Correspondence to: Cecilia Bouzat, Instituto de Investigaciones Bioquímicas, UNS-CONICET, Camino La Carrindanga Km 7, 8000 Bahía Blanca, Argentina. Fax:54-291-4861201 E-mail:inbouzat{at}criba.edu.ar.
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Abstract |
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The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated T422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of
T422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of
T422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.
Key Words: patch clamp, kinetic analysis, nicotinic acetylcholine receptor channel gating, fourth transmembrane domain, hydrogen bond
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INTRODUCTION |
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The nicotinic acetylcholine receptor (AChR)1 is a pentamer of homologous subunits with composition 2ß
in fetal muscle and
2ß
in the adult. Each subunit contains an amino-terminal extracellular domain of ~210 amino acids, four transmembrane domains (M1M4), and a short extracellular carboxy-terminal tail. The M2 domain of each subunit contributes to the cation-selective channel, and agonist binding triggers motion of M2 to initiate ion flow (
subunit of Torpedo californica AChR, making these the best candidates for residues in contact with the lipid bilayer (
subunit, we recently showed that T422 affects channel open duration (
Here we examine the structural and mechanistic contributions of the M4 domain to AChR activation by systematically mutating T422 of the mouse muscle AChR and evaluating changes in activation kinetics. Our results show that
T422 contributes through a hydrogen bond to both opening and closing steps.
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MATERIALS AND METHODS |
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Construction of Mutant Subunits
Mouse cDNAs were used subcloned into the cytomegalovirus-based expression vector pRBG4 ( subunits were constructed by bridging the naturally occurring sites BstX-1 and BspM-1 with synthetic double-stranded oligonucleotides (Bio-Synthesis Inc.), essentially as described previously (
Expression of AChR
Human embryonic kidney cells (HEK293) were transfected with (wild-type or mutant), ß,
, and
cDNAs using calcium phosphate precipitation at a subunit ratio of 2:1:1:1 for
:ß:
:
, respectively, essentially as described previously (
Patch-Clamp Recordings
Recordings were obtained in the cell-attached configuration (, and were coated with Sylgard (Dow Corning Corp.). Pipette concentrations of ACh ranged from 10 to 1000 µM. Single channel currents were recorded using an Axopatch 200 B patch-clamp amplifier (Axon Instruments, Inc.) and digitized at 94 kHz with a PCM adapter (VR-10B; Instrutech). Data were transferred to a computer using the program Acquire (Bruxton Corp.) and detected by the half-amplitude threshold criterion using the program TAC 3.0 (Bruxton Corp.) at a final bandwidth of 10 kHz. Open- and closed-time histograms were plotted using a logarithmic abscissa and a square root ordinate (
Open probability within clusters (Popen) was experimentally determined at each ACh concentration by calculating the mean fraction of time the channel is open within a cluster. The experimental Popen determinations were compared with theoretical doseresponse curves calculated from either Scheme 1 or Scheme 2, using the fitted rate constants in Table 2.
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Kinetic Analysis
At ACh concentrations >10 µM, clusters of openings corresponding to a single channel were identified as a series of closely spaced events preceded and followed by closed intervals greater than a specified duration; this duration was taken as the point of intersection of the predominant closed-time component and the succeeding component in the closed-time histogram. To minimize errors in assigning cluster boundaries at the lower ACh concentrations, we analyzed only recordings from patches with low channel activity. Only clusters containing >10 openings were considered for further analysis. In addition, any clusters showing double openings were rejected. For each recording, kinetically homogeneous clusters were selected based on their mean channel open duration and open probability distributions (
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RESULTS |
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We previously examined the M4 transmembrane domain by alanine-scanning mutagenesis, and found that T422 was unique in that its mutation markedly affected the rate of channel closing (
T422 to AChR activation, we replaced it by polar (serine and asparagine), aromatic (tryptophan and tyrosine), and nonpolar amino acids (alanine, valine, and cysteine). Then we transfected HEK293 cells with either wild-type or mutant
plus wild-type ß,
, and
subunit cDNAs and recorded single channel currents.
AChR channels were activated by a range of desensitizing concentrations of ACh (101,000 µM) to produce clear clusters of events corresponding to a single channel (
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For both wild-type and mutant AChRs, the predominant peak of the closed-time histograms moves to shorter duration with increasing ACh concentration (Fig 2). However, for mutant AChRs containing alanine, closed intervals are clearly longer than those of wild-type AChRs at all ACh concentrations (Fig 2). Similar results were obtained for receptors containing nonpolar (valine and cysteine) or aromatic (tryptophan and tyrosine) substitutions. The predominant open-time component of either wild-type or mutant AChRs does not change with changes in ACh concentration (Fig 2). However for T422A (Fig 2), as well as for
T422V,
T422W,
T422Y, and
T422C AChRs (not shown), open-time distributions shift to briefer durations compared with wild-type AChRs. In contrast, when T422 is replaced by serine (Fig 2) or asparagine (not shown), open- and closed-duration histograms are similar to those of wild type.
The observed changes in the kinetics of AChR activation could be due to changes in rate constants underlying either ACh binding or channel gating steps. To identify the kinetic steps affected by each M4 mutation, we fitted kinetic schemes to the open and closed dwell times. For wild-type AChRs, we used the classical activation scheme (Scheme 1), where two agonists (A) bind to the receptor (R) in the resting state with association rates k+1 and k+2 and dissociate with rates k-1 and k-2. Receptors occupied by one agonist open with rate ß1 and close with rate 1, and AChRs occupied by two agonist molecules open with rate ß2 and close with rate
2. At high agonist concentrations (>100 µM ACh) channel blockade is evident, requiring addition of the blocked state A2B.
To estimate the set of rate constants, Scheme 1 was fitted to the data using the program MIL (
For wild-type AChR, ß2 was constrained to its known value (
Rate constant estimates obtained for wild-type AChR, shown in Table 2, agree with those previously reported for mouse AChR (
We next applied kinetic analysis to AChRs containing mutations of T422 using clusters selected as described for wild-type AChRs (Table 1). Scheme 1 could not adequately describe data obtained from AChRs containing the hydrophobic/aromatic mutations
T422A,
T422V,
T422Y,
T422W, and
T422C. Scheme 1 predicts a minor open-time component, associated with AR*, that decreases in relative area with increasing ACh concentration, but these mutant channels show two open components whose relative areas do not change with changes in ACh concentration. To account for two concentration-independent open-time components, we fitted Scheme 2 to dwell times from mutant AChRs. The classical activation scheme (Scheme 1) is a subset of Scheme 2, which has the opening step separated into two sequential steps. An alternative branched scheme, containing two open states connected to the A2R closed state, also described the data, but was found to be less likely for all mutant AChRs, as judged by maximum likelihood analysis. For example, for the
T422A AChR, Scheme 2 was e110 times more likely than the branched scheme.
In fitting Scheme 2 to data from AChRs containing hydrophobic or aromatic mutations, the two binding sites were assumed to have equal microscopic rate constants, as found for wild-type AChR. In contrast to fitting Scheme 1 to data from the wild type, it was possible to allow ß2 to vary freely. For the hydrophobic and aromatic mutations, the unblocking rate constant (k-b) was constrained to the value used for wild-type AChRs because M4 does not contribute to the channel pore (2) (Table 2). Therefore, the doubly occupied mutant AChR opens with greater latency and reduced efficiency compared with wild-type AChRs. However, after opening, the mutant channels rapidly return to the closed state or, with low probability, reach a more stable open state, represented by A2R** in Scheme 2.
In contrast to the hydrophobic and aromatic mutations, AChRs containing the serine or asparagine mutations could be well fitted by Scheme 1 (Fig 2). Using the same constraints as in wild-type AChRs (ß2 = 49,000 s-1 and equivalent binding sites), the best-fit rate constants indicate that the primary effect of the serine and asparagine mutations is a twofold increase in the channel closing rate, 2 (Table 2). Because, as described above for wild-type AChRs, it was necessary to constrain ß2 in fitting data from
T422S and
T422N AChRs, and because the hydrophobic and aromatic mutations affected ß2, we systematically varied ß2 from 19,000 to 49,000 s-1. Based on likelihood, the best description of the
T422S and
T422N data was obtained when ß2 was 49,000 s-1, as found for wild-type AChR. For example, for the
T422S AChR, the fit using ß2 = 49,000 was e24, e81, and e235 times more likely than with ß2 equal to 39,000, 29,000, and 19,000 s-1, respectively.
Although dwell times from the hydrophobic and aromatic mutants were not well described by Scheme 1, dwell times from wild-type, T422S, and
T422N AChRs could be well described by either Scheme 1 or Scheme 2 (Table 2). To show that changes in rate constants due to the mutations are not scheme dependent, we also fitted Scheme 2 to data from wild-type as well as
T422S and
T422N AChRs (Table 2). Comparison of log likelihoods, obtained after fitting both schemes to wild-type and
T422N data, reveals that Scheme 1 provides the better description; Scheme 1 is e75 and e170 times more likely than Scheme 2 for wild-type and T422N AChRs, respectively. On the other hand, for the
T422S mutation, Scheme 2 is e72 more likely than Scheme 1. Fitting Scheme 2 to data from the serine and asparagine mutations reveals only slight changes in the gating steps, similar to results obtained with Scheme 1 (Table 2). Comparison of wild-type and M4 mutant rate constants, calculated on the basis of Scheme 2, also shows that
T422 contributes primarily to channel opening and closing, and that these steps are largely affected by the presence of hydrophobic or aromatic residues (Table 2). Thus, residues preserving the hydrogen bonding ability of the original threonine support nearly normal gating kinetics.
The channel gating equilibrium constant, 2, calculated as ß2/
2, significantly decreases in most of the mutants (Table 2). For each side chain at position 422, we calculated the net free energy change for the gating equilibrium, as well as the activation free energy changes for opening and closing transitions (Table 3). Free energy changes of ~2 kcal/mol in the gating equilibrium are observed when nonpolar or aromatic amino acids are substituted into position 422, whereas only slight changes occur when serine or asparagine are substituted. When threonine is replaced by either aromatic or nonpolar amino acids, the activation free energy for opening increases, whereas, for all mutants, activation free energy for closing decreases (Table 3).
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Rates of agonist association and dissociation (Table 2) and dissociation equilibrium constants (Table 4) are not significantly affected by these mutations. Thus, T422 selectively affects channel opening and closing transitions.
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To determine the overall consequences of each mutation for AChR activation, and to evaluate consistency of the fitted rate constants, we computed the open probability from the fitted rate constants and compared it with the experimentally determined open probability (Fig 3). For wild-type AChRs, open probability increases with increasing ACh concentration, reaching a maximum of ~0.9 at 100 µM and showing an EC50 of 40 µM (Fig 3). For most mutant AChRs, both the EC50 and maximum open probability change. The open probability curves are shifted to higher ACh concentrations, owing to decreased efficiency of channel gating. For the mutants T422A, T422V, T422W, T422Y, and T422C, open probability is <0.6 at 1,000 µM, and the EC50 values range from 200 to 300 µM (Fig 3). Thus, mutations of T422 affect both efficacy and EC50 for AChR activation. As expected, the profile for the T422N mutant is similar to that of wild-type AChR, and only slight changes are observed when threonine is replaced by serine. The EC50 and maximum open probability values for the T422S mutant are 80 µM and 0.8, respectively (Fig 3). The doseresponse curves computed from the fitted rate constants closely follow the experimentally determined open probability determinations, demonstrating overall consistency of the data.
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DISCUSSION |
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T422 of the M4 domain of the subunit is presumed to be located at the lipidprotein interface, owing to its susceptibility to labeling by a hydrophobic probe (
T422 is the most sensitive to mutation, with the alanine substitution decreasing the mean open time fivefold (
T422 to channel gating. Kinetic analysis of mutant
T422 AChRs disclose relatively small contributions to steps governing ACh binding to the resting, closed state of the AChR, but reveals significant contributions to steps underlying channel opening and closing.
Kinetic analysis requires that open and closed dwell times correspond to a single AChR channel. Clusters of opening events, corresponding to activation of the AChR between desensitized periods, were clearly distinguished at ACh concentrations >10 and 30 µM for wild-type and mutant AChRs, respectively. Because only clusters containing a single open conductance level were considered for kinetic analysis, it is possible to unequivocally assign the dwell times to sojourns in open and closed states of a single AChR. In addition, the clustering behavior observed for all mutant AChRs clearly demonstrates that, like wild-type, they can undergo cycles of desensitization and resensitization in the continued presence of ACh.
Based on gatings kinetics, one can distinguish the polar serine and asparagine from the nonpolar or aromatic amino acid mutations at position 422 as follows. (a) Kinetic changes due to replacement of T422 by serine or asparagine: recordings obtained from these mutant AChRs are similar to those from wild-type AChRs (Fig 1). Slight changes in the open- and closed-time histograms are observed (Fig 2). Data can be well fitted by the classical description of activation for wild-type AChR. The only detectable change is a twofold increase in the channel closing rate. (b) Kinetic changes due to replacement of T422 by nonpolar or aromatic residues: recordings show very brief openings and prolonged closings. The classical activation scheme does not fit the experimental data. However, expanding the classical scheme to include two sequentially connected open states (Scheme 2) satisfactorily describes the data. Kinetic analysis based on Scheme 2 reveals that the rate of opening to the first open state is slower than the analogous step in wild-type AChRs, but the closing rate from this state (
2) is much faster. Because
2 is faster than the competing rate ß3, the open channel (A2R*) has a much greater probability of closing than of reaching the second, more stable open state (A2R**). Thus, nonpolar or aromatic mutations dramatically impair the initial opening step in the gating pathway. Interestingly,
T422C shows a different behavior within this group of mutants; it is the only mutant in which the opening rate is much more affected than the closing rate. Comparison of rate constants of wild-type and M4 mutant AChRs, calculated on the basis of both Scheme 1 and Scheme 2, confirms that
T422 contributes to channel opening and closing regardless of the kinetic scheme used for analysis. Scheme 2 was recently used to explain the activation of an AChR mutated in the M3 domain associated with a congenital myasthenic syndrome (
V285I, also causes abnormally slow opening and rapid closing rates. Thus, Scheme 2 may represent a general mechanism that is not distinguished in wild-type AChR because the intermediate open state may be too short-lived to be detected, and the channel rapidly reaches the final open state. Thus, certain mutations that disrupt gating may have the ability to unmask intermediate open states.
The results also reveal the structural basis of the contribution of T422 to channel gating. Threonine and serine both contain polar hydroxyl groups, but differ by the presence of a methyl group attached to the beta carbon. Although the lack of a methyl group in serine slightly affects gating, T422S AChRs are kinetically similar to wild-type AChRs. However, if only the hydroxyl group of threonine is replaced by a methyl group, as in valine, gating is substantially disrupted. Moreover,
T422V AChRs behave similarly to those containing structurally unrelated amino acids, such as tryptophan. The asparagine side chain provides similarly electronegative atoms to the original threonine side chain, and the polar amide group is similar to the hydroxyl group in its propensity for donating hydrogen bonds; T422N AChRs are almost kinetically identical to wild-type AChRs. Although cysteine can form hydrogen bonds, the kinetics of T422C AChRs differ from those of wild type. This finding can be explained by the fact that the thiol group is a poor hydrogen bond donor and forms weaker hydrogen bonds compared with hydroxyl or amide groups. Because hydroxyl and amide groups can equally well accept hydrogen bonds, and because, in contrast, cysteine is a poor hydrogen bond acceptor (
subunit.
T422 is located in the last third of the M4 domain, closest to the outer leaflet of the bilayer (Fig 4). Whether the M4 domain is an helix (
T422. Although T422 is labeled by the hydrophobic probe TID, and therefore is probably exposed to the lipid, its hydroxyl group could interact with a hydrogen bond acceptor or donor within a transmembrane domain of the same or another subunit. The more energetic hydrogen bonding expected in a hydrophobic environment may account for the substantial functional importance of the hydrogen bond with which T422 is involved. Thus, the present work demonstrates that subtle interactions originating far away from the two main functional domains of the AChR, the ion pore and the binding site, significantly affect channel gating.
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Calculation of free energy changes for channel opening and closing disclosed only slight decreases in free energy for closing between serine and asparagine mutant AChRs, but revealed significant changes in free energy for both opening and closing steps for all other mutant AChRs. The free energy differences are on the order of those calculated for hydrogen bonds between uncharged residues in an aqueous solution (0.51.5 kcal/mol;
The significant role of a hydrogen bond at position 422 in channel gating demonstrated in the current work explains the high degree of conservation of T422 among subunits and species (Fig 4). Abnormal activation of AChR has been shown to underlie congenital myasthenic syndromes (CMS) (
Given the present understanding of the topology of the M4 domain, our results demonstrate that a lipid-exposed residue involved in hydrogen bonding is necessary for proper gating of the AChR.
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Footnotes |
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1 Abbreviations used in this paper: AChR, nicotinic acetylcholine receptor; M4, fourth transmembrane domain.
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Acknowledgements |
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We especially thank Dr. H.-L. Wang for advice and helpful discussion, Ana M. Roccamo, Dora Ortiz, and Horacio De Genaro for their expert technical assistance, and Nina Bren for providing two mutant subunits.
This work was supported by grants from Universidad Nacional del Sur, CONICET, and Agencia Nacional de Promoción Científica (C. Bouzat and F.J. Barrantes), National Institutes of Health grant NS-31744 (S. Sine), and Fogarty International Center grant 1R03 TW01185-01 (C. Bouzat and S. Sine).
Submitted: 6 December 1999
Revised: 27 March 2000
Accepted: 28 March 2000
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References |
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