Molecular Modulation of Human α7 Nicotinic Receptor by Amyloid-β Peptides

Abstract

Amyloid β peptide (Aβ) is a key player in the development of Alzheimer's disease (AD). It is the primary component of senile plaques in AD patients and is also found in soluble forms. Cholinergic activity mediated by α7 nicotinic receptors has been shown to be affected by Aβ soluble forms. To shed light into the molecular mechanism of this effect, we explored the direct actions of oligomeric Aβ_1-40 and Aβ_1-42 on human α7 by fluorescence spectroscopy and single-channel recordings. Fluorescence measurements using the conformational sensitive probe crystal violet (CrV) revealed that in the presence of Aβ α7 undergoes concentration-dependent conformational changes. Exposure of α7 to 100 pM Aβ changes CrV K_D towards that of the desensitized state. However, α7 is still reactive to high carbamylcholine (Carb) concentrations. These observations are compatible with the induction of active/desensitized states as well as of a novel conformational state in the presence of both Aβ and Carb. At 100 nM Aβ, α7 adopts a resting-state-like structure which does not respond to Carb, suggesting stabilization of α7 in a blocked state. In real time, we found that Aβ is capable of eliciting α7 channel activity either in the absence or presence of the positive allosteric modulator (PAM) PNU-120596. Activation by Aβ is favored at picomolar or low nanomolar concentrations and is not detected at micromolar concentrations. At high Aβ concentrations, the mean duration of activation episodes elicited by ACh in the presence of PNU-120596 is significantly reduced, an effect compatible with slow open-channel block. We conclude that Aβ directly affects α7 function by acting as an agonist and a negative modulator. Whereas the capability of low concentrations of Aβ to activate α7 could be beneficial, the reduced α7 activity in the presence of higher Aβ concentrations or its long exposure may contribute to the cholinergic signaling deficit and may be involved in the initiation and development of AD.

Keywords: Cys-loop receptor; amyloid peptide; crystal violet; nicotinic receptor; patch-clamp recordings; single-channel currents.

Figure 1

α7 conformational changes depend on Amyloid β peptide (Aβ) concentrations. (A) Crystal violet (CrV) titration curves obtained with muscle nicotinic acetylcholine receptor (nAChR) from T. californica rich membranes (circles) and with human α7 (squares) in the absence (gray, Resting) and presence of carbamylcholine (Carb; red, desensitized). The arrows indicate the changes from resting to desensitized conditions for each nAChR type. (B) Changes in K_D values of CrV by the exposure to low (0.1 nM) and high (100 nM) concentrations of Aβ_1–40. K_D values of CrV were calculated from the titration curves, in the absence and presence of 20 mM Carb (gray and red columns, respectively). Each column corresponds to the average ± SD of more than four independent experiments. Statistically significant differences are shown by asterisks, *p < 0.05.

Figure 2

Single-channel activity elicited by Aβ. Single-channel recordings were performed in the cell-attached patch configuration at −70 mV membrane potential. Representative α7 single-channel traces from a continuous recording in the presence of 100 μM acetylcholine (ACh) or 100 pM Aβ_1–40. For each condition, a trace at higher temporal resolution is shown (bottom traces). Channel openings are shown as upward deflections. Filter: 9 kHz.

Figure 3

Single-channel activity elicited by Aβ in the presence of N-(5-Chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596). Single-channel recordings were performed in the cell-attached patch configuration at −70 mV membrane potential. Representative α7 single-channel traces from a continuous recording in the presence of the type II positive allosteric modulator (PAM) PNU-120596 (1 μM) together with 100 μM ACh, 100 pM Aβ_1–40 or 100 pM Aβ_1–42. Channel openings are shown as upward deflections.

Figure 4

Aβ decreases the duration of activation episodes elicited by ACh and potentiated by PNU-120596. Single-channel recordings were performed in the cell-attached patch configuration at −70 mV membrane potential with 100 μM ACh and 1 μM PNU-120596 and in the absence or presence of Aβ. (A) Representative clusters are shown for each condition. Channels are shown as upward deflections. (B) Decrease of mean open and cluster durations by Aβ. Each value corresponds to the mean ± SD of different recordings. The number of recordings for each condition is shown in the bars. The cluster and open durations for each recording were obtained from the corresponding histograms and correspond to the slowest open components *p < 0.05.

Figure 5

Aβ decreases the duration of events elicited by ACh and potentiated by the type I PAM N-(5-Chloro-2-hydroxyphenyl)-N′-[2-chloro-5-(trifluoromethyl) phenyl]urea (NS-1738). Single-channel recordings were performed in the cell-attached patch configuration at −70 mV membrane potential with100 μM ACh and 10 μM NS-1738 and in the absence or presence of Aβ. (A) Representative channel currents are shown for each condition. (B) Decrease of mean open and burst durations by Aβ. Each value corresponds to the mean ± SD of n different recordings (shown in the bars). The open and burst durations for each recording were obtained from the corresponding histograms and correspond to the slowest open components *p < 0.05, ****p < 0.0001.