The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein

Abstract

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4β2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

Keywords: N2a; RVG; alpha7; electrophysiology; nAChR; nicotinic acetylcholine receptor; rabies virus; rabies virus glycoprotein.

Figure 1

The RVG peptide potently antagonizes α7 nAChRs. RVG responses on either un-injected (A) or α7 nAChR-expressing (B–D) Xenopus laevis oocyte currents were recorded using TEVC electrophysiology. (A) Application of 100 μM RVG peptide for 2s (blue drug application bar) to an un-injected oocyte failed to elicit a response (N = 3, n = 3). A lack of evoked response was also observed at lower RVG peptide concentrations. (B) Example trace showing a control 1s ACh (EC₉₀, 1.26 mM) (green drug application bar) α7 nAChR mediated response (B’) (green). Following a 1.5min wash, 30s pre-application of 10 nM MLA (red bar) prevented α7 nAChR activation via ACh (EC₉₀, 1.26 mM) stimulation (B”) (N=1, n = 3). (C) α7 nAChRs were initially activated with ACh (EC₉₀, 1.26 mM, green). To test for RVG peptide (blue) actions on α7 nAChRs, the RVG peptide (0.01 - 300 μM) was pre-applied for 30s, followed by a 1s ACh (EC₉₀, 1.26 mM) stimulation. Importantly, as seen in the compiled RVG peptide (0.01 - 300 μM) responses, no agonist responses were observed at any tested RVG peptide concentration (first 7s shown). (D) Concentration-response profile of RVG peptide antagonized α7 nAChR ACh-evoked responses. The IC₅₀ and n_H values are reported in Table 2 . Points are the mean ± S.D. (N = 6, n = 6).

Figure 2

The RVG peptide selectively inhibits the α7 nAChR subtype with comparatively high apparent potency. nAChR-expressing Xenopus laevis oocytes were pre-exposed to increasing concentrations of the RVG peptide for 30s, followed by 1s stimulation with the subtype specific ACh EC₉₀ (α4β2α5 20 µM; concatenated β3-α6-β2-α4-β2 40 µM; (α4β2)₂β2 100 µM; (α4β2)₂α4, α6/α3β2β3, (α3β2)₂β2 316 µM; (α3β4)₂β4 400 µM; (α3β4)₂α3 800 µM, α7 1.26 mM, (α3β2)₂α3 3.16 mM). (A) The α7 subtype was maximally inhibited by the RVG peptide while other isoforms lacking nAChR subtypes showed moderate inhibition (α7 nAChR data replicated from Figure 1 to facilitate comparison). (B) The RVG peptide inhibited the (α4β2)₂α4 isoform more than the (α4β2)₂β2 isoform. (C) The α3β2 isoforms were similarly inhibited by the RVG peptide. (D) The (α3β4)₂β4 isoform was minimally inhibited by the RVG peptide, while the (α3β4)₂α3 isoform was inhibited to a greater extent. IC₅₀, n_H, and p-values are reported in Table 2 . Points are the mean ± S.D. (N = 3 - 6, n = 3 - 7).

Figure 3

The α7 nAChR ACh-induced response was fully inhibited by 300 μM of the RVG peptide. The percent of ACh-induced current remaining post-RVG peptide exposure was determined for all tested nAChRs by performing concentration-response curves. The α7 nAChR was maximally inhibited by 300 μM of the RVG peptide, while the other subtypes were inhibited by varying amounts with 1000 μM RVG peptide. Significant changes in RVG peptide-induced inhibition are in reference to the α7 nAChR, and are indicated by the following statistical levels: β3-α6-β2-α4-β2 *p = 0.025, α4β2α5 **p = 0.0046, (α3β2)₂α3 **p = 0.0099, (α3β2)₂β2 **p = 0.0011, (α4β2)₂β2 ****p < 0.0001, (α3β4)₂α3 ****p < 0.0001, (α3β4)₂β4 ****p < 0.0001 (One-way ANOVA with Dunnett’s post-hoc test). Two-tailed unpaired t-tests were performed on the α4β2, α3β4, and α3β2 subtypes to assess differences between isoforms for each subtype. The (α4β2)₂α4 was inhibited significantly more than the (α4β2)₂β2 isoform (^ɫp = 0.0476). Similarly, the (α3β4)₂α3 was inhibited greater than its (α3β4)₂β4 counterpart (^ɫɫp = 0.0066). Values are mean ± S.D. with individual oocytes shown as symbols (N = 3 - 5, n = 4 - 6).

Figure 4

The RVG peptide competes with ACh for binding to the α7 nAChR. α7 nAChR-expressing Xenopus laevis oocytes were co-exposed to increasing concentrations of ACh and either the known competitive antagonists MLA or the RVG peptide. Two ACh Imax (10 mM) applications without MLA or RVG peptide were performed following the co-application responses. All responses were normalized to the second application of 10 mM ACh. (A) Co-application of 10 nM or 100 nM MLA significantly reduced ACh apparent potency without altering the maximal response. Sufficient ACh could not be applied to outcompete 1 µM MLA as ACh will block α7 nAChRs at concentrations greater than 10 mM. (B) The RVG peptide when co-applied with increasing concentrations of ACh also shifted the response curve to the right, reaching statistical significance with 50 µM. The maximal response was not altered at any RVG concentration tested. Data points are mean ± S.D. (N = 3 - 4, n = 9 - 14). IC₅₀, n_H, and p-values are reported in Table 3 .

Figure 5

RVG F192A, R196A, and R199A peptides inhibit the function of α7 nAChRs distinctly from the RVG peptide. α7 nAChR-expressing Xenopus laevis oocytes were pre-exposed to 30s of increasing concentrations of each altered RVG peptide, and inhibition of the ACh EC₉₀ response was measured. The α7 nAChR RVG peptide inhibition curve from Figure 1 is shown on each panel to facilitate comparison to the altered peptides. (A) The RVG F192A peptide concentration-response curve was shifted to the right relative to the RVG peptide. Application of 300 μM RVG F192A peptide evoked an agonist response. (B) The RVG R196A and (C) RVG R199A peptides showed reduced apparent potency on α7 nAChRs compared to the RVG peptide. (D) The RVG S195A peptide exhibited a similar concentration-response profile to the RVG peptide. IC₅₀, n_H, and p-values are reported in Table 4 . Points are mean ± S.D. (N = 3 - 5, n = 3 - 6).

Figure 6

RVGE robustly inhibits α7 nAChRs response to ACh with nanomolar apparent potency. (A) α7 nAChR-expressing oocytes were bath exposed to a single concentration of RVGE (1 - 300 nM) for 5min before EC₉₀ (1.26 mM) ACh application (1s). Example traces are shown. (B) Increasing concentrations of RVGE inhibited ACh-induced responses potently. Data were normalized to the control response. Non-linear regression curve fit to RVGE data with an IC₅₀ of 295 nM [CI 233, 399]. Data are mean ± S.D. (N = 2 - 3, n = 12 - 22).

Figure 7

The RVG-FITC peptide favorably labels cells expressing the α7 nAChR. Non-transfected and α7 nAChR-transfected N2a cells were treated with 30 µM RVG-FITC or 80 nM αBTX-AF647 for 24hr prior to live-cell confocal imaging. (A) Example image of RVG-FITC labeling using non-transfected N2a cells. (B) RVG-FITC robustly labeled α7 nAChR-expressing N2a cells. (A’, B’) Same as A and B images with the addition of the phase channel. (C) CTCF analysis of RVG labeling of transfected and non-transfected N2a cells (*p = 0.0180, two-tailed unpaired T-test). (D) αBTX-AF647 staining of endogenous α7 nAChRs expressed in non-transfected N2a cells. (E) α7 nAChR-transfected N2a cells labeled with αBTX-AF647. (D’, E’) Same as D and E images with the addition of the phase channel. (F) CTCF analysis of αBTX-AF647 labeling of transfected and non-transfected N2a cells (****p < 0.0001, two-tailed unpaired t-test). Images shown are representative of four separate experiments per group. CTCF data are mean ± S.D. (N = 4, n = 60 – 120).