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Address correspondence to Steven M. Sine, Department of Physiology and Biophysics, Mayo Clinic College of Medicine, 200 First St., SW, MSB 1-35, Rochester, MN 55905. Fax: (507) 284-9420; email: sine{at}mayo.edu
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ABSTRACT |
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D89 forms a highly conserved interdomain contact near T148, W149, and T150. Patch-clamp recordings show that the mutation D89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither T148L, T150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor D89. However substituting a negative charge at T148, but not at T150, counteracts the effect of D89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring W149 as principal hydrogen bond donors for D89. The specific effect of D89N on ACh association suggests that interdomain hydrogen bonding positions W149 for optimal interaction with ACh.
Key Words: acetylcholine receptor • ligand binding site • single channel kinetics • hydrogen bond • structural model
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit and domains D–G from the 
, 
, or 
subunits (for review see Sine, 2002
). Atomic-scale insight into the structure of the agonist recognition site emerged with x-ray structures of acetylcholine binding protein (AChBP; Brejc et al., 2001; Celie et al., 2004), a structural homologue to Cys-loop receptors, and with homology models derived from it (Sine et al., 2002a; Le Novere et al., 2002; Molles et al., 2002; Schapira et al., 2002).
In the muscle nicotinic receptor, the ACh binding site is a specialized pocket of aromatic and hydrophobic residues, as shown by AChBP, homology models of the major extracellular region, affinity labeling, and mutagenesis combined with functional measurements (for reviews see Corringer et al., 2000; Karlin, 2002; Sine, 2002). Among the multiple aromatic residues in the binding pocket, W149 emerged as a strong candidate for stabilizing the quaternary ammonium moiety of ACh (Zhong et al., 1998), which was confirmed by the x-ray structure of AChBP with bound carbamylcholine (Celie et al., 2004). Contained within recognition domain B of the binding site, W149 is part of a larger, highly conserved structural motif that includes hydroxyl side chains at positions T148 and T150 and the closely juxtaposed domain A, which harbors the invariant D89 (Fig. 1 D). In AChBP, the Asp equivalent to D89 hydrogen bonds with hydroxyl side chains of Ser and Thr residues flanking the Trp equivalent to W149, as well as with the main chain amide of the residue at the position equivalent to T150 (Celie et al., 2004; Fig. 1 C). These hydrogen bonds are likely critical for stabilizing the protein main chain of recognition domain B and consequently for determining the orientation and mobility of the key binding site Trp.
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D89 contributes to the function of the muscle nicotinic receptor by interacting with recognition domain B, we generated receptors containing mutations of D89, T148, and T150, recorded single channel currents, and measured rate constants underlying elementary steps in receptor activation. We find that D89 plays a key role in receptor activation by maintaining a high rate of ACh association and optimizing the rate of ACh dissociation; this is achieved by serving as a strong hydrogen bond acceptor for donors in domain B that in turn may optimize W149 for interaction with the agonist. |
MATERIALS AND METHODS |
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, ß, , and subunit cDNAs subcloned in the CMV-based mammalian expression vector pRBG4 (Lee et al., 1991) were described elsewhere (Ohno et al., 1996). Site-directed mutations were made using the QuickChange site-directed mutagenesis kit (Stratagene). The presence of each mutation and absence of unwanted mutations was confirmed by sequencing the entire cDNA insert.
Mammalian Cell Expression
All experiments were performed using the BOSC 23 cell line (Pear et al., 1993; Wang et al., 2000), which is a variant of the 293 HEK cell line. Cells were maintained in the Dulbecco's Modified Eagle Medium (DMEM) containing FBS (10% vol/vol) at 37°C until they reached 
50-70% confluence. Wild-type or mutant AChR cDNAs were then transfected by calcium-phosphate precipitation using final cDNA concentrations of 0.68 µg/ml, except the -subunit cDNA, which was 1.36 µg/ml. Patch-clamp and [125I]-bungarotoxin binding measurements were performed 2 and 3 d after transfection, respectively.
Patch-clamp Recordings
To record single channel currents, cells transfected with wild-type or mutant AChR cDNAs were rinsed with and maintained in the following bath solution (in mM): KCl 142, NaCl 5.4, CaCl2 1.8, MgCl2 1.7, and HEPES 10 (pH was adjusted to 7.4). The same solution was used to fill patch pipettes. ACh (Sigma-Aldrich) was kept as a 100 mM stock, dissolved in bath solution and stored at –80°C until use. Glass micropipettes (type 7052; Garner Glass Co.) were coated with Sylgard 184 (Dow Corning Co.) and heat polished to yield resistances of 5–8 M
. Single channel currents were recorded in the cell-attached configuration at 21°C using the Axopatch 200A (Axon Instruments, Inc.) at a membrane potential of –70 mV. Data were collected from two to four different patches for each ACh concentration, choosing only recordings in which channel activity was low enough to allow clear identification of activation episodes resulting from a single channel. The current signal was low pass filtered at 50 kHz and recorded to hard disk at 200 kHz using the program Acquire (Bruxton Co.).
Single Channel Kinetic Analysis
The digitized current signal was filtered using a 10-kHz digital Gaussian filter (Colquhoun and Sigworth, 1983), and channel events were detected by the half-amplitude threshold criterion using the program TAC using a dead time of 10 µs (Bruxton Co.). Precise determination of dwell time at threshold was achieved by cubic spline interpolation of the digital signal and correcting the measured dwell time at threshold for the effects of the Gaussian filter as previously described (Colquhoun and Sigworth, 1983). Open and closed time histograms were fitted by the sum of exponentials by maximal likelihood using the program TACFit (Bruxton Co.). Openings corresponding to a single receptor channel were identified by assigning a critical closed time defined as the intersection of the closed time component that depended on ACh concentration with the succeeding concentration-independent component due to rapid desensitization (Ohno et al., 1996). Because this method removes most but not all closed dwell times due to rapid desensitization, analyses were also performed in which the critical closed time was defined as the intersection of the longest closed time component, due to slow onset desensitization and the number of channels in the patch, with the adjacent component due to intermediate onset desensitization. To obtain kinetically homogeneous data, defined single channel episodes containing at least five openings were analyzed for open probability, mean open time, and mean closed time, and episodes within two standard deviations of the mean were accepted for further analysis (Wang et al., 1997). It should be mentioned that this processing removes a small population of brief openings flanked by long closed periods (Sine and Steinbach, 1987). For the receptors examined in this study, this selection typically retained 85–95% of the detected channel events, yielding 4,000–8,000 events per single patch recording. Kinetic analysis was performed on data obtained across a range of ACh concentrations simultaneously using MIL software (QuB suite, State University of New York), which employs a maximum likelihood method, corrects for missed events, and gives error estimates of the fitted parameters (Qin et al., 1996). An instrument dead time of 22 µs was uniformly applied to all recordings. For a given wild-type or mutant receptor, our global analysis included data from two to four patches for each ACh concentration, ACh concentrations spaced at half log-unit intervals and a 10- to 333-fold range of the ACh concentration. For each receptor mutant, the range of the ACh concentration spanned from the minimum to the maximum channel open probability (see Fig. 7).
[125I]-Bungarotoxin Binding Measurements
Cell surface expression of wild-type and mutant receptors was determined by measuring [125I]-bungarotoxin (125I--Btx; PerkinElmer) binding to intact cells. 3 d after transfection, cells were harvested using PBS containing EDTA (5 mM), and resuspended in high potassium Ringer solution buffered with 25 mM HEPES and supplemented with 0.3% BSA. The cell suspension was incubated with 5 nM 125I--Btx for 1 h and harvested (Brandel Inc.) to Whatman GF-B glass fiber filters (1 µm cutoff; Brandel Inc.). Nonspecific binding was determined in the presence of 100 µM d-tubocurarine. Total specific binding for each mutant was normalized to that of the wild-type receptor.
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RESULTS |
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Subunit). These recognition domains are stabilized by hydrogen bonds between the carboxylate moiety of D85 in the ß3-4 linker and hydroxyl side chains of S142 and T144 and the main chain amide of T144 in the ß7-8 linker (Celie et al., 2004; Fig. 1 C). Hydrogen bonding between these domains likely affects the orientation and mobility of both the main chain carbonyl oxygen and indole side chain of the key binding site residue W143. All nicotinic receptor subunits contain Asp at the position equivalent to D85, while in most subtypes and species of subunits, residues with hydroxyl side chains are found at positions equivalent to those flanking the key W143 (Fig. 1 A). A structural motif containing four residues equivalent to these is also present in our homology model of the major extracellular region from the human muscle receptor (Sine et al., 2002a; Fig. 1 D), but whether hydrogen bonds between domains A and B form and are functionally significant is not known. By analogy to AChBP, the D89 side chain in the receptor is a candidate for linking domains A and B through hydrogen bonding, which could affect conformation of W149 and the local main chain and thus the kinetics of receptor activation by ACh.
Single Channel Kinetic Analysis of the Adult Human Receptor
To provide a frame of reference for comparison with mutant receptors examined in this study, we first describe kinetic analysis of ACh-evoked single channel currents through the wild-type human receptor. To record these currents, we cotransfected , ß, , and subunit cDNAs in BOSC 23 cells and employed the cell-attached mode of the patch-clamp (see MATERIALS AND METHODS). Application of high concentrations of ACh in the patch pipette causes clustering of current pulses into epochs during which only one channel is active (Sakmann et al., 1980; Sine and Steinbach, 1987; Colquhoun and Ogden, 1988). At progressively increasing concentrations of ACh, closed periods between openings of a single receptor become briefer, and a corresponding closed time component shifts from long to brief times (Fig. 2). Distributions of open times change from bi- to mono-exponential as the ACh concentration is increased, but the mean duration of the major component of openings is relatively insensitive to ACh concentration. These qualitative features of the concentration dependence of dwell times are similar to those described previously (Sine et al., 1990; Zhang et al., 1995; Ohno et al., 1996; Hatton et al., 2003).
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). Our previous analyses of the adult human receptor identified channel events due to receptor activation as a series of openings flanked by closed intervals greater than a defined critical closed time (Ohno et al., 1996; Wang et al., 1999; Sine et al., 2002b); this was taken as the point of intersection of the component that depended on ACh concentration with the succeeding component due to fast desensitization (Fig. 2, arrow). Using this method to identify channel events due to receptor activation, we applied maximum likelihood analysis of the closed and open intervals according to kinetic schemes (Qin et al., 1996; see MATERIALS AND METHODS). The first scheme depicts receptor activation as sequential binding of ACh to receptors in the resting, closed state, followed by conformational change to the open state, and then block of the doubly occupied open receptor by ACh (Fig. 3, Scheme I). The second scheme also depicts sequential binding of ACh, but allows the initial binding event to occur at either binding site (Fig. 3, Scheme II). In both schemes, mono-liganded receptors can open, but in Scheme II only one of two possible mono-liganded openings is allowed; this restriction is necessary to obtain well-defined rate constants because only two components are present in the open time histograms (Fig. 2).
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; Wang et al., 1999; Sine et al., 2002b; Hatton et al., 2003), and demonstrate rapid and efficient opening of the doubly occupied receptor, distinct association and dissociation rate constants for each binding site, and transient, low affinity block of the open receptor channel by ACh.
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Functional Contribution of D89
To determine whether D89 contributes to the kinetics of receptor activation, we constructed the mutation D89N, cotransfected it with complementary ß, , and subunit cDNAs in BOSC 23 cells and recorded ACh-evoked single channel currents. The Asn substitution introduces a relatively conservative structural change, neutralizing the negative charge but changing only the hydroxyl group of the original side chain. Compared with wild-type receptors, the mutant receptors show markedly prolonged closed intervals between successive openings of a single channel, indicating impaired kinetics of receptor activation (compare Figs. 2 and 5). However when the concentration of ACh is increased sufficiently, closed intervals from single mutant receptors approach the brief durations observed for single wild-type receptors. These qualitative observations are born out in the closed time histograms where the shift of the distributions from long to short closed times requires greater concentrations of ACh for D89N compared with wild-type receptors (compare Figs. 4 and 5). Thus, D89 plays a pivotal role in receptor activation, predominantly affecting the dependence of receptor activation kinetics on ACh concentration.
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D89N, we analyzed open and closed dwell times according to Schemes I and II, as described for the wild-type receptor. Unlike the wild-type receptor, Scheme II yields a substantially higher likelihood than Scheme I (see legend to Fig. 5). The fitted rate constants show that D89N dramatically slows the rate of ACh association with receptors in the resting closed state, but has a smaller effect on the rates of ACh dissociation; the net result is a 10- to 20-fold reduction in ACh affinity at the two binding sites (Table II).
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D89N on ACh binding steps, channel gating steps are not discernibly affected by D89N (Table II). The rate constant for channel opening is given as a lower bound because a plot of log likelihood against channel opening rate plateaus at 30,000–40,000 s–1 (Fig. 6). The difficulty in placing an upper bound on the opening rate constant is likely due to bandwidth limitations; rapid dissociation of ACh, combined with rapid channel opening, predicts similar mean closed times for doubly liganded and ACh-blocked receptors, both of which approach the instrument dead time. The rate constant for channel closing, on the other hand, is well defined, although its magnitude depends on the choice of the opening rate constant beyond the lower bound. Thus D89N, although markedly affecting the kinetics of ACh binding, has no discernable effect on the kinetics of channel gating.
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D89N are further illustrated by plotting open probability within clusters of single receptor openings against ACh concentration. The mutation shifts the Popen curve 13-fold toward higher ACh concentrations (Fig. 7). The curves through the data are computed from the rate constants determined from kinetic fitting, and their close alignment with the data further supports the rate constant estimates. The overall findings show that D89 is essential for optimizing ACh binding to receptors in the resting closed state through a prominent effect on rate constants for ACh association and a smaller effect on rate constants for ACh dissociation (Table II).
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D89D89 and measured cell surface expression with radiolabeled -bungarotoxin (Fig. 8 A). The Ala substitution abolishes receptor expression on the cell surface, while Glu, Asn, Ser, Thr, His, and Lys all give low but measurable expression. Of these, the D89T and D89E mutations were examined for their effects on single channel kinetics. The Thr substitution, which neutralizes the negative charge but maintains a small side chain, impairs the kinetics of receptor activation even more than D89N; it causes a more pronounced slowing of the rate of ACh association but does not affect the kinetics of channel gating (Fig. 9; Table II). For the D89T mutation, adequate fitting could only be achieved by explicitly including two desensitized states, using the method described for the wild-type receptor, presumably because of the more pronounced slowing of ACh association and consequent overlap of closed time components due to activation with those due to desensitization.
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D89N and D89T. The kinetics of channel gating are not substantially affected by D89E, as observed for D89N and D89T (Table II). Thus kinetic analyses of D89N, D89T, and D89E receptors show that the negative charge is the most important structural feature at position 89 of the subunit, but side chain length is also essential for proper kinetics of receptor activation.
Main Chain Amide Groups in Recognition Domain B Are Hydrogen Bond Donors for D89
The full negative charge of the D89 side chain makes it a strong hydrogen bond acceptor. Hydrogen bond donors accessible to D89 include the hydroxyl side chains of T148 and T150 and the main chain amide groups of W149 and T150 (Fig. 1 D). To determine whether the hydroxyl side chains of T148 and T150 hydrogen bond to D89, as do the side chains of residues at equivalent positions in AChBP (Celie et al., 2004; Fig. 1 C), we substituted residues with hydrophobic side chains at these positions and measured rate constants for receptor activation. To determine optimal substitutions, we constructed a series of hydrophobic mutations at each position (Fig. 8 B). Substitution of Leu is strongly preferred at T148, whereas Ala is preferred at T150, suggesting that the environment of T148 is more hydrophobic than that of T150, in accord with the structures of AChBP and our model of the ligand binding site (Fig. 1, C and D). The mutation T148L shows kinetics like that of the wild-type receptor, while T150A has relatively modest effects on binding and gating steps (Fig. 10; Table II), showing that neither of these hydroxyl side chains is the sole hydrogen bond donor for D89. We then replaced hydroxyl side chains of both T148 and T150 with nonpolar ones by combining the optimal hydrophobic substitutions at both positions to yield the double mutation T148L plus T150A, which expresses 40% of cell surface receptors compared with wild type (Fig. 8 C). Single channel kinetic analysis reveals only modest changes in binding and gating rate constants for the T148L plus T150A double mutation (Fig. 10; Table II). Thus hydroxyl side chains of T148 and T150 do not form functionally important hydrogen bonds to D89. The remaining hydrogen bond donors accessible to D89 are the main chain amide groups of W149 and T150 (Fig. 1 D).
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D89ND89, we retained the D89N mutation but substituted Asp at positions proximal to the main chain of binding domain B. Restoring the local negative charge at T148 substantially reverses the slowing of the ACh association rate constants produced by D89N alone (Fig. 11; Table II). It also counteracts the 13-fold shift in the plot of Popen against ACh concentration, yielding an overall threefold shift for the D89N plus T148D double mutant compared with the wild-type receptor (Fig. 7). Rate constants for ACh association are increased markedly by the double mutation compared with D89N alone, with k+1 showing the greatest increase. By contrast, combining the negative charge at T150 with D89N shows no reversal of the plot of Popen against ACh concentration (Fig. 7), and smaller increases in association rate constants (Table II).
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T148D by itself slows ACh association to the first binding site, although much less than D89N, so if the two mutations were independent, the effect of combining them would be a further slowing of k+1. The observation that k+1 is nearly reversed in the double mutant indicates that the two mutations are interdependent, which, together with their close proximity, suggests they contribute to ACh association through a common mechanism. Normal or near normal ACh association is achieved only when a single negatively charged residue is present at either 89 or 148; association is greatly impaired when there is no negative charge (D89N), and moderately impaired when two charges are present (T148D or T150D). Thus the common structural feature associated with fast association is a single negative charge at either 89 or 148. This explanation is readily accommodated by the AChBP structure and our structural model of the ligand binding site (Fig. 1, C and D). T148 and the equivalent S142 in AChBP are situated in hydrophobic environments proximal to D89 or D85 and behind the main chain of recognition domain B (Fig. 1, C and D), enabling the negatively charged side chain to accept hydrogen bonds from the main chain amide donors W149 and T150. This hydrogen bonding whether via Asp at 89 or 148 restricts rotation of the main chain of recognition domain B. The negative charge may also polarize the main chain carbonyl moiety of W149, which may further stabilize the positive charge of the agonist (Celie et al., 2004).
The mutation T150D by itself also slows k+1, and should produce a further slowing when combined with D89N if the two residues are independent. The double mutant shows a greater association rate than expected from independence, indicating that these too are interdependent (Table II). However, the hydroxyl side chain of T150 is far from D89, and is in a hydrophilic rather than a hydrophobic environment (Fig. 8 B), both of which make it an unlikely hydrogen bond acceptor for the main chain amide group of W149 or Asn substituted at D89. Also the extent of reversal of k+1 is not as great as observed with T148D, and T150D alone or in combination with D89N has multiple effects not shown by T148D; T150D speeds ACh dissociation from the first binding site and slows channel opening (Table II). The interdependence between T150D and D89N may be due to the different ways in which they stabilize the quaternary ammonium moiety of the agonist. A negative charge introduced at T150 may enhance ACh association electrostatically, whereas a negative charge, at either 89 or 148, stabilizes the main chain of recognition domain B relative to domain A, polarizes the main chain carbonyl group of W149, or both. Stabilization of domain B is likely essential for positioning the indole side chain and main chain carboxyl groups of W149 for efficient encounter with ACh.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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D89 contributes to the kinetics of receptor activation by maintaining high rates of association between ACh and the two binding sites of the muscle nicotinic receptor, as well as by optimizing rates of ACh dissociation. Channel gating rate constants, on the other hand, are not affected by mutations of D89. Comparison of our structural model of the major extracellular domain of the receptor with the structure of AChBP suggests that recognition domains A and B are linked by hydrogen bonds between the carboxylate side chain of D89 and hydroxyl side chains of T148 and T150. This tertiary structural fold encompassing domains A and B contains highly conserved residues, and may represent a primordial structural motif essential for binding ACh. However contrary to expectations from the AChBP structure, we find that substituting residues with hydrophobic side chains at T148 and T150 does not substantially affect the kinetics of receptor activation, pointing to main chain amide groups in recognition domain B as the most likely hydrogen bond donors. In accord with our structural model (Sine et al., 2002a), main chain amide groups of W149 and T150 are in close proximity to the D89 side chain, suggesting a mechanism by which D89 contributes to a high rate of ACh association. Hydrogen bonds between the carboxylate moiety of D89 and main chain amide groups of W149 and T150 both positions W149 and restricts its mobility to promote efficient encounter between the binding site and ACh. Stabilization of the main chain by D89 may both position and polarize the main chain carbonyl of W149, which may further stabilize the quaternary ammonium moiety of ACh. Further support for stabilization of domain B by a negative charge is our charge reversal of the effect of D89N by substitution of Asp at T148; in our structural model, T148 is appropriately positioned for interaction with the main chain of domain B.
Patch-clamp recordings show that D89N prolongs the latency between successive openings of single receptor channels at low and intermediate concentrations of ACh, but this latency is comparable to that of wild-type receptors at high concentrations of agonist. Analyzed within the framework of kinetic schemes, these observations lead to the conclusion that D89 contributes to agonist binding steps but not to channel gating steps. The association rate constants for ACh are affected most, slowing 10- to 20-fold, while the dissociation rate constants are modestly affected, slowing twofold at one binding site and increasing threefold at the other site. Differences in functional consequences for ACh binding at the two sites likely owe to nonequivalence of the alpha-epsilon and alpha-delta interfaces. The selective effect of a mutation on agonist binding is often used as a criterion for direct contact between the agonist and the mutated site, but this is clearly not true for the muscle receptor studied here. D89 is located at the periphery of the binding site and has not been identified as a site of labeling by covalent affinity reagents, contrary to the labeling of aromatic residues within the binding pocket, including Y93, W149, Y190, Y198, and W55 (Dennis et al., 1988; Abramson et al., 1989; Galzi et al., 1990; Chiara et al., 1998). Further, the mutational consequences of D89N are like those of a small subset of functionally significant mutations of the receptor that selectively affect agonist binding such as G153S, N217K, and L221F (Sine et al., 1995; Wang et al., 1997; Hatton et al., 2003). This group of binding-specific mutations is distinct from mutations of aromatic residues in the binding pocket, which alter binding as well as channel gating (Chen et al., 1995; Akk et al., 1999; Akk, 2001).
The AChBP structure first revealed differences in secondary and tertiary structures between principal and complementary faces of the ACh binding site (Brejc et al., 2001). The principal face is composed of loops that separate ß strands, whereas the complementary face is composed primarily of ß strands. Thus, side chain disposition of residues on the principal face is not constrained by periodic main chain hydrogen bonds, whereas side chain disposition on the complementary face is strictly regulated by main chain hydrogen bonds that stabilize the ß sheet secondary structure (Sine et al., 2002a). The absence of a regular secondary structure on the principal face suggests that alternative linkages might be expected to form between conserved residues in proximal loops, such as those described here between the strong hydrogen bond acceptor D89 in recognition domain A and donors in domain B. The recent higher resolution x-ray structure of AChBP confirms the presence of hydrogen bonds between the homologous D85 and hydroxyl side chain donors in domain B (Celie et al., 2004; Fig. 1 C). The present data show profound functional consequences of mutating D89, suggesting that it interacts with chemical groups in domain B. However, unlike AChBP, the hydroxyl side chains of T148 and T150 in the receptor do not donate functionally significant hydrogen bonds to the negatively charged carboxylate group of D89, but rather the donors are main chain amide groups of W149, T150, or both.
The strategy employed here takes advantage of new opportunities for establishing structure–function relationships based upon the AChBP structure (Brejc et al., 2001). Now one can test predicted atomic-scale interactions for their functional significance, rather than relying on the linear sequence, residue conservation, ligand interactions, and solvent accessibility used before the AChBP structure was known. The present results for the muscle nicotinic receptor show that predicted hydrogen bonds between the invariant D89 and hydroxyl side chains of residues flanking W149 do not contribute to receptor function, but rather hydrogen bonding to main chain amide groups of W149 and T150 is most likely. Possible reasons the predicted hydrogen bonds are not evident include (a) the local structure of the receptor differs slightly from that of AChBP, (b) the hydrogen bonds are actually present in the receptor but not functionally significant despite the high degree of conservation, (c) the predicted hydrogen bonds form in the crystal but not in solution, and (d) hydrogen bonds form to both the main chain amide groups and the hydroxyl side chains in domain B, but these switch back and forth in a dynamic manner. These possibilities emerging from the present data cannot be distinguished by the current body of structural or functional knowledge. However, the overall results underscore the deeper understanding that can be achieved by testing the functional significance of predicted atomic-scale interactions in other parts of the muscle nicotinic receptor as well as in other members of the Cys-loop receptor superfamily.
The invariant W149 has long stood as the most likely candidate for the primary docking site for the quaternary ammonium moiety of ACh because the Phe substitution at this position profoundly impairs ligand binding affinity and the kinetics of receptor activation (Dennis et al., 1988; Zhong et al., 1998; Akk, 2001). The recent x-ray structure of AChBP in complex with carbamylcholine conclusively shows docking of the quaternary ammonium moiety of the agonist with the indole ring of the homologous W143, but also suggests that the negative charge of D85 polarizes the carbonyl group of W143 for additional stabilization of agonist (Celie et al., 2004). Within our structural model (Fig. 1 D; Sine et al., 2002a), the carboxylate moiety of D89 is 2.5–3 Å away from the main chain amides of W149 and T150, suggesting a structural explanation for the selective effect of D89N on elementary steps underlying ACh binding. Neutralizing the negative charge eliminates hydrogen bonding to the main chain of domain B, increasing mobility of the indole group of W149 and reducing polarization of its carbonyl group, which together impede the association of ACh. Our findings suggest future studies aimed at identifying additional functionally relevant contacts between proximal domains of the ACh binding site.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant NS31744 to S.M. Sine.
Olaf S. Andersen served as editor.
Submitted: 22 April 2004
Accepted: 21 September 2004
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subunit of the nicotinic receptor. J. Biol. Chem. 264:12666–12672.Y93 and W149 residues. J. Physiol. 535:729–740. subunit residue tyrosine 190. Biophys. J. 69:849–859.[Medline]-subunit of the Torpedo nicotinic acetylcholine receptor. FEBS Lett. 423:223–226.[CrossRef][Medline] subunit labeled by a photoaffinity ligand for the acetylcholine binding site. Biochemistry. 27:2346–2357.[CrossRef][Medline]-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites. J. Biol. Chem. 265:10430–10437.L221F, inferred from maximum likelihood fits. J. Physiol. 547:729–760. subunit of the nicotinic acetylcholine receptor interact to confer selectivity of Waglerin-1 for the – subunit interface site. Biochemistry. 41:7895–7906.[CrossRef][Medline] subunit. Neuron. 17:157–170.[CrossRef][Medline] subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity. Neuron. 15:229–239.[CrossRef][Medline] subunit decreases the rate of agonist dissociation. J. Gen. Physiol. 109:757–766.
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N. Mukhtasimova, C. Free, and S. M. Sine Initial Coupling of Binding to Gating Mediated by Conserved Residues in the Muscle Nicotinic Receptor J. Gen. Physiol., June 27, 2005; 126(1): 23 - 39. [Abstract] [Full Text] [PDF]
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