Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors

Abstract

Allosteric modulators of pentameric ligand-gated ion channels are thought to act on elements of the pathways that couple agonist binding to channel gating. Using α4β2 nicotinic acetylcholine receptors and the α4β2-selective positive modulators 17β-estradiol (βEST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pentameric ligand-gated ion channels. Previous studies have shown that the binding site for potentiating βEST is in the C-terminal (post-M4) region of the α4 subunit. Here, using homology modeling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top half of a cavity between the third (M3) and fourth transmembrane (M4) α-helices of the α4 subunit. We found that the binding sites for βEST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and Phe¹⁷⁰ of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Tyr³⁰⁹) and Phe¹⁶⁷, a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating βEST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop.

Keywords: C-terminal domain (carboxyl tail domain, CTD); Cys-loop receptor; nicotinic acetylcholine receptors (nAChR); signal transduction; transmembrane domain.

FIGURE 1.

Effects of βEST and dFBr on alternate α4β2 nAChRs. A, structure of βEST. B, concentration response effects of βEST on the ACh EC₁₀ current responses of (α4β2)₂α4 and (α4β2)₂β2 nAChRs. The data points represent the means ± S.E. of at least four experiments. The data were fit to the monophasic Hill equation, as described under “Experimental Procedures.” The effects of βEST were determined on ACh currents evoked by EC₁₀ (3 μm for (α4β2)₂β2) nAChRs and 10 μm for (α4β2)₂α4 nAChRs. C, structure of dFBr. D, potentiating effects of dFBr on the ACh responses of alternate (α4β2)₂α4 and (α4β2)₂β2 nAChRs. The concentration-responses curves were obtained as for those of βEST. The data points represent the means ± S.E. of five experiments. For B and D, functional expression of (α4β2)₂α4 or (α4β2)₂β2 nAChRs was achieved by expressing the concatenated forms of the alternate stoichiometries of the α4β2 nAChRs in Xenopus oocytes, as described under “Experimental Procedures.”

FIGURE 2.

M3 and M4 residues in the α4 nAChR subunit impact dFBr potentiation. A, full model of (α4β2)₂α4 with α4 in yellow and β2 in blue on the left, and a zoom on the potential dFBr site is on the right. Residues that might be involved in binding dFBr are shown as sticks. dFBr is shown in light blue. B, superimposition of the X-ray structure of (α4β2)₂β2 nAChR onto the homology model used in this study to predict the binding site for dFBr in the α4β2 nAChR. The homology model is shown in yellow, and the X-ray structure is in gray. Relevant residues are shown as sticks (dark pink in the homology model and gray in the X-ray structure). C, representative current responses elicited by ACh EC₁₀ in the absence or presence of dFBr from oocytes expressing mutant α4Y309Aβ2, α4F312Aβ2, or α4L617Aβ2 nAChRs. D, maximal dFBr potentiation of ACh EC₁₀ current responses from wild type or mutant (α4β2)₂α4 or (α4β2)₂β2 nAChRs. The alternate stoichiometries of the α4β2 nAChR were expressed using concatemeric constructs, as described under “Experimental Procedures.” Maximal potentiation by dFBr of ACh EC₁₀ current responses (I_{max pot}) was calculated as (I_{ACh EC}¹⁰ + dFBr)/I_{ACh EC}¹⁰ from concentration response curve data, as described under “Experimental Procedures.” The values represent the means ± S.E. of at least five independent experiments. Asterisks indicate that the change in I_{max pot} is statistically significant (**, p < 0.001, ***, p < 0.0001), as measured by one-way ANOVA with Dunnett's correction. The dotted line indicates a potentiation ratio of 1 (no potentiation). The cartoon underlying each column show how many copies of F312A (black dots) are present in the receptors. The red dotted line indicates a potentiation ratio of 1 (no potentiation).

FIGURE 3.

Effects of MTSET on dFBr potentiation of α4β2 nAChRs. A, representative current traces from α4F617Cβ2 receptors showing potentiation of ACh EC₁₀ currents before and after a 2-min application of 1 mm MTSET. B, scatter plot showing changes in dFBr potentiation after MTSET application to wild type or mutant α4β2 nAChRs containing α4L617C or α4F316C subunits. The percentage of change in dFBr potentiation was estimated using the equation: % Change = ((I_after/I_initial) − 1) × 100), where I_initial is the response to ACh EC₁₀ + dFBr EC₁₀₀ prior to MTSET application, and I_after is the response to ACh EC₁₀ + dFBr EC₁₀₀ after MTSET application. Asterisks indicate values that are significantly different from wild type (*, p < 0.05; ***, p < 0.0001). C, representative traces showing the rate of MTSET (20 μm) reaction with L617C in the absence or presence of EC₁₀₀ dFBr (10 μm). D, for protection assays using α4F617Cβ2 mutant receptors, the observed decreases in dFBr potentiation were plotted versus cumulative MTSET exposure in α4F617Cβ2 receptor. The data obtained from individual assays were normalized to the potentiation measured at t = 0 and fit to a single-exponential decay curve, as described under “Experimental Procedures.” ●, MTSET alone; ■, MTSET + dFBr. The data points are the means ± S.E. from at least three independent assays. Asterisks indicate values that are significantly different from wild type (***, p < 0.0001).

FIGURE 4.

Mutations in Cys loop affect dFBr effects on nAChRs. A, sequence alignment of the agonist-binding-channel gating coupling elements β1-β2 and Cys loop, M3 and M4 transmembrane α-helices, and the post-M4 region of nAChR α and β2 subunits. Residues that impact dFBr potentiation in α4β2 nAChRs are highlighted in bold. As shown in the alignment, these residues are conserved in the nAChR family. The post-M4 region is highlighted for all the sequences aligned. Unlike, the residues of the β1-β2, Cys loop, and M3, M4 and post-M4 are highly variable in the nAChR family (1). Sequences were aligned using the T-Coffee sequence alignment tool. B, concentration-response curve for dFBr on wild type α3β2 nAChRs (●) or α3F310Aβ2 (○) nAChRs. The data points represent the means ± S.E. of at least four experiments. The data were fit to the monophasic Hill equation, as described under “Experimental Procedures.” The inset shows representative ACh current responses traces of wild type or mutant α3β2 nAChRs in the absence or presence of increasing dFBr concentrations (10, 30, and 100 μm). C, maximal dFBr potentiation of ACh EC₁₀ current from wild type and β1-β2 or Cys loop mutant α4β2 receptors. dFBr potentiation was calculated as (I(ACh EC₁₀ + dFBr)/IACh EC₁₀). The dashed line indicates wild type levels of potentiation, and the dotted line indicates a potentiation ratio of 1 (no potentiation). Asterisks indicate values that are significantly different from wild type (**, p < 0.001).

FIGURE 5.

Post-M4 is a critical determinant of dFBr potentiation. A, maximal effects of dFBr on wild type and mutant α4β2 nAChRs. Mutations were introduced individually on α4 post-M4. dFBr potentiation was calculated as I(ACh EC₁₀ + dFBr)/IACh EC₁₀. The data are the means ± S.E. from at least three oocytes from two or more batches. The dashed line indicates wild type levels of potentiation, whereas the dotted line indicates a potentiation ratio of one (no potentiation). Asterisks indicate values that are significantly different from wild type (**, p < 0.001). B, representative traces of the maximal effects of dFBr on α3PPWLAGMIβ2 nAChRs. α3 subunit in wild type receptors has a longer and more hydrophilic post-M4 (LMAREDA). Wild type α3β2 receptors are insensitive to potentiation by dFBr (see Fig. 3B). C, concentration-response curves for the effects of dFBr on post-M4 mutants of the α3β2 nAChR. The post-M4 domain of the α3 subunit was changed to that of the α4 subunit, first keeping the α3 PQ motif preceding post-M4 (α3PQWLAGMI) and then substituting PQ for the PP motif found in the α4 subunit (α3PPWLAGMI). F310A effects on the effects of dFBr on α3PPWLAGMIβ2 receptors was also determined. The data were fit by non-linear regression as described under “Experimental Procedures.”

FIGURE 6.

Cys loop and post M4 interactions affect dFBr potentiation. A, homology model of α4β2 showing the Cys loop and adjacent M3, M4, and post-M4 regions of a α4 subunit. Note that PP and post-M4 are shown to be helical because the homologous region in the template used (5-HT3 X-ray structure) was found to be helical (36); however, the PP motif most likely disturbs the helicity. Also, note that only the PPWL motif of post-M4 (blue cartoon) is included in the model because the template was missing for the latter four residues of post-M4, the AGMI motif. B–D, representative current responses elicited by ACh EC₁₀ traces in the presence and absence of EC₁₀₀ dFBr from α4β2 nAChRs containing the following mutant α4 subunits: α4F170I, α4I626F, and α4F170I/I626F (B); α4F167Y, α4L305F, and α4F167L-L305F (C); and α4F167Y, α4Y309F, and β4F167Y/Y309E) α4β2 nAChRs (D). E, histogram of peak EC₁₀ ACh currents in the presence of dFBr obtained from oocytes expressing wild type or mutant (α4F170I, α4I626F, α4F170I/I626F, α4F167Y, α4Y309F and β4F167Y/Y309E) α4β2 nAChRs. The data represent the means ± S.E. of at least three independent experiments. Statistical significance was determined by one-way ANOVA with a Dunnett's post-test. *, p < 0.05; *** p < 0.001. The dashed line indicates the wild type level of dFBr potentiation, and the dotted line indicates a potentiation ratio of 1 (no response).

FIGURE 7.

Cys loop and post-M4 interactions affect βEST potentiation. A and B, representative traces of ACh current responses in the presence and absence of EC₁₀₀ βEST from wild type and mutant receptors. The mutant α4 subunits tested were as follows: α4F170I, α4I626F, and α4F170I/I626F (A) and α4F167Y, α4Y309F, and β4F167Y/Y309E (B). C, histogram of peak EC₁₀ ACh currents in the presence of EC₁₀₀ βEST obtained from oocytes expressing wild type or mutant (α4F170I, α4I626F, α4F170I/I626F, α4F167Y, α4Y309F, and β4F167Y/Y309E) α4β2 nAChRs. The data represent the means ± S.E. of at least three independent experiments. Statistical significance was determined by one-way ANOVA with a Dunnett's post-test. **, p < 0.001. The dashed line shows the wild type level of βEST potentiation, and the dotted line indicates a potentiation ratio of one (no potentiation).