The novel α7β2-nicotinic acetylcholine receptor subtype is expressed in mouse and human basal forebrain: biochemical and pharmacological characterization

Abstract

We examined α7β2-nicotinic acetylcholine receptor (α7β2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7β2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7β2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using β2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7β2- versus α7-nAChRs. This study represents the first direct confirmation of α7β2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7β2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7β2-nAChR subunit isoforms with different α7/β2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7β2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites.

Fig. 1.

Western blot analysis of nAChR subunit content in α-Bgtx–purified receptors prepared from 2% Triton X-100 extracts of WT and β₂ KO mouse hippocampi (A) and basal forebrain samples (B). (A) α-Bgtx–purified receptors were prepared from mouse hippocampi by incubating extracts with Sepharose 4B covalently bound with α-Bgtx. The bound receptors were recovered from the beads using Laemmli sample buffer. Western blot analysis of 10 µg 2% Triton X-100 extracts of the hippocampus before (lane 1) and after α-Bgtx purification (lane 2; supernatant), and 1/20 of the corresponding α-Bgtx purified receptors (lane 3; recovered from beads). The blots were probed with an anti-α₇ Ab (top) or β₂(1) Ab (bottom). (B) α-Bgtx–purified receptors were prepared as described above. Western blot analysis of 10 µg 2% Triton X-100 extracts of the basal forebrain before (lane 1) and after α-Bgtx purification (lane 2; supernatant), and 1/10 of the corresponding α-Bgtx–purified receptors (lane 3; recovered from beads). The blots were probed with an anti-α₇ Ab (top) or β₂(1) Ab (bottom).

Fig. 2.

Western blot analysis of α-Bgtx–purified nAChRs prepared from human basal forebrain and cerebellum. α-Bgtx–binding nAChRs were purified from the same volume of 2% Triton X-100 extracts of basal forebrain and cerebellum by incubating them with Sepharose 4B covalently bound with α-Bgtx. The bound receptors were eluted using sample buffer and an identical volume of purified receptors was loaded on the gel. The Western blots were probed with anti-α₇ Ab (top) or anti-β₂ Ab (bottom).

Fig. 3.

Representative traces and maximum function (I_max) comparison for α₇*-nAChR pentameric concatemer constructs. Oocytes were injected with mRNA encoding unlinked α₇-nAChR subunit monomers (A), concatenated α₇ homopentamers (B), α₇β₂-nAChRs with the β₂ subunit in position 3 (C), or α₇β₂-nAChRs with the β₂ subunit in positions 2 and 4 (D). Representative TEVC recordings are shown in each case for ACh concentration-response determinations (see Materials and Methods for details). Black bars above each trace represent 5-second applications of ACh at a range of concentrations. The time course of receptor desensitization/inactivation during stimulation with a maximally effective dose of ACh (10 mM) was also investigated for each nAChR construct using additional groups of oocytes. In each case, the time course was best fit by a double-exponential decay. The fast time constants (τ_fast) for desensitization/inactivation were statistically indistinguishable by one-way ANOVA across all four groups (unlinked α₇, 436 ± 85 milliseconds; α₇-only concatemer, 214 ± 80 milliseconds; α₇β₂(p3), 312 ± 55 milliseconds; α₇β₂(p2,4), 247 ± 35 milliseconds; F_[3,11] = 2.06, P = 0.16; and n = 3 in each group). By contrast, the slow time constant (τ_slow) for desensitization/inactivation of the α₇β₂(p2,4) construct was significantly longer that of the other groups. No other differences were detected by a Tukey post hoc comparison (P < 0.05). Values were as follows: unlinked α₇, 5109 ± 800 milliseconds; α₇-only concatemer, 3130 ± 585 milliseconds; α₇β₂(p3), 5073 ± 638 milliseconds; α₇β₂(p2,4), 6318 ± 365 millisecond; F_[3,11] = 5.29, P = 0.02; and n = 3 in each group. (E) Summary of maximal function (I_max) measured in each concatemeric nAChR group by stimulation with the full agonist ACh (10 mM). Bars represent mean ± S.E.M. (n = 3). I_max values were as follows: α₇ only, 83.9 ± 18.6 nA; α₇β₂(p3), 285 ± 11 nA; and α₇β₂(p2,4), 216 ± 45 nA. Analysis using one-way ANOVA with a Tukey post hoc comparison showed that incorporation of β₂ subunits resulted in a statistically significant increase in I_max (F_[2,6] = 12.7, P = 0.007; denoted by the asterisk). The I_max values obtained from the two α₇β₂-nAChR constructs were statistically indistinguishable from each other. ANOVA, analysis of variance.

Fig. 4.

Agonist concentration-response profiles for α₇- and α₇β₂-nAChRs. Oocytes were injected with mRNA encoding unlinked α₇ subunits (○), concatenated α₇ homopentamers (●), or concatenated α₇β₂ pentameric concatemers (□ indicates α₇β₂-nAChR with the β₂ subunit in position 3; ▪ indicates α₇β₂-nAChR with the β₂ subunit in positions 2 and 4). Oocytes were perfused with the following nAChR agonists: ACh (10^−5.5 to 10⁻²; n = 6) (A), choline (10^−4.25 to 10⁻²; n = 3) (B), nicotine (10^−5.5 to 10⁻³; n = 3) (C), sazetidine-A (10^−7.5 to 10⁻⁴; n = 3) (D), or CC4 (10^−6.5 to 10⁻³; n = 3) (E). All responses within each group were normalized to an initial control stimulation with 10 mM ACh. Data points represent the mean ± S.E.M. Drug potency and efficacy parameters were calculated by nonlinear least-squares curve fitting to the Hill equation (see Materials and Methods). The resulting pharmacological parameters and statistical analyses are summarized in Table 4.

Fig. 5.

Antagonist concentration response profiles for α₇- and α₇β₂-nAChRs. Oocytes were injected with mRNA encoding unlinked α₇ subunits (○), concatenated α₇ homopentamers (●), or concatenated α₇β₂ pentameric concatemers (□ indicates α₇β₂ nAChR with the β₂ subunit in position 3; ▪ indicates α₇β₂ nAChR with the β₂ subunit in positions 2 and 4). Before antagonists were applied to each oocyte, a control 10 mM ACh-evoked response was measured. Oocytes were preperfused with the following nAChR antagonists: DHβE (10^−6.25 to 10⁻³; n = 3) (A), methyllycaconitine (10^−10.5 to 10⁻⁷; n = 3) (B), mecamylamine (10^−7.25 to 10⁻⁴; n = 3) (C), or α-Cbtx (10⁻¹⁰ to 10⁻⁷; n = 3) (D). The magnitudes of subsequent 10 mM ACh stimulations were compared with that of the initial control. Data points represent the mean ± S.E.M. Drug potency and efficacy parameters were calculated by nonlinear least-squares curve fitting to the Hill equation (see Materials and Methods). The resulting pharmacological parameters and statistical analyses are summarized in Table 5.