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Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics.

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 αT422, 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 αT422 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 αT422 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.

The nicotinic acetylcholine receptor (AChR) 1 is a pentamer of homologous subunits with composition ␣ 2 ␤␥␦ in fetal muscle and ␣ 2 ␤⑀␦ in the adult. Each subunit contains an amino-terminal extracellular domain of ‫ف‬ 210 amino acids, four transmembrane domains (M1-M4), 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 (Unwin, 1995). The locations and functional roles of the M1, M3, and M4 transmembrane domains are not as well understood. The M1 domain appears closely associated with both the ion conducting pathway and the bilayer, as revealed by substituted cysteine accessibility and labeling by hydrophobic reagents (Akabas and Karlin, 1995;Blanton and Cohen, 1994). The M3 domain has been shown to have some contact with lipid (Blanton and Cohen, 1994) and to contribute to channel opening and closing rates . The M4 domain is the least conserved among the transmembrane domains, is the most hydrophobic, and has been extensively labeled by hy-drophobic probes Cohen, 1992, 1994), suggesting that it is the outermost of the transmembrane domains. Labeling by the photoactivatable hydrophobic probe [ 3 H]TID {[ 3 H]3-(trifluoromethyl)3m-(iodophenyl)diazirine} occurs at C412, M415, C418, T422, and V425 of the ␣ subunit of Torpedo californica AChR, making these the best candidates for residues in contact with the lipid bilayer (Blanton and Cohen, 1994). Mutagenesis studies suggest that the M4 domain is essential for proper AChR activation (Lee et al., 1994;Ortiz-Miranda et al., 1997). By alanine scanning of the lipid-exposed residues in the M4 domain of the ␣ subunit, we recently showed that T422 affects channel open duration (Bouzat et al., 1998).
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.

Construction of Mutant Subunits
Mouse cDNAs were used subcloned into the cytomegalovirusbased expression vector pRBG4 (Sine, 1993). Mutant ␣ 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 (Bouzat et al., 1998). Single-stranded oligonucleotides were purified by PAGE 664 ␣ T422 AChR in M4 Contributes to Channel Opening and Closing and annealed before ligation. Restriction mapping and dideoxy sequencing on polyacrylamide gels confirmed all constructs.

Patch-Clamp Recordings
Recordings were obtained in the cell-attached configuration (Hamill et al., 1981) at a membrane potential of Ϫ 70 mV and at 20 Њ C. The bath and pipette solutions contained 142 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl 2 , 1.7 mM MgCl 2 , and 10 mM HEPES, pH 7.4. Patch pipettes were pulled from 7052 capillary tubes (Garner Glass), to produce final resistances of 5-7 M ⍀ , and were coated with Sylgard (Dow Corning Corp.). Pipette concentrations of ACh ranged from 10 to 1000 M. 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 halfamplitude 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 (Sigworth and Sine, 1987) and fitted to the sum of exponentials by maximum likelihood using the program TACFit (Bruxton Corp.). For each patch corresponding to a given ACh concentration, the number of opening events ranged from 4,000 to 10,000.
Open probability within clusters ( P open ) was experimentally determined at each ACh concentration by calculating the mean fraction of time the channel is open within a cluster. The experimental P open determinations were compared with theoretical dose-response curves calculated from either Scheme I or II, using the fitted rate constants in Table II.

Kinetic Analysis
At ACh concentrations Ͼ 10 M, 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 Ͼ 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 (Wang et al., 1997). For each cluster within a recording, we calculated the P open and mean open duration and plotted their distributions. Histograms were constructed and fitted by a Gaussian function, which yielded a mean and a standard deviation for each distribution. Typically, the distributions contained a dominant, approxi-  Table I). The similarity of the evaluated parameters before and after selection indicates no qualitative changes in the dwell-time distributions due to the selection procedure. The resulting open and closed intervals, from single patches at several ACh concentrations, were analyzed according to a kinetic scheme using the program MIL (QuB suite, www.qub.buffalo.edu). In brief, MIL simultaneously fits recordings obtained at different ACh concentrations, and estimates rate constants in a kinetic scheme using a maximum likelihood method that corrects for missed events (Qin et al., 1996). We typically included recordings obtained at 10, 30, 50, 100, 200, and 300 M ACh in the analysis of each mutant AChR. The dead time was typically 30 s, but the fitted rate constants were stable over a range of dead times ranging from 18 to 40 s (data not shown). Probability density functions of open and closed durations were calculated from the fitted rate constants and instrumentation dead time and superimposed on the experimental dwell-time histogram, as described by Qin et al. (1996). Calculated rate constants were accepted only if the resulting probability density functions reasonably superimposed upon the experimental open-and closed-duration histograms at all ACh concentrations. To check the final set of fitted rate constants, open and closed intervals were simulated according to Scheme I or II, using the program SIMU (QuB suite). Simulated channel events were analyzed and the resulting dwell times were fitted with the program MIL. The original rate constants were recovered with 10-15% accuracy. Comparison of the fits to different schemes was evaluated by the log likelihood ratio (LLR). For example, a LLR of 50 indicates that the probability of the model with the higher LL is e 50 times more than that of the scheme with the lower LL (Salamone et al., 1999).

R E S U L T S
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 (Bouzat et al., 1998). To further examine the structural contribution of ␣ 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 ␤ , ⑀ , and ␦ subunit cDNAs and recorded single channel currents.
AChR channels were activated by a range of desensitizing concentrations of ACh (10-1,000 M) to produce clear clusters of events corresponding to a single channel (Sakmann et al., 1980). Wild-type as well as mutant AChRs open in clusters of well-defined activation episodes at ACh concentrations Ͼ 10 M. Each activation episode begins with the transition of a single receptor from the desensitized to the activatable state and terminates by returning to the desensitized state ( Fig. 1). For both wild-type and mutant AChRs, closed intervals within clusters become progressively briefer with increasing ACh concentration (Fig. 2). However, compared with wild-type, most of the mutant AChRs show both briefer openings and prolonged intracluster closings ( Fig. 1).
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 I), where two agonists (A) bind to the receptor (R) in the resting state with association rates k ϩ 1 and k ϩ 2 and dissociate with rates k Ϫ 1 and k Ϫ 2 . Receptors occupied by one agonist open with rate ␤ 1 and close with rate ␣ 1 , and AChRs occupied by two agonist molecules open with rate ␤ 2 and close with rate ␣ 2. At high agonist concentrations ( Ͼ 100 M ACh) channel blockade is evident, requiring addition of the blocked state A 2 B.
To estimate the set of rate constants, Scheme I was fitted to the data using the program MIL (Qin et al., 1996). MIL computes the likelihood of the experimental series of open and closed times given a set of trial constants, and then changes the rate constants to maximize the likelihood. We simultaneously analyzed recordings obtained at multiple ACh concentrations (10-300 M), with the aim of representing all the states in Scheme I in the fitting. The procedure for kinetic analysis requires selection of clusters to produce kinetically homogeneous data (MIL, QUB suite; Salamone et al., 1999). For each recording, kinetic homogeneity was de-  Table I) indicates that the selected clusters are representative of the entire population of events for each recording. In addition, open-and closed-time histograms constructed with selected data are very similar to those constructed with the original events (not shown). Data of open and closed intervals corresponding to the selected clusters were subsequently used to fit Scheme I.
For wild-type AChR, ␤ 2 was constrained to its known value (Sine et al., 1995;Wang et al., 1997;Salamone et (SCHEME I) Figure 1. Clusters of single-channel currents from wild-type and mutant AChRs. Channels activated by 100 M ACh were recorded from HEK cells expressing mouse wild-type (␣ 2 ␤⑀␦) and mutant ␣T422A, ␣T422V, ␣T422W, ␣T422Y, ␣T422C, ␣T422N, and ␣T422S AChRs. Currents are displayed at a bandwidth of 9 kHz with channel openings as upward deflections. Membrane potential, Ϫ70 mV. al., 1999) because brief closings due to gating and channel blocking became indistinguishable at high ACh concentrations (Wang et al., 1997;Salamone et al., 1999). When ␤ 2 was allowed to vary freely, MIL failed to converge to a well-defined set of rate constants and approached a value of ‫ف‬ 100,000 s Ϫ 1 (data not shown). Similar observations were recently reported by Salamone et al. (1999). Although channel blocking is too brief to be completely resolved with our instrumentation dead time of 30 s, we retain channel block in Scheme I because significant numbers of blockages are resolved at our highest ACh concentrations of 300 M. Moreover, the unblocking rate constant obtained under the present conditions is similar to values previously reported (Maconochie and Steinbach, 1995;Wang et al., 1997). Finally, association and dissociation rate constants were assumed to be equal at both binding sites, as previously described for adult mouse AChRs (Akk and Auerbach, 1996;Wang et al., 1997;Salamone et al., 1999).
Rate constant estimates obtained for wild-type AChR, shown in Table II, agree with those previously reported for mouse AChR (Wang et al., 1997;Salamone et al., 1999). The fitting analysis confirms that singly occupied AChRs open slowly and with low efficiency, but doubly occupied receptors open rapidly and efficiently.
We next applied kinetic analysis to AChRs containing mutations of ␣ T422 using clusters selected as described for wild-type AChRs (Table I). Scheme I could not adequately describe data obtained from AChRs containing the hydrophobic/aromatic mutations ␣T422A, ␣T422V, ␣T422Y, ␣T422W, and ␣T422C. Scheme I 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 concentrationindependent open-time components, we fitted Scheme II to dwell times from mutant AChRs. The classical activation scheme (Scheme I) is a subset of Scheme II, which has the opening step separated into two sequential steps. An alternative branched scheme, containing two open states connected to the A 2 R 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 II was e 110 times more likely than the branched scheme.
In fitting Scheme II 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 fit- (SCHEME II) ting Scheme I to data from the wild type, it was possible to allow ␤ 2 to vary freely. For the hydrophobic and aromatic mutations, the unblocking rate constant (k Ϫb ) was constrained to the value used for wild-type AChRs because M4 does not contribute to the channel pore (Blanton and Cohen, 1994), and because we found the channel conductance to remain constant for all M4 mutations. The resulting fitting analysis establishes that the T422V, T422A, T422Y, T422W, and T422C mutations markedly affect the kinetics of channel gating (Table II). The steps primarily affected are channel opening and closing, with changes of two-to sevenfold in the opening rate (␤ 2 in Scheme II) and four-to ninefold in the closing rate (␣ 2 ) (Table II). 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 A 2 R** in Scheme II.
In contrast to the hydrophobic and aromatic mutations, AChRs containing the serine or asparagine mutations could be well fitted by Scheme I (Fig. 2). Using the same constraints as in wild-type AChRs (␤ 2 ϭ 49,000 s Ϫ1 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 II). Because, as described above for wild-type AChRs, it was necessary to constrain ␤ 2 in fitting data from ␣T422S and ␣T422N AChRs, and because the hydrophobic and aromatic mutations affected ␤ 2 , we systematically varied ␤ 2 from 19,000 to 49,000 s Ϫ1 . Based on likelihood, the best description of the ␣T422S and ␣T422N data was obtained when ␤ 2 was 49,000 s Ϫ1 , as found for wild-type AChR. For example, for the ␣T422S AChR, the fit using ␤ 2 ϭ 49,000 was e 24 , e 81 , and e 235 times more likely than with ␤ 2 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 I, dwell times from wild-type, ␣T422S, and ␣T422N AChRs could be well described by either Scheme I or Scheme II (Table II). To show that changes in rate constants due to the mutations are not scheme dependent, we also fitted Scheme II to data from wild-type as well as ␣T422S and ␣T422N AChRs (Table II). Comparison of log likelihoods, obtained after fitting both schemes to wild-type and ␣T422N data, reveals that Scheme I provides the better description; Scheme I is e 75 and e 170 times more likely than Scheme II for wild-type and T422N AChRs, respectively. On the other hand, for the ␣T422S mutation, Scheme II is e 72 more likely than Scheme I. Fitting Scheme II to data from the serine and asparagine mutations reveals only slight changes in the gating steps, similar to results obtained with Scheme I (Table II). Comparison of wild-type and M4 mutant rate constants, calculated on the basis of Scheme II, 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 II). Thus, residues preserving the hydrogen bonding ability of the original threonine support nearly normal gating kinetics. The channel gating equilibrium constant, 2 , calculated as ␤ 2 /␣ 2 , significantly decreases in most of the mutants (Table II). 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 III). Free energy changes of ‫2ف‬ 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 III).
Rates of agonist association and dissociation (Table  II) and dissociation equilibrium constants (Table IV) are not significantly affected by these mutations. Thus, T422 selectively affects channel opening and closing transitions.
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 T A B L E I I Gating ⌬(⌬G) is the change between mutant (m) and wild-type (wt) AChRs in the energy of channel gating, calculated by ⌬(⌬G) ϭ ϪRTln ( 2 m/ 2 wt) (where R is the gas constant and T is the absolute temperature). 2 is the initial channel opening equilibrium constant shown in Table II. ⌬(⌬G) is the change between mutant and wild-type AChRs in the energy that the channel must overcome to make the transition from the biliganded closed state to the main open state (Opening) and from this open to the closed state (Closing). The differences in free energies are calculated as ⌬(⌬G) ϭ ϪRTln (␤ 2 m/␤ 2 wt) and
with the experimentally determined open probability (Fig. 3).  For the mutants T422A, T422V, T422W, T422Y, and  T422C, open probability is Ͻ0.6 at 1,000 M, and the EC 50 values range from 200 to 300 M (Fig. 3). Thus, mutations of ␣T422 affect both efficacy and EC 50 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 EC 50 and maximum open probability values for the T422S mutant are 80 M and 0.8, respectively (Fig. 3). The dose-response curves computed from the fitted rate constants closely follow the experimentally determined open probability determinations, demonstrating overall consistency of the data.

D I S C U S S I O N
T422 of the M4 domain of the ␣ subunit is presumed to be located at the lipid-protein interface, owing to its susceptibility to labeling by a hydrophobic probe (Blanton and Cohen, 1994). Although M4 is the least conserved of the four transmembrane domains, the high degree of conservation of T422 among subunits and species indicates that it might play a structural or functional role. We previously reported that among residues in M4, ␣T422 is the most sensitive to mutation, with the alanine substitution decreasing the mean open time fivefold (Bouzat et al., 1998). Here we elucidated both mechanistic and structural bases for the contribution of ␣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 Ͼ10 and 30 M 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 closedtime histograms are observed (Fig. 2). Data can be well fitted by the classical description of activation for wildtype AChR. The only detectable change is a twofold in- Equilibrium constants were obtained with data from Table II.  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 hy-droxyl 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 (Pal and Chakrabarti, 1998), an alternative interpretation would be that the side chain at position 422 acts as a hydrogen bond acceptor. In addition, the lower pKa of cysteine compared with threonine, or the greater hydrophobicity of cysteine compared with threonine, serine, or asparagine may contribute to the impaired channel gating. The observed gating kinetics of T422Y AChRs indicate that tyrosine does not provide the correct hydrogen bonding capability, perhaps owing to delocalization of electron density from the hydroxyl oxygen to the aromatic ring, or to the bulk of the aromatic ring. Interestingly, the tyrosine hydroxyl group is a poorer hydrogen-bond acceptor than an aliphatic or water hydroxyl group (Fersht et al., 1985). In summary, our results show that correct gating requires a hydrogen bond involving the side chain at position 422 in the M4 domain of the ␣ 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 (Blanton and Cohen, 1994) or a ␤-sheet (Unwin, 1993) remains controversial. In addition, current models of the AChR place M4 with a tilt of ‫03ف‬Њ with respect to the membrane (Ortells et al., 1997). Thus, atomic structure resolution of the AChR will be needed to unequivocally identify the hydrogen-bonding partner for ␣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. 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 (0.5-1.5 kcal/mol; Fersht et al., 1985), or for other weak interactions such as dipole-dipole interactions. Our observation that a hydrogen bond is required for proper gating agrees with the calculated free energy changes. However, the correlation between the free energy of a hydrogen bond and free energy of activation for a state transition is not necessarily strict, given that disruption of a chemical bond can have global consequences.
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) (Engel et al., 1998). Although mutations in the M4 domain have not been identified in CMS, our findings indicate that if they naturally occurred they could lead to a CMS.
Given the present understanding of the topology of the M4 domain, our results demonstrate that a lipidexposed residue involved in hydrogen bonding is necessary for proper gating of the AChR.