Amyloid beta(1-42) peptide alters the gating of human and mouse alpha-bungarotoxin-sensitive nicotinic receptors

Abstract

The beta-amyloid(1-42) peptide (Abeta(1-42)), a major constituent of the Alzheimer's disease amyloid plaque, specifically binds to the neuronal alpha-bungarotoxin (alpha-BuTx)-sensitive alpha7 nicotinic acetylcholine receptor (alpha7 nAChR). Accordingly, Abeta1-42 interferes with the function of alpha7 nAChRs in chick and rodent neurons. To gain insights into the human disease, we studied the action of Abeta(1-42) on human alpha7 nAChRs expressed in Xenopus oocytes. In voltage-clamped oocytes expressing the wild-type receptor, Abeta(1-42) blocked ACh-evoked currents. The block was non-competitive, required over 100 s to develop and was partially reversible. In oocytes expressing the mutant L248T receptor, Abeta(1-42) activated methyllycaconitine-sensitive currents in a dose-dependent manner. Peptide-evoked unitary events, recorded in outside-out patches, showed single-channel conductances and open duration comparable to ACh-evoked events. Abeta(1-42) had no effect on the currents evoked by glutamate, GABA or glycine in oocytes expressing human or mouse receptors for these transmitters. Muscle nAChRs are also alpha-BuTx-sensitive and we therefore investigated whether they respond to Abeta(1-42). In human kidney BOSC 23 cells expressing the fetal or adult mouse muscle nAChRs, Abeta(1-42) blocked ACh-evoked whole-cell currents, accelerating their decay. Outside-out single-channel recordings showed that the block was due to a reduced channel open probability and enhanced block upon ACh application. We also report that the inverse peptide Abeta(42-1), but not Abeta(40-1), partially mimicked the effects of the physiological Abeta(1-42) peptide. Possible implications for degenerative neuronal and muscular diseases are discussed.

Figure 1. Block of WT α7 nAChRs by Aβ_1–42

A, Aβ_1–42 at concentrations of 0.01, 0.1 and 10 nm (open bars) fails to elicit current responses in an oocyte sensitive to ACh (100 µm, filled bar). Traces representative of 8 experiments, where Aβ_1–42 concentrations were applied in random order. B, inward currents evoked by 100 µm ACh (filled bars) in an oocyte expressing human WT α7 nAChRs in standard solution (left), after 180 s preincubation with 100 nm of Aβ_1–42 (open bar, middle), and 35 min after wash-out (right). Note the incomplete recovery of I_ACh. C, block of I_ACh by increasing doses of Aβ_1–42. In each of 5 oocytes, currents evoked by ACh (100 µm) plus Aβ_1–42 after 180 s preincubation with the peptide were normalised to the response to ACh alone (-0.67 ± 0.11 µA, 5/1). Best fitting to the Hill equation yielded an IC₅₀ of 90 nm. Inset, histogram representing the effects of 100 nm Aβ_1–42, Aβ_42–1 and Aβ_40–1. I_ACh was measured after 180 s incubation with the peptides and normalised to the control value in each cell (bar labelled ACh). Bars represent mean ± s.e.m. of 5–11 oocytes (3 donors). *Statistically not different from control (Student's t test, P = 0.2). ACh concentration, 100 µm. Note the reduced effect of Aβ_42–1 as compared to Aβ_1–42 and the ineffectiveness of Aβ_40–1. D, Aβ_1–42 (0.4 µm for 180 s) is unable to block currents evoked by AMPA (50 µm plus cyclothiazide 50 µm), kainate (200 µm), GABA (1 mm) or glycine (1 mm), while blocking α7 nAChRs in Xenopus oocytes. Filled bars represent mean ± s.e.m. from 4 oocytes injected with mouse brain membranes. Inward current amplitude was: 0.08–0.68 µA (GABA); 0.01–0.03 µA (glycine); 0.09–0.13 µA (kainate); 0.3–0.44 µA (AMPA). Open bars represent mean ± s.e.m. of 5 oocytes injected with cDNAs encoding human homomeric GluR1 or α7 nAChRs, as indicated. Current ranged from 0.2–0.4 µA (hGluR1, activated by AMPA as above), from 0.2–1.0 µA (α7 nAChR, ACh 100 µm). Holding potential was −80 mV; the protocol of Aβ_1–42 treatment was as in B. *Statistically not different from control (Student's t test, P > 0.23).

Figure 2. Non-competitive nature of Aβ_1–42-induced block of WT α7 nAChRs

A, ACh dose-response relationships obtained from 4 oocytes (1 donor) in standard solution (○) and after 180 s preincubation with 100 nm Aβ_1–42 (•). I_ACh was normalised to values obtained at 2 mm ACh (-2.07 µA for ○, −1.02 µA for •). Best fitting with the Hill equation yielded: ○, EC₅₀= 108 µm, n_H= 1.48; •, EC₅₀= 112 µm, n_H= 1.20. Inset, typical currents evoked by 2 mm ACh (filled bars) in an oocyte representative of four (2 donors) in standard solution (left) and after a preincubation with 100 nm Aβ_1–42 (right). Note the same percentage of block as in Fig. 1A. B, currents evoked by ACh (100 µm), alone (filled bar, trace labelled C) or plus Aβ_1–42 (800 nm, open bar, trace labelled T). Between trace C and T, oocytes were treated (150 s) with Aβ_1–42 (800 nm), alone (top), together with ACh (1 mm, middle) or with ACh (1 mm) alone (bottom), then washed with normal Ringer (240 s). Note the same percentage of Aβ_1–42 -induced inhibition in the T traces, independent of the presence of ACh during treatment period. All the traces were recorded from one oocyte, representative of three experiments.

Figure 3. Activation of L248T α7 nAChRs by Aβ_1–42

A, currents activated by Aβ_1–42 (400 nm, filled bars), in an oocyte (representative of 15 oocytes, 4 donors) expressing L248T α7 nAChRs. Note the complete block by 0.2 µm MLA (≈40 s preincubation, open bar). This particular response was slightly smaller than average. B, currents evoked by Aβ_1–42 at the indicated concentrations in two other oocytes. Note the sustained response during agonist application. C, histogram comparing the agonism of Aβ peptides (as indicated), normalised to the response evoked by 100 µm ACh in each oocyte. Each bar represents the mean ± s.e.m. of 6–8 oocytes (4 donors) expressing L248T α7 nAChRs.

Figure 4. Single-channel properties of L248T α7 nAChRs activated by Aβ_1–42

A, spontaneous and Aβ_1–42-evoked single-channel activity, blocked by MLA (0.2 µm), in an outside-out patch from an oocyte expressing L248T α7 nAChRs. Inset, part of the trace on an expanded time scale, to show three classes of channel conductance in Aβ_1–42-evoked channel openings (γ_L= 39.7 pS, γ_M= 53.2 pS, γ_H= 67 pS). Spontaneous channels in the same patch had matching conductances. Inward currents downwards. B, open time distributions and sample traces for Aβ_1–42-activated channels, recorded from a different patch. Superimposed lines: best fitting exponential curves with time constants (weight): τ_o1= 0.29 ms (28 %), τ_o2= 1.02 ms (56 %), τ_o3= 4.11 ms (16 %), τ_op= 1.86 ms (n = 1689). All recordings were performed at −50 mV. Aβ_1–42 concentration, 1 µm.

Figure 5. Aβ_1–42 blocks muscle nAChRs in transiently transfected BOSC 23 cells

A, typical inward currents evoked by ACh (1 µm) in BOSC 23 cell expressing γ-AChR (left) or ε-AChR (right), before (C) or after (Aβ) 120 s of application of Aβ_1–42 (100 nm). Note the accelerated decay of I_ACh during Aβ_1–42 application. B, time course of I_ACh block by Aβ_1–42 or Aβ_42–1 (both 100 nm) in two different cells expressing γ-AChR. Bars, Aβ applications. Note the lack of recovery 15 min after Aβ_1–42 wash-out, as compared to the prompt recovery upon Aβ_42–1 removal. ACh concentration, 1 µm. I_ACh normalised to control current amplitude. C, plot of I_ACh amplitude (normalised to the control in each cell) vs. the duration of Aβ_1–42 (100 nm) application. The continuous line represents the linear regression of the data, with a slope of −0.005 % s⁻¹ (R = 0.05), indicating that the block of I_ACh is independent of the duration of Aβ_1–42 application. All the data obtained for γ-AChR, irrespective of ACh concentration (0.2–20 µm), are included in this plot. D, activation of γ-nAChR-channels by ACh (1 µm) in an outside-out patch, before (trace C), during (Aβ) and after (W) the application of Aβ_1–42. τ_cl, 26 ms (C), 184 ms (Aβ), 60 ms (W). Aβ_1–42 (100 nm) application began 60 s before recording trace Aβ, terminated 5 min before recording trace W. Traces were filtered at 200 Hz for display purposes. Insets, expanded traces beginning 750 ms after ACh application (filter, 1 kHz). Single-channel conductance (35.7 pS) and open duration (3.5 ms) were not affected by Aβ_1–42. E, plot of the average NP_op, normalised to the control value of each patch, in 5 outside-out patches, measured over 1-s intervals during the first 10 s of ACh (1 µm) application. Data were sampled before (□), in the continuous presence of Aβ_1–42 (30–120 s preincubation, (•), or 30–120 s after Aβ_1–42 wash-out (○).