Concentration-response curves of nAChR agonists on human α7 and α7β2 nAChRs in <i>Xenopus</i> oocytes.

<p>Acetylcholine (ACh), carbachol, choline, epibatidine and compound B were applied in various concentrations to <i>Xenopus</i> oocytes expressing α7 (filled circles) and α7β2 (1:10, empty circles) nAChRs. All responses were normalized to the peak amplitude of a 1 mM control ACh-induced ion current in the respective oocyte.</p

Effect of antagonists on human α7 and α7β2 nAChRs in <i>Xenopus</i> oocytes.

<p>Inhibition curves for dihydro-β-erythroidine (DhβE) and methyllycaconitine (MLA) on α7 (filled circles) and α7β2 (1:10, empty circles) nAChRs. Co-expression of α7β2 nAChR subunits lead to a significantly decreased IC₅₀ for DhβE (<i>P</i><0.01), and an increased IC₅₀ for MLA (<i>P</i><0.05) compared to α7 nAChR homomers.</p

α7-containing nAChRs form heteromers with β2 nAChR subunits in human cortex.

<p>Magnetic beads covalently coupled with α-bungarotoxin (α-Bgt) or uncoupled beads (PBS) were incubated with homogenates from human temporal cortex (two separate identical experiments are shown) and the isolated proteins were separated using gel electrophoresis. Subsequent detection Western blotting demonstrated the presence of the α7 and β2 from samples isolated using α-Bgt-coupled, but not uncoupled beads. The α4 subunit was not detected in any of the isolates, confirming that the presence of β2 protein is not due to non-specific isolation of α4β2 nAChRs.</p

Potentiation of human α7 and α7eβ2 nAChRs in Xenopus oocytes by the allosteric potentiator PNU120596.

<p>A) A control response was evoked by applying 1 mM ACh to an α7 nAChR expressing oocyte. After a 3 min wash period 3 μM PNU120596 was applied to the same oocyte and after 1 min PNU120596 was co-applied with 1 mM ACh. In the presence of PNU120596 the peak of the 1 mM ACh-induced response was largely potentiated. B) A control response was evoked by applying 1 mM ACh to an α7β2 nAChR expressing oocyte. After a 3 min wash period 3 μM PNU120596 was applied to the same oocyte and after 1 min PNU120596 was co-applied with 1 mM ACh. In the presence of PNU120596 the peak of the 1 mM ACh response was largely potentiated.</p

Bead-coupled α-bungarotoxin selectively purifies α7-containing nAChRs from mouse cortex.

<p>(A) Affinity purification was performed with bead-coupled α-bungarotoxin (α-Bgt) on cortical homogenates from α7^+/+, α7^-/-, β2^+/+, and β2^-/- mice and the isolated proteins were separated using gel electrophoresis. Subsequent detection using Western blotting demonstrated the presence of the α7 and β2 in the isolates from α7^+/+ and β2^+/+ mice. In isolates from α7^-/- mice none of these proteins were detected, demonstrating that α-Bgt specifically isolates α7-containing nAChRs, and that the detection of β2 is dependent on the presence of α7 protein. In isolates from β2^-/- mice, there was no detection of β2 protein, confirming the identity of the band isolated using α-Bgt as being β2. The α4 subunit was not detected in any of the isolates, confirming that the presence of β2 protein is not due to non-specific isolation of α4β2 nAChRs. In the original tissue lysates α7, β2, and α4 protein was readily detectable, except that β2 is not detected in β2^-/- lysates, demonstrating the specificity of the antiserum. A 55 kDa protein was detected in both α7^+/+ and α7^-/- lysates, as has previously been shown for several other α7 antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130572#pone.0130572.ref026" target="_blank">26</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130572#pone.0130572.ref027" target="_blank">27</a>]. (B) α-Bgt affinity purification on cortical homogenates from α7^+/+ and a mixture of α7^-/- and β2^-/- mice. β2 subunits were not detected in the latter. (C) Detection of β2 is evident in HEK293 cells transfectd with the human β2 gene (HEK β2), but not in untransfected cells (HEK). Similarly transfection of GH4 cells with human α7 (GH4 α7) does not alter detection of the band corresponding to β2.</p

Summary of agonist and antagonist concentration-response curves on human α7 and α7β2 nAChRs expressed in <i>Xenopus</i> oocytes.

<p>All responses were normalized to the peak amplitude of a 1 mM control ACh-induced ion current. Antagonists were first pre-applied for 1 min followed by co-application with 1 mM ACh. During curve fitting of agonist data the bottom of the curves were constrained to 0 and curve fitting of antagonist data was performed by constraining the top and the bottom of the curves at 100 and 0%, respectively. T-tests on EC50 and IC50 data was performed on log EC50 and log IC50 values. Statistical significance</p><p>* <i>P</i><0.05</p><p>** <i>P</i><0.005.</p><p>Summary of agonist and antagonist concentration-response curves on human α7 and α7β2 nAChRs expressed in <i>Xenopus</i> oocytes.</p