Tang JS , Xie BX , Bian XL , Xue Y , Wei NN , Zhou JH , Hao YC , Li G , Zhang LR , Wang KW .

	Figure 1. Chemical structure of Br-IQ17B and its dose-dependent activation of human α7 nAChR channels expressed in Xenopus oocytes. (A) Chemical structure of N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, named Br-IQ17B, molecular weight of 348.24. (B) Representative α7 currents recorded from an oocyte expressing human α7 nAChR in response to 300 μmol/L ACh (a near half-maximal concentration) and increasing concentrations of Br-IQ17B (0.1–30 μmol/L). Oocytes were held at −90 mV, and traces were consecutively acquired with 7-min lapses. (C) Curve with open squares represents concentration-response relationship of inward α7 nAChR currents induced by Br-IQ17B. The maximal response evoked by Br-IQ17B is 64.3%±3.6% of ACh (3 mmol/L) with an EC50 of 1.8±0.2 μmol/L and a Hill coefficient of 1.2±0.1 (n=6 for all data points). Curve with fitted triangles represents concentration-response of EVP-6124 with an EC50 of 0.45±0.08 μmol/L and a Hill coefficient of 1.2±0.2 (n=6 for all data points). The maximal response compared to ACh is 30.6%±1.8%. Curve with fitted circles represents concentration-response of natural agonist ACh, yielding an EC50 of 284.2±14.6 μmol/L and a Hill coefficient of 1.1±0.1 (n=6 for all data points). Peak current amplitudes were measured and normalized to the current evoked by 3 mmol/L ACh. (D) The α7 current evoked by 30 μmol/L Br-IQ17B was inhibited by 10 nmol/L α7 antagonist MLA. The inhibitory effect could be reversed after a 10-min washout.
	Figure 2. Dose-dependent activation of α7 current by Br-IQ17B agonist in the presence of the PAM PNU-120596 and desensitization induced by pre-incubation of Br-IQ17B. (A) Representative traces induced by increasing concentrations of Br-IQ17B co-applied with the selective α7 type II PAM PNU-120596 (1 μmol/L). Oocytes were held at −90 mV, and traces were consecutively acquired with 7-min lapses. (B) Concentration-peak current of α7 induced by Br-IQ17B in the presence of 1 μmol/L PNU-120596 and fitted by the Hill equation (line with open squares), with an EC50 of 93.6±6.5 nmol/L and a Hill coefficient of 2.4±0.1 (n=5 for all data points); partial activation of α7 by Br-IQ17B alone with EC50 of 1.8±0.2 μmol/L (line with filled circles). (C) Representative current traces depicting the response to ACh (100 μmol/L) after 1-min sustained exposure to low concentrations of Br-IQ17B (3–300 nmol/L). Oocytes were held at −90 mV, and consecutive acquisitions of traces were made with 7-min lapses. (D) Fitting of concentration-dependent inhibition of the ACh (100 μmol/L)-evoked response after sustained exposure to Br-IQ17B. Peak current amplitudes were measured and normalized with respect to the amplitude of current elicited by 100 μmol/L ACh alone. Data points were fitted to a Hill equation, yielding an IC50 of 28.5±1.9 nmol/L and a Hill coefficient of 2.8±0.3 (n=6 for all data points).
	Figure 3. Displacement of [3H]-MLA ligand binding in crude membranes from rat brain. Displacement of the binding of the radio-ligand α7 antagonist [3H]-MLA to membranes of rat brain hippocampus in the presence of increasing concentrations of Br-IQ17B (line with open squares) and natural agonist ACh (line with filled triangles) with Kis of 14.9±3.2 nmol/L and 3.9±0.3 μmol/L, respectively. The line with filled circles shows the dose-dependent inhibition of [3H]-MLA binding by the orthosteric agonist PNU-282987 as positive control, yielding a Ki of 34.1±4.3 nmol/L.
	Figure 4. Selectivity assessments of Br-IQ17B. (A) Representative current traces showing the effects of Br-IQ17B on an oocyte expressing rat α4β2 nAChR in the presence of 100 μmol/L ACh (left), 100 μmol/L Br-IQ17B (middle), and the co-application of ACh and Br-IQ17B (right). (B) Representative current traces showing the effects of Br-IQ17B on an oocyte expressing α3β4 nAChR subunits in the presence of ACh (300 μmol/L) (left), or different concentrations (3–3000 μmol/L) of Br-IQ17B alone (middle), or the co-application of different concentrations of Br-IQ17B ranging from 3–3000 μmol/L (only 3 traces for concentrations of 10, 100 and 1000 μmol/L are shown) with a fixed concentration of ACh (300 μmol/L) (right). (C) Concentration-response curve for inhibition of Br-IQ17B on rat α3β4; the peak current amplitudes were plotted as a function of Br-IQ17B concentration (3–3000 μmol/L) normalized to ACh (300 μmol/L), with an IC50 of 381.5±28.6 μmol/L and a Hill coefficient of 0.9±0.1 (n=5 for all data points). (D) Representative current traces from oocytes expressing 5-HT3A receptors in response to 100 μmol/L Br-IQ17B alone (first trace), 10 μmol/L 5-HT alone, and the co-application of increasing concentrations of Br-IQ17B (0.3–30 μmol/L) with 5-HT (10 μmol/L). (E) Concentration-response relationship for antagonist properties of Br-IQ17B against human 5-HT3A; plot of the peak current as a function of the Hill equation of Br-IQ17B (0.1–100 μmol/L) and normalized to 5-HT (10 μmol/L), with an IC50 of 3.74±0.64 μmol/L and a Hill coefficient of 2.1±0.1 (n=6 for all data points).
	Figure 5. Activation of native α7 nAChR in hippocampal neurons by Br-IQ17B. (A) Representative current traces evoked by agonist ACh (1 mmol/L) and Br-IQ17B (10 μmol/L) in hippocampal neurons. The current elicited by Br-IQ17B was blocked by the inhibitor MLA (10 nmol/L) (n=3). The neurons were held at −80 mV. (B) Representative current traces evoked by a low concentration of ACh (10 μmol/L) or Br-IQ17B (100 nmol/L) in the presence or absence of PNU-120596 (1 μmol/L), respectively (n=3). Current induced by the co-application of Br-IQ17B (100 nmol/L) and PNU-120596 (1 μmol/L) is significantly larger than Br-IQ17B (100 nmol/L) alone. The neurons were held at −80 mV.
	Figure 6. Activation of α7 nAChR by Br-IQ17B enhances pERK signaling in PC12 cells. (A) In the upper panel, Western blot analysis of PC12 cells stimulated with different concentrations of Br-IQ17B (0.01–10 μmol/L) for 7 min after pre-incubation of 1 μmol/L PNU-120596 for 10 min. Lower panel, semi-quantitative analysis of pERK/tERK from upper panel data. Data were normalized to that of control without any treatment and expressed as the mean±SEM. Co-application of PNU-120596 (1 μmol/L) and Br-IQ17B significantly increased the ratio of phosphorylated ERK to total ERK (n=3, cP<0.01 vs control, paired t-test). (B) In upper panel, the Br-IQ17B-increased phosphorylation of ERK1/2 was blocked in the presence of the α7 blocker MLA. Western blot analysis of PC12 cells pretreated with or without 100 nmol/L MLA for 10 min followed by the addition of 1 μmol/L PNU-120596 and agonists (10 μmol/L for nicotine; 1 μmol/L for Br-IQ17B, and 1 μmol/L for PNU-282987). Lower panel, semi-quantitative analysis of pERK/tERK from upper panel data. Data were normalized to that of control without any treatment and expressed as the mean±SEM. MLA significantly reversed the increased ratio of phosphorylation ERK caused by either Br-IQ17B or PNU-282987 (n=3, bP<0.05, paired t-test).
	Figure 7. α7 nAChR agonist Br-IQ17B enhances GABAergic synaptic transmission. (A) Representative raw traces showing that Br-IQ17B (10 μmol/L) reversibly enhanced IPSC (inhibitory postsynaptic current) in the presence of PNU-120596 (1 μmol/L). Superfusion of PNU-120596 alone for 10 min had no detectable effect on spontaneous IPSC (sIPSC); further application of Br-IQ17B (10 μmol/L) increased both the frequency and amplitude of sIPSC. Enhanced sIPSC induced by the co-application of PNU-120596 and Br-IQ17B can be washed out. The area of the dashed rectangle (left panels) is expanded to show a faster timescale at right. The neurons were held at −70 mV. (B) Peak amplitude distributions of all IPSC events detected in the left panel of (A). Co-application of Br-IQ17B and PNU-120596 significantly increased IPSC amplitudes (cP<0.01, compared with control, PNU-120596 and wash, one-way ANOVA). (C) Normalized average IPSC peak amplitudes from all recordings. Co-application of Br-IQ17B and PNU-120596 increased IPSC amplitude of each individual neuron (n=5, record duration for 1 min, one-way ANOVA). (D) Statistical analysis of average IPSC frequency from all neurons recorded (n=6, record duration for 1 min). Co-application of Br-IQ17B and PNU-120596 significantly increased average IPSC frequency (bP<0.05 vs control, cP<0.01 vs PNU-120596 or wash, paired t-test).
	Figure 8. Selectivity assessments of Br-IQ17B on subtypes of GABAA receptors. Representative current traces recorded in oocytes expressing human α1β3γ2 (A), α2β3γ2 (B), α3β3γ2 (C), and α5β3γ2 (D) subtypes showing the lack of effects of Br-IQ17B on GABAA activity in the presence of 1 μmol/L GABA (left), 100 μmol/L Br-IQ17B (middle), or the co-application of GABA and Br-IQ17B (right) (n=3 for all subtypes).

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