Molecular dynamic modeling reveals potential sites of N-Aβ terminal interactions at α7*-nAChR
Informed by our set of findings using single-channel recordings, we carried out independent molecular dynamic simulations starting from different docking conformations of the natural N-Aβ fragment placed at α7+/α7- and α7+/β2- binding interfaces (Figure 6). We calculated that N-Aβ fragments with different poses appeared to remain bound in the binding site during the simulation up to 400 ns. The residues of N-Aβcore hexapeptide, including Y10, H13, H14 (equivalent to Y625, H628, H629 in APP sequence) and negatively charged Q15 (corresponding to Aβ10-15) in the Aβ sequence, can form π-stacking interactions and salt bridges with the aromatic residues (e.g., Y92, W148, Y187, Y194) and charged residues (e.g., K142, K144, R185 and K191) of α7- subunit in both α7+/α7- and α7+/β2- interfaces (Figures 6B-B” and D-D”). This is consistent with the previously published data as R185 and Y187 in our work are equivalent to R208 and Y210 in Roberts et al.’s work, and Y187 is also equivalent to Y188 in Forest et al.’s work. The aforementioned N-Aβcore residues also have interactions with the residues (e.g., W54, K75 and H114) of α7+ subunit in α7+/α7- interface and the residues (e.g., W57, F119 and K163) of β2+ subunit in α7+/β2- interface. The simulation results suggest that several residues of N-Aβcore (Y10, H13, H14 in full-length Aβ) are critical for the agonist-like interactions with α7*-nAChR canonical binding sites that are rich in charged and aromatic residues, although the interacting residue pairs are not specific. This is consistent with the experiment data showing that the mutant N-InAβcore 10-SEVAAQ-15 is inactive on α7*-nAChR single-channel kinetics.
DISCUSSION
In this study, we identified for the first time the activation of human homomeric α7- and heteromeric α7β2-nAChR single-channel openings by the human N-Aβ fragment (N-terminal 15 amino acid Aβ peptide fragment) and by the N-Aβcore (essential core sequence hexapeptide within the N-Aβ fragment), as we have done for oligomeric Aβ1-42(oAβ42) (George et al., 2021). Specifically, we demonstrated that N-Aβ fragment and N-Aβcore differentially alter the biophysical properties of α7*-nAChR subtypes in comparison to activation by ACh and oAβ42 (summarized in Figure 7).
Here, we provide evidence that N-Aβ fragment exposure alone increases α7-nAChR component 2 (C2) open-dwell time within bursts and burst duration relative to effects of ACh alone. Co-administration the N-Aβ fragment plus ACh increased α7-nAChR C2, burst duration and Popen relative to effects of ACh alone, while the α7-nAChR burst duration was higher in the presence of N-Aβ fragment alone or co-administered with oAβ42 than upon exposure to oAβ42 alone. However, N-Aβ fragment plus oAβ42 exposure reduces α7β2-nAChR open-dwell times within bursts (component 1; C1) relative to effects of oAβ42 alone. Importantly, at α7β2-nAChR, N-Aβcore exposure when co-applied with oAβ42 reduces C1, C2, burst duration, and Popen from elevated levels seen for oAβ42 treatment alone to those seen upon exposure to ACh, Aβ core or N-Aβ alone. The current observations, therefore, substantially expand our understanding of the functional interactions of α7*-nAChR with Aβ (Grassi et al., 2003; Liu et al., 2009; Wang, Lee, D’Andrea, et al., 2000; Wang, Lee, Davis, & Shank, 2000) and the N-terminal domain Aβ peptides at the single-channel level.
Several studies have investigated agonist-like actions of Aβ1–42 at α7*-nAChR with varying effects (Dineley, Bell, Bui, & Sweatt, 2002; Fu & Jhamandas, 2003; Lasala, Fabiani, Corradi, Antollini, & Bouzat, 2019). Mutational and base docking analyses suggest that a site of interaction between Aβ1-42 and α7*-nAChR is dependent upon key aromatic residues in loops A, B, and C in the extracellular, ACh-binding domain (Espinoza-Fonseca, 2004a, 2004b; Tong, Arora, White, & Nichols, 2011). This is in accordance with other molecular modeling simulations suggesting similar docking conformations on the α7-nAChR-AChBP (acetylcholine binding protein) for N-Aβ fragment and N-Aβcore (Forest et al., 2018; Tong et al., 2011) at the α7*-nAChR ACh-binding pocket. More specifically, computational modeling has suggested binding of the N-Aβ fragment at the C-loop of α7+/α7- interfaces (Roberts, Stokoe, Sathler, Nichols, & Kim, 2021). Roberts and colleagues identified three key C-loop residues (R208, Y210 and E211) critical for Aβ1-42 binding at α7-nAChR. Forest et al. modeled the N-Aβcore into the known ligand-activation binding pocket in the α7-nAChR-AChBP chimera and identified that tyrosine (Y188), which is equivalent to Y210 in Roberts et al.’s work, is critical for Aβ-mediated activation of the rodent α7*-nAChR (Forest et al., 2018).
A mechanistic interpretation, for the common action of the N-Aβ fragment and the N-Aβcore to elicit α7*-nAChR single-channel openings is that they act at both receptor subtypes at orthosteric sites between subunit interfaces. An implication is that the N-Aβcore could represent a minimal template for α7*-nAChR activation via the N-terminal domain of oAβ42 and hence the N-Aβ fragment. Uniquely, N-Aβ fragment exposure alone selectively prolonged homomeric α7-nAChR open-dwell times (component 2; c2) within bursts and increases total burst duration, relative to effects of ACh alone. More strikingly, α7-nAChR burst duration, open-dwell time within bursts, and Popen were elevated relative to effects of ACh alone when the N-Aβ fragment was applied in the presence of ACh. The identification of residues within α7*-nAChR canonical agonist binding site (i.e., α7+/α7- and/or α7+/β2- interfaces) for oAβ42, N-Aβ fragment or N-Aβcore does not discount the possibility that these ligands interact with α7*-nAChR at other sites.
Consistent with previous studies (George et al., 2021), full-length oAβ42 exposure enhanced α7β2-nAChR Popen, single-channel open-dwell time within bursts and total burst duration compared to effects of ACh alone, or to the effects of N-Aβ fragment or N-Aβcore alone. On the other hand, oAβ42 was not significantly different in effect on these parameters for homomeric α7-nAChR from that of ACh, while the N-Aβ fragment but not the N-Aβcore enhanced open-dwell time and bursts of the α7-nAChR. That neither N-Aβ fragment nor N-Aβcore mimic the effects of oAβ42 on α7β2-nAChR of oAβ42 is noteworthy, especially as both N-Aβ fragment and N-Aβcore alone activate α7β2-nAChR and α7-nAChR channel openings, to a similar extent as that of ACh, the natural ligand. This difference may explain, in part, why the N-Aβ fragment and the N-Aβcore are non-toxic while being neuroprotective (Forest et al., 2018).
From another perspective, the absence of N-Aβ fragment effects alone or in combination with ACh on open-dwell time, burst duration or Popen at α7β2-nAChR is potentially informative. ACh occupancy at only one of five orthosteric sites in homomeric α7-nAChR produces maximal current responses (Andersen et al., 2016). ACh occupancy at a single orthosteric site coupled with N-Aβ fragment agonisms and positive allosteric modulation (agoPAM) activity at one or more remaining orthosteric or perhaps peri-orthosteric sites on α7-nAChR may explain the potentiation of Popen in the presence of both ACh and N-Aβ fragment. Such action would not occur if the single α7+/α7- orthosteric site in α7β2-nAChR is occupied by ACh, leaving no possibility for N-Aβ fragment to also do so, or just allowing for one or the other ligand to occupy that functional interface. This interpretation is consistent with prior and current modeling implicating extended domains for Aβ interactions with α7*-nAChR and would explain agonist-like action of N-Aβ fragment at both α7- and α7β2-nAChR in addition to observed N-Aβ fragment agoPAM activity in α7-nAChR in the presence of ACh.
In view of the findings that co-exposure to N-Aβcore also neutralizes the ability of oAβ42 to enhance α7β2-nAChR but not α7-nAChR Popen, single-channel open-dwell time within bursts, and total burst duration, another reasonable hypothesis is that the N-Aβcore sterically hinders oAβ42 interactions at a non-orthosteric sites involved in the modulatory enhancement of α7β2-nAChR single-channel kinetics that is not accessed by ACh. This could imply that oAβ42 residues beyond those present in N-Aβ fragment or N-Aβcore are involved in potentiation of α7β2-nAChR channel opening. Indeed, the ability of N-Aβ fragment alone and/or in combination with ACh to enhance features of α7-nAChR, but not α7β2-nAChR, channel opening resembles that of a PAM (Andersen et al., 2016; Williams, Wang, & Papke, 2011). This agoPAM mechanism is consistent with studies suggesting that oAβ42 enhance α7-nAChR channel opening by acting both orthosterically and allosterically (Lasala et al., 2019). The possibility of allosteric modulation of α7β2-nAChR by the N-Aβ fragment and the N-Aβcore raises the question as to what extent this non-orthosteric regulation contributes to the reversal of Aβ-linked deficits in synaptic plasticity by these Aβ-derived, neuroprotective peptides.
Lastly, it is important to consider the biologically and/or pathologically relevant concentrations of these reagents and their forms (e.g., aggregation state). Whereas definitive knowledge of these factorsin situ , particularly at vicinal critical sites (e.g., synapses) remains to be determined, oAβ42 concentrations from the low pM range as physiological to high nM-low µM range as neurotoxic have been reported (Forest et al., 2018). Our previous studies used oAβ42 at 100 nM, where it enhances basal forebrain cholinergic neuronal excitability in organotypic cultures (George et al., 2021), which coincides with a previous study demonstrating that nAChR-sensitive neurons to cellular toxicity at this concentration (Arora, Alfulaij, Higa, Panee, & Nichols, 2013). In regard to single-channel activity of the α7*-nAChR, studies show that oAβ42 at high nM elicits α7*-nAChR channel activity, preferentially modulating α7β2-nAChR single-channel kinetics by enhancing α7β2-nAChR open-dwell times within single-channel bursts. These findings contrast with evidence suggesting that high concentrations of oAβ42 may lead to functional blockade of α7*-nAChR (Lasala et al., 2019). However, long-term exposure may convert from agonism to downregulation of α7*-nAChR, contributing to deficits in cholinergic neurotransmission and to cognitive deficits observed in AD. The impact of chronic elevated oAβ42 as well as the N-terminal Aβ peptides on α7*-nAChR channel function remains to be studied.
In conclusion, our research has expanded our understanding of the range of Aβ peptides impacting human α7*-nAChR, which could play a role in both neuronormal biological processes and neurodegenerative conditions. We speculate that oAβ42/α7β2-nAChR interactions manifest early in AD development, suggesting that targeting or reducing them could hold therapeutic value, particularly if the natural responses of α7*-nAChR to ACh remain unaffected. Together, these findings offer novel insights into potentially effective strategies for selectively safeguarding against Aβ-induced neurodegeneration, particularly in the pre-clinical stages of AD.
FIGURE LEGENDS
Figure 1. Design and functional expression of human α7*nAChR. (A) Schematic representation of human α7- and α7β2-nAChR DNA constructs. Top row: Concatenated α7-nAChR homopentamer (containing only α7 subunits) harboring the sequence for the mCherry fluorophore and fused to the α7 nAChR subunit gene in position 5 (P5); Middle row: Concatenated α7β2-nAChR (containing two β2 subunits in position 2 and 4) harboring the sequence for the mCherry fluorophore and fused to the α7 nAChR subunit gene in position 5 (P5). Bottom row: Construct for the human nAChR chaperone protein NACHO containing the sequence for the ZsGreen1 fluorophore. (B) Illustration of assembly and stoichiometry of concatenated human α7-nAChR homopentamer and concatenated human α7β2-nAChR co-expressed with the nAChR chaperone NACHO + ZsGreen1. (C) Fluorescent imaging of the α7-nAChR homopentamer transiently expressed in SH-EP1 cell lines. Left panel represents NACHO expression (as indicated by ZsGreen1 expression), middle panel represents α7-nAChR expression (as indicated by mCherry expression), and bottom panel represents cells co-expressing NACHO and α7-nAChR (merged; yellow). (C’) Fluorescent imaging of the α7β2-nAChR heteropentamer transiently expressed in SH-EP1 cell lines. Left panel represents NACHO expression (as indicated by ZsGreen1 expression), middle panel represents α7β2-nAChR expression (as indicated by mCherry expression), and bottom panel represents cells co-expressing NACHO and α7β2-nAChR (merged; yellow). Arrows indicated cells targeted for cell-attached, single-channel recordings. Scale bar represents 50 µm.
Figure 2. α7*-nAChR single-channel burst amplitude and burst rates. α7- and α7β2-single-channel burst amplitudes (Panels A and B; pA) and burst rates (Panels C and D; bursts/sec) were analyzed for each treatment. No significant differences in single-channel burst amplitudes or burst rates were observed across α7*-nAChR subtypes. Differences in single-channel amplitude and burst rate were determined using two-way ANOVA with Tukey’s posthoc test for multiple comparisons. Data represented as mean ± SEM.
Figure 3. Selective enhancement of α7*-nAChR single-channel open probability (Popen) by oAβ42, N-Aβcore, and the N-Aβ fragment. (A) Co-application of ACh + N-Aβ fragment enhanced single-channel Popen of homomeric α7-nAChR compared to α7-nAChR exposed to ACh alone, N-Aβ fragment alone, ACh + N-Aβcore, or oAβ42 + N-Aβ fragment. (B) Heteromeric α7β2-nAChR Popen was enhanced in the presence of oAβ42 alone compared to α7β2-nAChR exposed to ACh alone, N-Aβcore alone, N-Aβ fragment alone, or oAβ42 + N-Aβcore. Further, co-application of oAβ42 + N-Aβ fragment enhanced α7β2-nAChR Popen compared to groups treated with oAβ42 + N-Aβcore, ACh + N-Aβ fragment, or N-Aβ fragment alone. No differences in Popen were observed between α7β2-nAChR exposed to oAβ42 alone or oAβ42 + N-Aβ fragment. Differences in α7*-nAChR single-channel Popen were determined using two-way ANOVA with Tukey’s post-hoc test for multiple comparisons. Data represented as mean ± SEM. *p<0.05, ***p<0.001, ****p<0.0001.
Figure 4. Homomeric α7-nAChR single-channel open-dwell times within bursts and total burst duration. (A) α7-nAChR open-dwell times within bursts (ms) are best fit with two open-dwell time components indicating two distinct open-dwell time distributions within bursts (red lines indicate individual components and black line represents overall fit). Representative traces demonstrate a binary closed or open state at uniform amplitudes and various burst durations and accompany each single-channel open-dwell time histogram. Closed (c) and open (o; dashed line) states are indicated for each trace. (B) No significant difference was observed between treatment groups was observed in single-channel open-dwell time component 1 (C1). (C) Significant differences in single-channel open-dwell time component 2 (C2) were observed between treatment groups. α7-nAChR subtypes exposed to ACh + N-Aβ fragment exhibited the greatest increase in C2 single-channel open-dwell time compared to ACh alone, N-Aβ fragment alone, ACh + N-Aβcore, or oAβ42 + N-Aβ fragment. (D) α7-nAChR single-channel burst duration. Significant differences in α7-nAChR bust duration were observed for α7-nAChR exposed to N-Aβ fragment alone compared to α7-nAChR exposed to ACh or oAβ42 alone, N-Aβcore alone, ACh + N-Aβ fragment, or oAβ42 +N-Aβ fragment. Further, α7-nAChR single-channel burst duration (ms) was enhanced when the N-Aβ fragment was co-applied with ACh or oAβ42 when compared to α7-nAChR exposed to ACh or oAβ42 alone, indicating the ability of N-Aβ fragment to enhance α7-nAChR burst duration. Differences in α7-nAChR single-channel open-dwell times and/or burst duration were analyzed using a two-way ANOVA with Tukey’s posthoc test for multiple comparisons. Data represented as mean ± SEM. *p<0.05, ****p<0.0001.
Figure 5. Heteromeric α7β2-nAChR single-channel open-dwell times within bursts and total burst duration. (A) α7β2-nAChR open-dwell times within bursts (ms) are best fit with two open-dwell time components indicating two distinct open-dwell time distributions within bursts (red lines indicate individual components and black line represents overall fit). Representative traces demonstrate a binary closed or open state at uniform amplitudes and various burst durations and accompany each single-channel open-dwell time histogram. Closed (c) and open (o; dashed line) states are indicated for each trace. (B) Significant differences in single-channel open-dwell time component 1 (C1) were observed for α7β2-nAChR events elicited with oAβ42 compared to α7β2-nAChR exposed to ACh alone, N-Aβcore alone, N-Aβ fragment alone, oAβ42 + N-Aβcore, or oAβ42 + N-Aβ fragment. (C) Comparison of α7β2-nAChR single-channel open-dwell time component 2 (C2). C2 open-dwell times were enhanced for α7β2-nAChR in the presence of oAβ42 alone and were greater than α7β2-nAChR C2 open-dwell times induced by ACh alone, N-Aβcore alone, N-Aβ fragment alone, or oAβ42 + N-Aβcore. Further, C2 α7β2-nAChR open-dwell times were similar between α7β2-nAChR exposed to oAβ42 alone and co-application of oAβ42+ N-Aβ fragment. Application of oAβ42 + N-Aβcore normalized C2 open-dwell times compared to α7β2-nAChR events elicited with oAβ42 + N-Aβ fragment. (D) α7β2-nAChR burst duration. Single-channel burst durations were significantly greater for α7β2-nAChR events elicited with oAβ42 alone or those elicited with oAβ42 + N-Aβ fragment. Differences in oAβ42-induced α7β2-nAChR burst duration were significantly greater than those elicited with ACh alone, N-Aβcore alone, N-Aβ fragment alone, or oAβ42 + N-Aβcore. Data represented as mean ± SEM. Significance was determined using 2-way ANOVA as follows: *p<0.05, ***p<0.001, ****p<0.0001.
Figure 6. Aβ/α7*-nAChR molecular dynamic simulations. (A) Simulated binding pose of a single N-Aβ fragment (highlighted in red) placed within the homomeric α7-nAChR (α7 subunits represented in yellow). (A’) Close up of the conical binding site formed between two subunits at the α7-/α7+ subunit interface. Structural elements with the orthosteric binding site are labeled and N-Aβ fragment binding restricted within the square: Loop C (cyan) of α7+ subunit, loop E (purple) and loop D (magenta) of α7- subunit. (B-B”) Side view of representative snapshots extracted from three (out of ten; see methods) simulation runs where N-Aβ fragment remained bound in the binding site after the systems were simulated for 400 ns. The residues of N-Aβ fragment (colored cyan and magenta; magenta representing the N-Aβcore) are indicated in red, the residues of α7+ subunit are indicated in blue, and the residues of α7- subunit are indicated in purple. (C) Simulated binding pose of a single N-Aβ fragment (highlighted in red) placed within the heteromeric α7β2-nAChR (α7 and β2 subunits represented in yellow and green, respectively). (C’) Close up of the conical binding site formed at the α7-/β2+ subunit interface. Structural elements within the orthosteric binding site are labeled: Loop C (cyan) of α7+ subunit, loop E (purple) and loop D (magenta) of β2- subunit. (D-D”) Side view of representative snapshots extracted from three (out of ten) simulation runs where N-Aβ fragment remained bound in the binding site after the systems were simulated for 400 ns. The residues of N-Aβ fragment are indicated in red, the residues of α7+ subunit are indicated in blue, and the residues of β2+ subunit are indicated in purple.
Figure 7. Summary of Aβ-induced alterations in α7*-nAChR single-channel kinetics.
Supplemental Figure 1 (S1). α7*-nAChRs single-channel closed-dwell time distributions. α7- and α7β2-nAChR closed-dwell times were best fit to with 4 closed-dwell time components for all treatments. (A) α7- nAChR and (B) α7β2-nAChR closed-dwell times. Representative traces (below each histogram) are included for all experimental groups. Dashed lines represent closed (c) states. Bursts of single-channel activity (identified using Qub single-channel software; see methods) are highlighted within each trace for α7-nAChR (red) and α7β2-nAChR (blue). At a constant transmembrane voltage, both α7- and α7β2-nAChR closed-dwell times could be best fit with four (4) distinct closed-dwell time components. For each recording, single-channel bursts were defined as a series of openings separated by closures shorter than the minimum inter-burst closed duration (or Tcrit ). Each closed-dwell time component and minimum inter-burst closed time was calculated for each of the following ligand exposure conditions and summarized in Table 1: ACh or oAβ42 alone, N-Aβ fragment or N-Aβcore alone, ACh + N-Aβcore, oAβ42 + N-Aβcore, ACh + N-Aβ fragment, or oAβ42 + N-Aβ fragment. An analysis of closed-dwell time components revealed a significant interaction between α7*-nAChR subtype and treatment (F(49,192) = 2.4; P<0.0001). Compared to responses to ACh alone, no differences in α7-nAChR closed-dwell time components C1-C3 were observed. However, closed-dwell time component 4 (C4) was reduced for α7-nAChR exposed to N-Aβcore alone (P < 0.001) or to oAβ42 + N-Aβ fragment (P< 0.0001) when compared to α7-nAChR exposed to ACh alone. Conversely, α7-nAChR exposed to ACh + N-Aβcore showed a significant increase in C4 closed dwell-times compared to responses to ACh alone (P < 0.05). No differences in any closed-dwell time components were observed for α7β2-nAChR across ligand exposure conditions (F(21,96) = 1.072; P = 0.39).All single-channel closed-dwell times were analyzed using a one-way ANOVA for within-group comparisons and Dunnett’s post-hoc test for multiple comparisons (ACh as control) and summarized in Table 1.
Supplemental Figure 2 (S2). α7- and α7β2-nAChR closed dwell-times within bursts. (A) Closed-dwell time distributions within bursts for α7-nAChR. (B) Closed-dwell time distributions within bursts for α7β2-nAChR. (C &D) An analysis of α7*-nAChR closed-dwell times revealed a significant interaction between receptor subtype and treatment (F(7,140) = 2.1; P < 0.05). Post hocanalysis revealed a significant enhancement in the α7β2-nAChR mean closed-dwell time within bursts in the presence of N-Aβcore alone, but only relative to oAβ42 + N-Aβ fragment condition (P < 0.01). Otherwise, no differences in closed-dwell times within bursts were evident between α7- and α7β2-nAChR across all treatment groups (F(1,140) = 0.97; P = 0.05). Differences in single-channel closed-dwell times within bursts were determined using two-way ANOVA with Tukey’s posthoc test for multiple comparisons. Data represented as mean ± SEM. **p<0.01
Supplemental Figure 3 (S3). Cross-group comparisons between α7- and α7β2-nAChR amplitude, burst rate, open-dwell times within bursts, open probability (Popen), and burst duration. Data represented as mean ± SEM. Significance was determined using 2-way ANOVA as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Supplemental Figure 4 (S4). The effect of the control Inactive N-Aβcore hexapeptide (In-Aβcore) on α7*-nAChR single-channel kinetics.
Table 1. α7*-nAChR single-channel closed-dwell time distributions. Differences in single-channel closed-dwell times were determined using one-way ANOVA followed by Dunnet’s post hoc comparison (against ACh as control): *p<0.05, ***p<0.001, ****p<0.0001.
Table 2. α7*-nAChR single-channel amplitudes and burst rates. Differences in single-channel amplitudes and burst rates were determined using one-way ANOVA followed by Dunnet’s post hoc comparison (against ACh as control).
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