Tryptophan Substitutions at Lipid-exposed Positions of the Gamma M3 Transmembrane Domain Increase the Macroscopic Ionic Current Response of the Torpedo californica Nicotinic Acetylcholine Receptor (original) (raw)
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Journal of Biological Chemistry, 2004
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 ␥M4 transmembrane domain (␥TM4) by introducing tryptophan, at residues 451-462, along the ␥TM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [ 125 I]␣-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC 50 , and Hill coefficient values for WT are 1.8 ؎ 0.4 fmol, 30.3 ؎ 1.1 M, 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 EC 50 (19.5 ؎ 1.7, 11.4 ؎ 0.7, 16.4 ؎ 3.8, and 19.1 ؎ 2.6 M, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC 50 (42.8 ؎ 6.8 M) 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 (EC 50 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 ␥TM4.
Biochemistry, 1996
Our previous amino acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the RC418 position. A tryptophan substitution on the RC418 produced a 3-fold increase in normalized macroscopic response to acetylcholine in voltage-clamped Xenopus laeVis oocytes (Lee et al., 1994). This result was explained by a 23-fold decrease in the closing rate constant measured from single-channel analysis (Ortiz-Miranda et al., 1996). In this study, we introduce more tryptophan substitutions at different positions of this postulated lipidexposed segment M4 in order to examine functional consequences at the single-channel level. From a series of amino acid substitutions at RG421, only phenylalanine and tryptophan produced a substantial increase in the open time constant. The lack of response from a tyrosine substitution at the RG421 suggests that the side chain volume is not the main structural element responsible for the effect of tryptophan on the stabilization of the open state of the channel. Three multiple mutants, RC418W/G421A, RC418W/ G421W, and RC418W/ C447W, were constructed in order to establish the correlation between the number of lipid-exposed tryptophans and the channel open time constant. The RC418W/G421A double mutant demonstrated that when both previous mutations are combined the open time constant was increased 1.5-fold relative to the RC418W. When the two mutants (RC418W and RG421W) were combined in a single mutation, a functional receptor was expressed and the open time constant of the new double mutant increased to 33.4 ms, an 80-fold increase relative to wild type. Estimations of free energy changes calculated from the rate constant for the opening transition suggest that each tryptophan contributes to the stabilization of the open state of the channel by about 0.8 kcal/mol, and the effect of tryptophan substitutions on the free energy is additive. This result suggests that in the channel gating mechanism of the AChR, each subunit contributes independently to the energy barrier between the open and closed state. At selected positions within the postulated lipid surface of the AChR, tryptophan substitutions could establish hydrophobic and perhaps dipole interactions that may play a dramatic role in the channel gating mechanism.
Biochemistry, 2000
Previous amino acid substitutions at the M4 domain of the Torpedo californica and mouse acetylcholine receptor suggested that the location of the substitution relative to the membrane-lipid interface and perhaps to the ion pore can be critical to the channel gating mechanism [Lasalde, J. along this postulated lipid-exposed segment M4 so that we can examine functional consequences on channel gating. The expression levels of mutants C412W, G421W, S424W, and V425W were almost the same as that of the wild type, whereas other mutants (M415W, L416W, C418W, I419W, I420W, T422W, and V423W) had relatively lower expression levels compared to that of the wild type as measured by iodinated R-bungarotoxin binding ([ 125 I]-R-BgTx). Two positions (L416W and I419W) had less than 20% of the wild type expression level. I417W gave no detectable [ 125 I]BgTx binding on the surface of oocyte, suggesting that this position might be involved in the AChR assembly, oligomerization, or transport to the cell membrane. The RV425W mutant exhibited a significant increase in the open channel probability with a moderate increase in the macroscopic response at higher ACh concentrations very likely due to channel block. The periodicity for the alteration of receptor assembly and ion channel function seems to favor a potential R-helical structure. Mutants that have lower levels of expression are clustered on one side of the postulated R-helical structure. Mutations that display normal expression and functional activity have been shown previously to face the membrane lipids by independent labeling studies. The functional analysis of these mutations will be presented and discussed in terms of possible structural models.
Biochemistry, 2003
The functional role of the RM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within RM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [ 125 I]-R-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipidexposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the RM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC 50 . Taken together, our results suggest that RM3 contributes to the gating machinery of the nicotinic ACh receptor and that RM3 is comprised of a mixture of two types of helical structures.
Biochemistry, 2003
The functional role of the alphaM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within alphaM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [(125)I]-alpha-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipid-exposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the alphaM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptoph...
Biochemistry, 2003
The functional role of the RM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within RM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [ 125 I]-R-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipidexposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the RM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC 50. Taken together, our results suggest that RM3 contributes to the gating machinery of the nicotinic ACh receptor and that RM3 is comprised of a mixture of two types of helical structures.
Biochemistry, 2004
We used tryptophan substitutions to characterize the beta M3 transmembrane domain (TM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the TM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [ 125 I]-R-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282-296 of the TM3 domain was consistent with an R-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed R-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica subunit.
Journal of Biological …, 2007
Membrane proteins constitute a large fraction of all proteins, yet very little is known about their structure and conformational transitions. A fundamental question that remains obscure is how protein domains that are in direct contact with the membrane lipids move during the conformational change of the membrane protein. Important structural and functional information of several lipid-exposed transmembrane domains of the acetylcholine receptor (AChR) and other ion channel membrane proteins have been provided by the tryptophan-scanning mutagenesis. Here, we use the tryptophan-scanning mutagenesis to monitor the conformational change of the ␣M3 domain of the muscle-type AChR. The perturbation produced by the systematic tryptophan substitution along the ␣M3 domain were characterized through two-electrode voltage clamp and 125 Ilabeled ␣-bungarotoxin binding. The periodicity profiles of the changes in AChR expression (closed state) and ACh EC 50 (openchannel state) disclose two different helical structures; a thinner-elongated helix for the closed state and a thicker-shrunken helix for the open-channel state. The existence of two different helical structures suggest that the conformational transition of the ␣M3 domain between both states resembles a spring motion and reveals that the lipid-AChR interface plays a key role in the propagation of the conformational wave evoked by agonist binding. In addition, the present study also provides evidence about functional and structural differences between the ␣M3 domains of the Torpedo and muscle-type receptors AChR.
Biochemistry, 2004
We used tryptophan substitutions to characterize the beta M3 transmembrane domain (TM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the TM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [ 125 I]-R-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282-296 of the TM3 domain was consistent with an R-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed R-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica subunit.
Biophysical Journal, 1994
Site-directed mutagenesis was used to mutate aCys418 and (3Cys447 in the M4 domain of Torpedo califomica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both a subunits to aTrp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 pM to 13 pM. Patch measurements of single-channel currents revealed that the aTrp418 increased channel open times -28-fold at 1 30C with no effect on channel conductance. All of our measured functional changes in the aTrp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.