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* This work was supported by NIGMS, National Institutes of Health Grants GM56371-05 and GM08102-27. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported by a Postdoctoral Supplement to Grant GMRO156371. ¶ Supported by Minority Access to Research Careers-National Institutes of Health Grant 5T34GM07821 and the SCORE Program.
The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the γM4 transmembrane domain (γTM4) by introducing tryptophan, at residues 451–462, along the γTM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [125I]α-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC50, and Hill coefficient values for WT are 1.8 ± 0.4 fmol, 30.3 ± 1.1 μm, and 1.8 ± 0.3, respectively. Mutations L456W, F459W, and G462W induce a significant increase in nAChR expression (2.8 ± 0.5, 3.6 ± 0.6, and 3.0 ± 0.5 fmol, respectively) when compared with WT. These data suggest that these residues are important for AChR oligomerization. Mutations A455W, L456W, F459W, and G462W result in a significant decrease in EC50 (19.5 ± 1.7, 11.4 ± 0.7, 16.4 ± 3.8, and 19.1 ± 2.6 μm, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC50 (42.8 ± 6.8 μm) or loss in function when compared with WT. The Hill coefficient values were the same for WT and all of the mutations studied. The periodicity in function (EC50 and macroscopic peak current) and nAChR expression reveals an average of 3.3 and 3.0 amino acids respectively, thus suggesting a helical secondary structure for the γTM4.
). The five subunits cross the membrane and form a ring around the ion channel in the center. Each subunit consists of a large hydrophilic N terminus, four transmembrane domains (TM) of 20–30 amino acids denoted M1–M4 (see Fig. 1), an intracellular loop between M3 and M4, and a short extracellular C terminus (
). The M2 transmembrane domain (TM2) from each subunit lines the channel pore in the closed state conformation, whereas TM1 and TM2 contribute to the formation of the ion channel pore in the open state of the AChR (
). Consequently, the model proposed in the latter study has become the best approximation to the AChR structure. Other alternative approaches to crystallization have been utilized to probe both AChR structure and function. Examples of these techniques include Fourier transformed infrared spectroscopy (
Tryptophan scanning and/or site-directed mutagenesis have been used to gather valuable data about the structure and function of various transmembrane domains of the AChR, with emphasis on the lipid-exposed residues (
). These mutations resulted in a substantial increase in channel open time and demonstrated the important role of these residues in the AChR channel gating. Tryptophan scanning of several TM domains of the AChR has been successfully used to assess the structure and function of the AChR. This approach has been used for the αTM4 (
). Moreover, analysis of the changes in AChR function and expression as a function of the mutation position or periodicity has given valuable information used to predict the secondary structure of various transmembrane domains of the AChR. The periodicity for αTM3-TM4 and βTM3 are all consistent with a helical structure for these transmembrane domains (
In this study, we extend the tryptophan scanning mutagenesis to the γTM4 transmembrane domain of the AChR from T. californica. The purpose of this study is to determine the role of the γTM4 transmembrane domain on the AChR function and to use the data gathered to make predictions about the secondary structure of γTM4. Eleven residues in the middle of γTM4, Cys451–Gly462, were selected for this study. Two of these residues, Cys451 and Ser460, have been reported to be exposed to the membrane lipids (
). We expressed WT and mutated AChRs in Xenopus oocytes. The nAChR expression was determined using [125I]α-BTX binding assays, and functional studies were performed using two electrode voltage clamp to determine ACh-induced currents. We found that mutations L456W, F459W, and G462W resulted in a significant increase in nAChR expression when compared with that of WT. The rest of the mutations showed nAChR expression levels similar to WT. All of the mutations that increased nAChR expression and A455W produced an increase in function, as evidenced by a decrease in EC50 relative to WT. Seven mutations, I454W, A455W, L456W, L457W, F459W, I461W, and G462W resulted in an increase in macroscopic response (Imax) relative to WT. Mutations I454W, A455W, L457W, and L458W caused a significant increase in the normalized macroscopic response (Imax/fmol) induced by ACh. The average periodicity calculated for nAChR expression, EC50, and Imax suggest a helical secondary structure for γTM4.
Mutations in the γTM4 Transmembrane Domain—A QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to replace the existing amino acids with tryptophan, at positions 451–462 of the γ subunit of the AChR. The PCR product was used to transform Epicurian coli XL1 supercompetent cells. The DNA was then purified using a QIAprep spin miniprep kit (Qiagen) and sequenced to ensure a successful mutation.
nAChR Expression in Xenopus laevis Oocytes—mRNA transcripts were synthesized in vitro. The mRNA transcripts for α, β, γ, and δ subunits were microinjected into X. laevis oocytes at a 2:2:1:1 ratio. The volume of mRNA mix injected was varied depending upon the concentration of the mRNA mix.
Voltage Clamp on Xenopus Oocytes—ACh-induced currents were recorded 3 days after mRNA microinjection using a two-electrode voltage clamp. The electrodes were filled with 3 m KCl (resistance, <2 mΩ). Impaled oocytes were perfused at a rate of 0.5 ml/s with MOR-2 buffer (82 mm NaCl, 2.5 mm KCl, 5 mm MgCl2, 1 mm Na2HPO4, 5 mm HEPES, and 0.2 mm CaCl2, pH 7.4). The membrane potential of each oocyte was clamped at -70 mV during the experiment. The membrane currents were digitized and filtered at 2 kHz using an Axon DigiData interface (Axon Instruments) and recorded using the Whole Cell Analysis Program, version 3.2.9 (provided by John Dempster, University of Strathclyde, UK). This program is installed on a Pentium III-based computer. Dose-response data were collected from peak currents (I) observed for each of the six ACh concentrations used (1, 3, 10, 30, 100, and 300 μm). The GraphPad Prism software (GraphPad Software Incorporated, San Diego, CA) was used to perform a nonlinear regression fit with a sigmoidal dose-response (variable slope) equation,
where I is the macroscopic current at a given ACh concentration, Imin and Imax are the smallest and the largest current observed, respectively, EC50 is the concentration required to achieve half-maximal response, and the Hill Slope is the slope of the linear portion of the sigmoidal curve.
[125I]α-Bungarotoxin Binding Assay—We performed [125I]α-bungarotoxin (Amersham Biosciences) binding assays to determine the membrane expression of AChR in oocytes. The oocytes were incubated in 10 nm [125I]α-bungarotoxin with 5 mg/ml albumin serum bovine in MOR-2 without EGTA at room temperature for 1.5 h. Noninjected oocytes were also incubated in [125I]α-bungarotoxin to measure nonspecific binding (Beckman Gamma 5500). Excess toxin was removed by washing each oocyte with 15 ml of MOR-2 without EGTA. A standard curve was obtained by plotting the counts/min against [125I]α-bungarotoxin concentration (0.5–20 fmol). The equation for the linear regression obtained from this graph of counts/min versus volume (μl) was used to calculate the AChR expression (fmol) present in the membrane of each oocyte. The nAChR expression was determined for two groups of oocytes. One of these groups consists of oocytes used in the dose-response experiments, whereas the other group of oocytes belonged to experiments performed solely to calculate nAChR expression as a function of mutation. The nAChR expression value for the first group of oocytes was used to calculate the normalized channel response to ACh for each mutation, which is defined as the ACh-induced current in nanoamperes divided by femtomole of surface α-bungarotoxin binding. The expression calculated for the second group of oocytes was used to plot nAChR expression as a function of mutation along the γTM4 domain.
Statistical Analysis—We compared nAChR expression level (fmol), EC50, Hill coefficient, and maximal current values between wild type and each mutation. The data were analyzed using the SPSS general linear model procedure for one-way analysis of variance to perform planned comparisons between these groups.
Calculation of Periodicity for Both nAChR Expression and EC50—The expression of each mutant was normalized to the change in amino acid volume (Å3) caused by each tryptophan substitution. Tryptophan being the largest amino acid, this change corresponds to the volume of tryptophan (Å3) minus the volume of the WT amino acid replaced with tryptophan. The amino acid volumes used in this study were obtained from the calculations of Chothia (
). We then constructed plots of the normalized nAChR expression values as a function of their position along the γTM4 domain. Separate plots were created using EC50 and macroscopic response (Imax) values, for both WT and mutants, as a function of residue in the γTM4 domain. A cubic spline function (GraphPad Software Incorporated, San Diego, CA) was found to best fit the fluctuations induced by the tryptophan replacements, in normalized nAChR expression, EC50, and Imax data. To calculate the periodicity, we first determined the amino acid positions in which the first derivative of the cubic spline function is zero and then calculated the distance between the zeros.
Topological Representation of the γTM4 —The structural model for the γ subunit presented in Fig. 6 corresponds to that proposed by Miyazawa et al. (
nAChR Expression of γTM4 Mutants—Tryptophan scanning mutagenesis was performed on the γTM4 of Torpedo AChR, from residues Cys451–Gly462 (Fig. 1), as explained under “Experimental Procedures.” Subsequently, Xenopus oocytes were injected with mRNA encoding the mutated γ subunit and the wild type α, β, and δ subunits. [125I]α-BTX binding assays were performed 3 days after injection to determine the effect of each mutation on the nAChR expression level of AChR. The results of these binding assays are summarized in Fig. 2. The average WT expression was 1.8 ± 0.4 fmol (Fig. 2). Three of the mutations studied, L456W, F459W, and G462W exhibited nAChR expression levels significantly higher than that of WT. Specifically, L456, F459W, and G462W resulted in 1.6-, 2.0-, and 1.7-fold increases in nAChR expression, respectively (Fig. 2). The rest of the mutations resulted in expression levels similar to that of WT, thus suggesting that the secondary structure of γTM4 is loose enough to accommodate a large hydrophobic amino acid, such as tryptophan, without major consequences to the AChR assembly. Moreover, the increase in nAChR expression detected for three of the mutants suggests that, at those positions, tryptophan facilitates the AChR assembly and/or oligomerization. The tryptophan scanning results obtained with γTM4 are in contrast with those found with αTM4 (
), in which 23 of a total of 40 mutations examined either prevented or markedly decreased the expression of the AChR. Moreover, from these 40 mutations, only αI286W and βM285W resulted in a significant increase in nAChR expression when compared with WT (
Functional Effects of γM4 Trp Scanning of the AChR; ACh-induced Currents—The TM4 domain is the most lipophilic of all of the subunits of the AchR, and some of the residues in this TM domain are known to be exposed to the membrane lipids (
). The purpose of these studies is to determine the role of γTM4 on channel function and to assess the role that lipid-protein interactions may have on the AChR function. We used the two electrode voltage clamp technique to determine the dose response for ACh, using ACh concentrations ranging from 1 to 300 μm. Fig. 3 illustrates representative current traces induced by the different ACh concentrations used for both mutants and WT. All of the mutations studied yielded functional AchRs, and some mutations (e.g. I454W, A455W, and L456W) resulted in a gain in function (Fig. 3). Table I contains the average maximal current (Imax) or current induced by 300 μm for each oocyte. Mutations I454W, A455W, L456W, L457W, F459W, I461W, and G462W resulted in macroscopic response (Imax) values significantly higher than that of WT, with n-fold values ranging from 2.3–3.4-fold. When the Imax values were normalized to the nAChR expression level (fmol) for each oocyte (normalized macroscopic response), mutations I454W, A455W, L457W, and F458W were found to display normalized currents significantly higher than WT, with n-fold increase values of 3.4-, 10.7-, 7.0-, and 4.5-fold, respectively. No mutation resulted in a decrease of either Imax of Imax/fmol. These results contrast our previously reported findings with αTM4 (
). In those studies, 21 of a total of 40 mutations studied resulted in a decrease in Imax, and five mutations resulted in nonfunctional receptors. In contrast, eight of the total of 40 mutations examined in αTM4 (
) resulted in normalized currents higher than the controls (WT).
Table IFunctional parameters of wild type and γTM4 mutants Comparison of the functional parameters of wild type and all the γTM4 mutants generated in this study. The values are given as the means ± S.E.
We used the dose-response data (Fig. 4) to calculate the EC50 for ACh and the AChR Hill coefficient for each mutation. The average for the EC50, Hill coefficients, and Imax for each condition are presented in Table I. Four mutations, A455W, L456W, F459W, and G462W, resulted in a significant decrease in EC50 (i.e. gain in function), when compared with WT. In contrast, one mutation, L458W, resulted in an increase in EC50, thus demonstrating a loss in function as a result of a tryptophan substitution at that position. There was no significant difference between the Hill coefficient values of WT and mutants.
Overview—The purpose of this study was to determine the role of the γTM4 transmembrane domain on the AChR function. To that end, we performed tryptophan scanning mutagenesis on 11 residues, Cys451–Gly462, of the γTM4 transmembrane domain from the AChR of T. californica. Results from independent studies (
) suggest that two of these residues, Cys451 and Ser460, are exposed to membrane lipids. The AChR receptor was expressed in Xenopus oocytes and the effect of each tryptophan substitution on nAChR expression and channel function was assessed. Receptor expression was determined with [125I]α-BTX binding assays, whereas functional studies were performed using two electrode voltage clamp to determine ACh-induced currents for both WT and mutants.
We found that mutations L456W, F459W, and G462W resulted in a significant increase in nAChR expression when compared with that of WT. The rest of the mutations showed nAChR expression levels similar to WT. All of the mutations that caused increased nAChR expression and mutation A455W produced an increase in function, as evidenced by a decrease in EC50 relative to WT. Seven mutations, I454W, A455W, L456W, L457W, F459W, I461W, and G462W, resulted in an increase in macroscopic response (Imax) relative to WT. Mutations I454W, A455W, L457W, and L458W caused a significant increase in the normalized macroscopic response (Imax/fmol) induced by ACh. An analysis of the functional and structural implications of these results follows.
Expression of AChRs with Mutations on γTM4 —Our results demonstrate that the introduction of tryptophan, the largest and most lipophilic amino acid, did not result in a decrease in nAChR expression in any of the mutants studied. Moreover, three of the mutations studied, L456W, F459W, and G462W, resulted in significant 1.6–2-fold increase in nAChR expression relative to WT (Fig. 2). These results differ markedly from those found in αTM4 (
) from T. californica. Taken together, these three studies evaluated the effect of a total of 40 tryptophan replacements at different residues of each of the TM domains. From these, 23 such replacements resulted in either prevention or a marked decrease in nAChR expression when compared with WT and only two mutations, αI286W and βM285W, resulted in an increase in nAChR expression (
). The results obtained with αTM3, αTM4, and βTM3 thus demonstrate that the steric hindrance caused by the introduction of a large amino acid drastically decreases the efficiency of receptor assembly and suggest that these subunits are more tightly packed than γTM4. Consequently, it is possible that γTM4 has a larger membrane crossing angle than α and β subunits, thereby allowing the introduction of a tryptophan residue without major consequences to the AChR assembly. We thus hypothesize that the high tolerance for tryptophan replacements exhibited by γTM4 is the result of a loosely packed arrangement of its residues and/or a wide crossing angle in the membrane.
In contrast to the results obtained with α and β subunits, the results presented in this study are consistent with those reported for γTM3 from T. californica (
). In this study, a total of eight tryptophan substitutions were performed in this transmembrane domain, and all of the mutations resulted in nAChR expression levels similar to WT. These results demonstrate that γTM3 and γTM4 can accommodate the introduction of tryptophan without major consequences to the efficiency of nAChR assembly. Moreover, in the case of γTM4, introducing tryptophan at three residues (L456W, F459W, and G462W) increased the expression of the AChR and thus suggests that, at these positions, a large and lipophilic amino acid facilitates receptor assembly. The results obtained with αTM3-TM4, βTM3, and γTM3-TM4 are all consistent with the hypothesis that the transmembrane segments of the γ subunit are less tightly packed toward the interior of the AChR and/or have a wider crossing angle than α or β subunits.
Functional Correlations—The functional parameters calculated for the L456W, F459W, and G462W reveal a notable finding. These three mutations resulted in increased nAChR expression and displayed a gain in function when compared with WT, as evidenced by their lower EC50 and increased macroscopic response (Imax) values (Table I). Of the aforementioned mutations, L456W results in the highest gain in function and a 3.3-fold increased macroscopic response. These results suggest that, at these residues, the increased efficiency in channel assembly induced by tryptophan replacement also lead to a stabilization of the open state of AChR and consequently a gain in channel function. Moreover, these results suggest that these three residues play an important role in AChR gating.
Four additional mutations, I454W A455W, L457W, and L458W resulted in a significant increase in channel function and/or macroscopic response. L457W and I454W resulted in no change in EC50 but displayed a significant increase in both macroscopic and normalized macroscopic responses. The most dramatic increase in response was detected for mutation A455W, which resulted in a significant decrease in EC50 (p < 0.0001), a 3-fold increase in macroscopic response, and an 11-fold increase in normalized macroscopic response. These results suggest that introducing tryptophan at residue Ala455 results in the stabilization of the open channel conformation of the AChR, and therefore this position plays an important role in the AChR gating. The macroscopic responses detected in mutations A455W, L457W, and L458W constitute the largest observed of all of the tryptophan substitutions that we have reported so far. Future single-channel characterization of these mutants will provide important information necessary to understand the mechanism of action of tryptophan at these positions.
The L458W is the only mutation that displayed a significant increase in EC50 from all of the γTM4 mutants examined in the present study. According to a previous photolabeling study (
) places this position facing the γTM1 domain (see Fig. 6). The moderate increase in EC50 observed for the L458W mutation is the only indication of a possible inhibitory effect; however, the normalized current was significantly larger than that of WT. Furthermore, the nAChR expression level of this mutant is not significantly different from that of WT. These data indicate that the volume increase at this position does not affect nAChR assembly yet results in a minor increase in EC50.
The functional data obtained for γTM4 differs noticeably from that obtained for αTM4 (
) for T. californica. Taken together, these studies found that in 83% of the cases, a tryptophan replacement at these TM domains results in a decrease in either macroscopic or normalized macroscopic responses. In fact, tryptophan replacement in residues like αI417W (
) resulted in a complete loss of AChR assembly. Finally, the results for γTM4 are consistent with those obtained with γTM3, in which only one of seven tryptophan substitutions resulted in a decreased normalized macroscopic response, whereas 43% of the mutations resulted in an increase in normalized macroscopic response (
). These functional results are consistent with the trend seen with the nAChR expression data (see above), in that they demonstrate that tryptophan replacements in both α and β subunits result in a decrease in function, whereas in the γ subunit these substitutions result in either an increase or no change in function, relative to WT.
Structural Interpretation—Even though a high resolution structure from a crystallized AChR has not been reported yet, several research groups have used various alternative approaches in an effort to gain insight on the secondary structure of the TM domains of the AChR. Hydrophobicity plots of the transmembrane domains of the AChR from T. californica suggest that the secondary structure for these domains is an α-helix (
). Photoaffinity labeling studies conducted to delineate the lipid-exposed residues of different TM domains from T. californica indicate that the labeling pattern for various TM domains is consistent with that of an α helix (
). In one of the initial cryomicroscopy reports, a structure at 9-Å resolution was reported and led the author to conclude that only the TM2 domains lining the pore are α-helical, whereas TM1, TM3, and TM4 are β-sheets (
) concludes that the secondary structure of all of the transmembrane domains of the AChR is α-helical, yet no explanation is given for such a fundamental change in their model.
Another approach that has proven useful to predict protein structural patterns, as they relate to function, is tryptophan scanning mutagenesis. This approach has been used to characterize transmembrane segments from several proteins. These include the MotA proton channel (
) from T. californica. In this study, we have performed tryptophan scanning mutagenesis along the central portion of the γTM4 transmembrane domain from T. californica.
The detection of the surface AChR is not dependent upon AChR activation; thus the periodicity of nAChR expression reveals structural information about the closed or resting state. In contrast, the periodicity of functional parameters reveals structural information about the open state of the channel, because EC50 values are determined from the peak macroscopic response, or Imax, induced by ACh. Fig. 5A illustrates the periodicity of nAChR expression for each of the tryptophan replacements studied. We have found that normalizing the nAChR expression level of each mutant to the change in amino acid volume (fmol/ΔVol) at each residue provides a better representation of the steric disruption caused by each tryptophan substitution (
) and thus an improved approximation of the degree to which the net amino acid volume change affects the expression of the AChR. We have proposed that the reason for this observation is that transmembrane segments of the AChR are tightly packed, and thus side chain volume of residues in the transmembrane domain is the most critical property for heteropentamer assembly and consequently ion channel function (
). Moreover, in the case of the γM4 positions selected for this study, there are only two polar residues (Ser460 and Cys451), whereas the other nine residues (three leucines, two phenylalanines, two isoleucines, one alanine, and one glycine) are hydrophobic, with the exception of glycine, which is nonpolar aliphatic. Because of the homogeneous nature of the R groups of the amino acids in γTM4 and the tightly packed nature of transmembrane domains, it is reasonable to propose that the predominant effect of the tryptophan replacement is that caused by a net increase in amino acid side chain volume. The normalized nAChR expression data in Fig. 5A reveals three phases with an average periodicity of 3 amino acids for phases I, II, and III. The periodicity for the EC50 (Fig. 5B) and macroscopic response (Imax) values (Fig. 5C) display a similar trend with an average periodicity of 3.3 amino acids for both of these functional parameters. These data suggest a helical secondary structure for γTM4.
The data presented in this study are consistent with a recently published secondary structure of the AChR (
), which proposes that all of the TM domains are helical. Using this model reveals that the only mutation that resulted in an increase in EC50, L458W, faces the interior of the protein, specifically γTM1 (Fig. 6). A tryptophan replacement at this position may cause considerable steric hindrance, thus resulting in a mild increase in EC50. Two of the mutations that resulted in increased nAChR expression and gain in function, L456W and F459W, face the lipid according to this model (Fig. 6). Similarly, A455W, which resulted in a remarkable gain in function and increased macroscopic and normalized macroscopic response, faces the lipid interface (Fig. 6). It is thus possible that the introduction of a lipophilic amino acid, such as tryptophan, in these lipid-exposed residues increases the efficiency of oligomerization and/or channel function.
The fact that expression and function of the AChR are not negatively affected by tryptophan replacements in γTM4 is consistent with the hypothesis that this domain is more loosely packed and/or has a wider crossing angle than the other AChR TM domains studied. To accommodate γTM4 in a wide crossing angle may require this TM domain to be more stretched along the lipid bilayer, thus resulting in a periodicity of less than 3.6. Our calculations yield average periodicity values of 3 amino acids for nAChR expression and 3.3 amino acids for nAChR function. The periodicity analysis presented here is consistent with the hypothesis that γTM4 has a wider crossing angle than the other subunits. Analysis of the model proposed by Miyazawa (
), however, does not reveal drastic differences in crossing angles for the TM domains within each subunit.
In summary, we have used tryptophan scanning mutagenesis to assess the role of γTM4 on the function of the AChR from T. californica. We have found that three mutations resulted in an increase in nAChR expression, whereas the others did not induce any change in nAChR expression relative to WT. A similar trend is apparent in the functional data, because four mutations resulted in an increase in function (decreased EC50 for ACh), whereas the others did not change in function relative to WT. The only exception for this was mutation L458W, which resulted in a modest increase in EC50. These data suggest that this γTM4 is not as tightly packed as the other TM domains studied so far (i.e. αTM3, αTM4, and βTM3), because steric disruptions caused by the introduction of a large and lipophilic amino acid either do not affect or increase AChR oligomerization or function. Finally, the periodicity of changes, in both structure and function, suggest a helical secondary structure for the γTM4 of the T. californica AChR.
We especially thank Alexis Torres and Jose A. Lizardi for help with software-related problems. We also thank Dr. Manuel Navedo for help with the molecular modeling software and Dr. Madeline Nieves for help with computer hardware. We are grateful for the excellent technical help provided by Juan C. Galloza.