of cholinergic agonists is the occurrence of fast blockade. Due to this phenomenon, the current amplitude is reduced and the excess open-channel noise is increased. With respect to the amplitude decrease, both 20 mM choline and 2 mM ACh reduced the current amplitude of S
T receptors by ~50% (3.2 pA at approximately -100 mV). The current amplitude in the presence of blocker (iB) is given by Equation 8:
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(8) |
where io is the current in the absence of blocker, [B] is the blocker's concentration, and KB is its dissociation equilibrium constant from the pore-blocking site. Therefore, KB is ~20 mM for choline and ~2 mM for ACh (
Although the effect of 20 mM choline and 2 mM ACh on the current amplitude was approximately the same, the magnitude of the open-channel noise was different for these two agonists (Fig 3). The excess open-channel noise of S
T AChR in the presence of 20 mM choline (
ex = 0.560 pA; i = 3.4 pA) was less than one third that in the presence of 2 mM ACh (
ex = 1.818 pA; i = 3.4 pA), and comparable with that in the presence of 5 µM ACh (
ex = 0.606 pA; i = 7.4 pA), measured at the analysis bandwidth of 18 kHz. The lower noise in the presence of 20 mM choline is most likely due to the faster dissociation of choline from the blocking site.
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The excess noise, expressed as a fraction of the single-channel current amplitude (ex/i), for the wild type and all the mutants studied here is listed in Table 1. Mutations did not substantially affect the magnitude of the excess noise. Also, during the process of cluster definition, we could not detect any additional fast component in the closed-time distribution that could have been attributed to dwellings in the blocked state. In summary, fast blockade by 20 mM choline neither affected the closed-time distributions nor significantly compromised the single-channel signal idealization process.
Since blocking events were not detected as discrete dwellings in the zero-current level, the idealized open dwell times include sojourns in both the open and blocked states. The mean duration of these apparent openings () is given by:
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(9) |
where and
B are the closing rate constants of the unblocked and blocked channel, respectively. In our particular case, we showed above that KB
[B] and, therefore, Equation 9 reduces to Equation 10:
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(10) |
If blocked channels could not close (i.e., B = 0), then the mean duration of apparent openings would be twice as long as that of true openings in the absence of block (1/
). This is the usual assumption, and corresponds to the sequential scheme for blockade (C
CA
CA2
OA2
OA2B;
B =
), then the mean duration of openings in the presence or absence of block would be the same. It has been shown that muscle AChRs can close while blocked by QX-222 (
2. As no obvious differences between constructs were observed in the single-channel amplitude reduction, we conclude that KB is similar for all of them and, hence, that all the closing rate constant were underestimated by the same factor.
Effects of 12' ST and T
S Mutations
The probability of being open within a cluster of diliganded openings was higher in S
T AChRs than in the wild type because the unliganded gating equilibrium constant increases with the mutation (
T in
and T
S in
, ß, and
subunits. Table 2 shows the results of the kinetic analysis.
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Gating is most affected by mutations in . Mutations in ß and
increase the diliganded gating equilibrium constant (
2) to a smaller extent but while the former increases the opening rate and decreases the closing rate, the latter increases both rate constants. A more detailed analysis of this difference in kinetics will be given below. Mutations in both
subunits result in a minor reduction in the gating equilibrium constant. This effect is more evident when the mutation is engineered on a background receptor that has a higher
2. The addition of the
T
S mutation to constructs having a single mutation in either the ß,
, or
subunit yields receptors with somewhat lower
2 values. This effect is even clearer when the ß +
+
mutant combination with and without the mutation in
is compared. Thus, the effect of mutations in the
subunits (a slight decrease in
2) differs qualitatively from those of mutations in the other subunits (an increase in
2).
If the effect of a given mutation is independent of the background receptor, as hinted above, then the effects of the multiple mutations should be additive (2 values, and calculating the corresponding coupling energies (Equation 5). These can be calculated for any pair of mutations by designing double-mutant thermodynamic cycles (
T and T
S constructs studied here) that test for deviations from independence between pairs of single mutations. Table 3 gives estimates of such deviations calculated as coefficients of variation (Equation 6 and Equation 7) and as free energies of coupling (Equation 5). The small magnitude of the latter suggests that the effects of the different mutations on the equilibrium properties of mutant AChRs can be considered to be essentially additive. Indeed, in the context of allosteric transitions and protein folding, values of
Gºcoupling < 0.2 kcal/mol are considered negligible (
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Prediction of Agonist Efficacies using Double-Mutant Cycles
Finding regions of a protein that make independent contributions to function is a powerful tool in protein engineering. The right combination of single-point mutations necessary to modify a given function to a desired extent can be chosen to tailor a multiply mutated protein based on the behavior of the single mutants. In our case, we found that this approach can be used to expand the variety of chemical structures that can be examined in agonist structurefunction relation studies. For example, when bound to wild-type AChRs, choline is such a low-efficacy agonist that clusters are hard to define and, therefore, the ability to estimate 2 is compromised. However, choline's efficacy (
2) is easily measured when bound to M2 12' mutants. Because any combination of T
S and S
T mutations at the M2 12' position have additive effects (Fig 4 and Fig 5, and Table 3), the efficacy of choline on the wild type could be predicted based on the
2 values of choline on mutant receptors. Fig 7 shows all the double-mutant cycles, having the wild type in one of the vertices, that can be formed with the set of mutations in Table 2. In each case, the efficacy of choline on the wild type was assumed to be unknown and was calculated according to Equation 6. The values calculated from the 13 cycles and their average are listed in Table 4 along with the average of experimentally determined efficacies. The fact that these two values are almost identical confirms the experimental value of
2 for choline on the wild type (~0.05) and suggests the applicability of this protein-engineering approach to other very-low-efficacy agonists.
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Since the closing rate constant values were underestimated (because of fast blockade), the 2 values displayed throughout this paper should not be taken as absolute efficacies of choline on the various constructs. The ratios between these values, however, should be good estimates of the choline's relative efficacies.
Contribution of the 12' Position to Gating
The results in Fig 4 and Table 2 indicate that the ST mutation in
has a greater effect on the equilibrium properties of gating than T
S changes in the other subunits. In an attempt to better understand the structural basis of the role of the
12' position during gating, different amino acid residues were substituted by site-directed mutagenesis. Fig 8 shows single-channel clusters of the wild type and a set of
12' mutants in the presence of 20 mM choline, displayed in increasing order of
2. The results of the kinetic analysis are displayed in Table 5 and show that, as
2 increases, the closing rate constant decreases and the opening rate constant increases, although to a smaller extent (Fig 9).
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Fig 10 plots log 2 (a measure of the free-energy change upon gating) against some properties of the substituted amino acids. As far as the volume is concerned, in the range between ~60 and 150 Å3, the bulkier a residue, the larger is
2. However, this general trend is lost above ~160 Å3 (i.e., for Ile, Tyr, and Trp). With respect to hydrophobicity, highly hydrophilic residues such as Lys, Gln, and Asn have the largest
2 values. However, among less hydrophilic residues, this parameter is not correlated with
2. A lack of correlation was also found between
2 and the
helical propensity, both in polar and nonpolar environments, of the
12' residues. We conclude that the relationship between the gating equilibrium constant of
12' mutant channels and the physicochemical properties of the mutated residues is complex.
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Linear-free Energy Relationships
In contrast with the complexity noted above, Fig 9 reveals a very regular relation between kinetics and equilibrium in 12' mutants. As the gating equilibrium constant increases, the opening rate constant increases, and the closing rate constant decreases almost monotonically. This type of behavior, which is not demanded by any law of thermodynamics, is usually referred to as an "extrathermodynamic" relationship (
G
-vs.-
Gº plot), where the points are fitted best with a straight line. This type of plot is common in physical organic chemistry and is known as a "Brønsted plot" (
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The Brønsted relationship can be written as:
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(11) |
where , the slope, reflects the relative position of the local environment of the mutated residue along the reaction pathway at the time that the transition state is reached. Therefore,
ranges from 0 (closed-like) to 1 (open-like). An analogous expression holds for
2, the slope being (
- 1). It is remarkable that the linearity of this relationship holds for a three-order-of-magnitude range of
2 values (from ~0.035 in
S
A to ~35 in
S
K). As show in Fig 11 A, at
12',
= 0.275 ± 0.023.
There has been some debate as to the statistical validity of a Brønsted plot (log k vs. log k/k') as a measure of the existence of a linear relation (
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(12) |
Fig 11 B shows such a plot for the 12' mutant series. From this plot, we estimate that
= 0.268 ± 0.022, which is very close to the value estimated from the Brønsted plot. We conclude that, at the transition state of diliganded gating, the interactions of the
12' position with the rest of the protein are 27% like those in the open state and 73% like those in the closed state.
The solid lines in Fig 11A and Fig B, are the results of the linear regressions through all the data points (), with the exception of that corresponding to the S
P mutant (
). The behavior of this mutant was considered to be an outlier based on the analysis shown in Table 6. The different behavior of the Pro mutant is also evident from a cursory examination of the data in Table 5. The increase in ß2 upon the S
P mutation is ~ 3.4-fold larger than the one expected from the observed 1.25-fold decrease in
2 and a
-value of 0.275.
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We can go back now to Table 2 and analyze the ST and T
S mutations from an LFER perspective. The
S
T mutant is a member of the mutant series plotted in Fig 11. As compared with the wild type, opening is faster and closing is slower in this mutant, as expected from a mutation that alters the stability of the transition state to an extent that is intermediate between the effects on the stability of the ground (closed and open) states. The slope,
, between
S
T and the wild type (only considering these two points) is ~0.357. The mutation ßT
S also increases the opening rate and decreases the closing rate, but the slope between this mutant and the wild type is
= 0.663. This suggests that the transition state has a more open-like character (i.e., the stabilization of the transition state follows that of the open state more closely) at ß12' than at
12'. Although more mutations are needed to confirm the different slope of ß12', the different values of
suggest that the movement of the ß subunit precedes that of the
subunit during the opening reaction.
Free-Energy Relationships Are Not Always Linear
The TS perturbation in the
subunit alters the behavior of the channel in a different manner than when engineered in the ß or the
subunit. In this case, both opening and closing rate constants get faster and, thus, an LFER does not hold. Therefore, this mutation has a "catalytic" effect stabilizing the transition state to a greater extent than either ground state. In qualitative terms, this effect of the
T
S mutation is also observed when the mutation is engineered on other background AChRs (
T
S, ßT
S, or
S
T; Table 2). Regardless of the background receptor, the increase in the opening rate is far more pronounced than the increase in the closing rate.
The effect of the T
S mutation (in both subunits) on the kinetics of gating is more difficult to assess because, like its effect on the equilibrium constant (see above), the changes were modest. Nevertheless, when coexpressed with other mutant subunits, this effect becomes evident. On the background of the single mutants ßT
S or
S
T, or of the triple mutant ßT
S +
S
T +
T
S, the
mutation also increases both rate constants (Table 2). This suggests that the slightly larger values of the opening and closing rate constants of
T
S receptors, as compared with the wild type, represent genuine changes as well. It is interesting to note that the
T
S mutation does not increase the rates of the
T
S mutant when both mutations are coexpressed. This contrasts with the results in Table 3, which show that the effects of these two mutations on
2 are additive. If the effects of the
T
S and
T
S mutations on the rate constants had been additive as well, the opening and closing rate of the double mutant would have been 2,880 and 19,953 s-1, respectively (from Equation 6, replacing equilibrium constants with rate constants). As compared with the effect of the T
S mutation in the
subunit (the other "catalytic" mutation), the T
S substitution in
affects the opening and closing rate constants to a smaller and more even extent.
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DISCUSSION |
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Choline as an Agonist: An Alternative Approach
Extracting rate constants from single-channel data can be difficult. The observed open and closed interval durations are often related in a complex way with the underlying rate constants, which can only be estimated through the application of elaborate algorithms (for example,
The use of a saturating concentration of choline, a low-efficacy agonist that supports a slow opening rate constant, is a very useful tool to reliably estimate the gating rate constants of 12' M2 mutants. None of the drawbacks associated with the use of a blocking concentration of agonist posed a serious problem. Instead, most closures were detected and the kinetic complexity of the channel was reduced to that of a closed open reaction scheme.
It was somewhat surprising that the open times followed a single exponential considering that spontaneous (
We envisage that this weak-agonist approach will become a useful tool to characterize the gating structurefunction relationships of other regions of the receptor that, like M2 12', increase the diliganded opening rate constant (ß2) upon mutation. Mutations that affect KB (the dissociation equilibrium constant of the agonist from the pore-blocking site), however, could not be dealt with by this approach because the underestimation of the closing rate constant (2) will no longer be the same for the entire mutant series. In these cases,
2 should be estimated at concentrations of agonist that are low enough to prevent blockade, yet high enough to minimize the occurrence of unliganded and monoliganded openings.
Additivity of Mutational Effects: Lack of Interaction between 12' Residues
We investigated whether interactions between the five 12'-position residues change upon (and thus contribute to the free-energy change of) gating. During wild-type diliganded gating, there is a net loss of ~2.0 kcal/mol (2
30) in going from the closed to the open conformation, whereas during unliganded gating there is an uphill change of ~6.5 kcal/mol [
0
1.5 x 10-5, from
While considerable progress has been made in identifying the amino acid residues that interact with the agonist at the transmitter binding sites (reviewed in 2 values of various constructs in the context of a double-mutant cycle analysis (
T (in
) and T
S (elsewhere) 12' mutations on
2 turned out to be additive (Table 3). Also, structural data suggest that cysteines engineered in
and ß 12' do not face the lumen of the channel (
12'). Taken together, we suggest that interactions between 12' residues do not exist at all, in either the closed or open conformations of the channel and, thus, do not contribute to the
Gº of gating.
Additivity of Mutational Effects: Application to Agonist StructureFunction Studies
The diversity of chemical structures that can be examined in agonist structurefunction relation studies is limited by the nature of the receptor used. For example, the efficacy of ACh on the wild-type receptor is so high that more efficacious ligands could hardly be identified if tested on the same receptor. Likewise, agonists with very low efficacy would elicit currents that only seldom get clustered, an absolute requirement for this type of analysis. Application of the "protein engineering" method described here should significantly broaden the range of molecules that can be analyzed in these structurefunction studies. By selecting the right combination of independent mutations, the efficacy of virtually any molecule on the wild type could be known. Here, we showed how this approach was used in the case of choline, an agonist of very low efficacy on muscle AChRs. A similar procedure, engineering additive mutations that slow channel opening, could be applied to test for ligands with higher efficacy than ACh.
Structural Aspects of the 12' Position
The M2 12' position is very well conserved among the members of the superfamily of nicotinoid receptors (see first table in , ß,
/
, and a Ser in
.
From a physicochemical viewpoint, ST and T
S mutations are rather subtle substitutions. Nevertheless, the asymmetry of the muscle AChR's M2 12' position is very well conserved across species, even though only a single-nucleotide mutation is needed to turn the
12' Ser (TCT) into a Thr, or the
(ACC) or ß/
/
(ACT) 12' Thr into a Ser. It was interesting then to test the functional effects of S
T and T
S mutations.
The results in this paper indicate that even these conservative mutations affect gating, being the most sensitive and
the least sensitive subunit (even less considering that both
subunits were mutated). In addition, we showed that the three constructs having Ser only in
, ß, or
subunits (Thr elsewhere) have
2 values that are ~10-, ~51-, and ~52-fold higher than the wild-type's value. In the context of synaptic transmission, these receptors would give rise to slowly decaying end-plate currents much like congenital myasthenic-syndrome mutants do (assuming that the mutations do not affect the kinetics of agonist dissociation). Therefore, there seems to be a tight requirement for a single Ser in 12' for normal function, and this Ser has to be in
. With respect to different side chains in
12', only an S
A or S
G mutation would still be compatible with normal synaptic transmission.
The marked functional asymmetry of this ring of residues leads us to propose that the 12' positions of the different subunits face different environments. This would be the case if, for example, the orientation of the M2 helices around the central pore differed between subunits. Alternatively, this could also happen if the five M2 12' positions, having the same orientation, were packed against the transmembrane segments M1/M3, whose amino acid sequences vary from subunit to subunit.
The relationship between 2 and several physicochemical properties of the residue occupying
12' is not straightforward, at least when these properties are considered one at a time, as in Fig 10. However, the response to volume and hydrophobicity can, to some extent, be rationalized in the framework of the 12' residues being packed against M1 (
helices than in aqueous-based ones (
helix (
A mutation. We hypothesize, then, that a combination of steric hindrance and hydrophobic interactions in the local environment of
12' can explain the observed effects of mutations. Side chains bulkier than the -CH2-OH of Ser [but no larger than the -CH-(CH3)2 of Val] would cause steric repulsion in both the closed and open states. However, this repulsion should be larger in the closed channel conformation to account for the net destabilization of the closed with respect to the open state (i.e., the increase in
2) that accompanies the mutations. This further suggests that, upon opening, there has to be an increase in the volume between
12' and the residue/s against which it packs to explain how an increase in the volume of the side chain causes less strain in the open than in the closed state. It is not clear why the three bulkiest residues tested (Ile, Tyr, and Trp) deviate from this trend. That side chains smaller than that of Ser, like the -CH3 of Ala and the -H of Gly, do not substantially decrease
2 suggests that the cavity where the wild-type Ser is located has the right dimensions to hold the -CH2-OH side chain and that there is not much steric hindrance to be relieved.
We also observed that receptors having Lys, Asn, or Gln at 12' (the three more hydrophilic residues tested) displayed the largest
2 values. These residues are expected to weaken the hydrophobic interactions that contribute to the putative helixhelix packing in both the closed and open conformations. As these mutations increase
2, the packing seems to be tighter in the closed than in the open state, where a looser association of transmembrane domains might relieve the strain of having to accommodate a highly hydrophilic side chain.
In summary, both volume and hydrophobicity considerations lead us to speculate that there is an expansion in the volume around 12' upon channel opening.
Linear Free-Energy Relationships
The fact that mutational effects in 12' conform to linear free-energy relationships is firm evidence that the tested side chains (with the probable exception of Pro) affect the structure of the AChR in a qualitatively similar way, as if the resulting structural changes were part of a continuum (
2). Nevertheless, single point mutations usually cause only local rearrangements (
2 are needed to observe the predicted deviations from linearity (
2 values would be too extreme to be estimated correctly and, therefore, that most LFER applications to gating will be in the linear range.
It is not at all obvious that an LFER should always hold. Such relations are only expected to occur for positions in the protein where the change in the transition state's free energy upon mutation is the linear combination of the free-energy changes of the ground states (S substitution in
or
deviate from such behavior, suggesting that these mutations affect local interactions of the
and
12' sidechains that are present only at the transition state. As a consequence, the position of the
and
12' residues along the reaction coordinate of gating cannot be revealed by the LFER approach, at least when a T
S mutation is used as the perturbation.
As mentioned earlier, the behavior of the S
P receptor was also anomalous. With our results so far, it is not possible to assert that the reason why this mutant is an outlier (Fig 11 and Table 6) lies on the proline's different backbone properties (Pro can act as a hydrogen-bond acceptor but not as a donor). To unequivocally address this issue, the wild-type Ser should be replaced with its corresponding
-hydroxi acid (glyceric acid) so as to preserve the side chain while changing the hydrogen-bonding pattern to that of Pro. This can be done by resorting to the use of unnatural amino acid mutagenesis (
-helix hydrogen bonds contributed by M2
12' play an important role during the process of gating.
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Footnotes |
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1 Abbreviations used in this paper: AChR, acetylcholine channel; LFER, rate-equilibrium linear free-energy relationship; M2, second transmembrane segment; rms, root mean square.
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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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