Proteins that could affect the life-cycle of α7-nAChRs.

<p>A total of twenty-one identified proteins have functions that could affect the life-cycle of the α7-nAChR, e.g., receptor biogenesis, modulation of intracellular and plasma-membrane expressed receptor pools, as well as receptor turnover, autophagy, or apoptosis related. These proteins are grouped based on their reported cellular compartment localization. The activity of these proteins may be localized to the endoplasmic reticulum (A), the Golgi complex (B), or the cytosol (C&D). Cytosolic proteins can either be involved in the mobilization of internal pools of α7-nAChRs through kinase and phosphatase activity (C) or be associated with protein turnover, autophagy, and apoptosis-related processes (D).</p

Summary analysis of Ric-3-mediated proteins with literature citations implicating functional interactions with nAChRs.

<p>Summary analysis of Ric-3-mediated proteins with literature citations implicating functional interactions with nAChRs.</p

¹²⁵I-α-bgtx binding to affinity immobilized protein.

<p>Detergent solubilized membrane extracts were incubated with α-bgtx-affinity beads for 4 hours at 4°C. Protein-α-bgtx-affinity bead complexes were incubated with 5 nM ¹²⁵I-α-bgtx for 1 hour at room temperature. Non-specific binding was determined in controls by the inclusion of 1 μM unlabeled α-bgtx to preparations prior to the addition of ¹²⁵I-α-bgtx. Following incubation with ¹²⁵I-α-bgtx, beads were washed three times with solubilization buffer and measured. Comparable ¹²⁵I-α-bgtx binding activity of protein-α-bgtx-affinity bead complexes isolated from SH-EP1-hα7-Ric-3 (56 ± 15 fmol/mg, in blue) and SH-EP1-hα7 (49 ± 9 fmol/mg, in green) was observed (Student’s <i>t</i> test, p = 0.40) while SH-EP1 preparations (in purple) did not show α-bgtx binding activity. No ¹²⁵I-α-bgtx binding to protein-α-bgtx-affinity bead complexes was observed in samples treated with 5 μM MLA confirming α7-nAChR specificity (Student <i>t</i> test, p < 0.05). SH-EP1-hα7-Ric-3 and SH-EP1-hα7 ¹²⁵I-bgtx binding activity was analyzed with five independent biological replicates. MLA treated samples and SH-EP1 ¹²⁵I-α-bgtx binding activity were analyzed with three independent biological replicates.</p

Ric-3 immunoreactivity in SH-EP1-ha7-Ric-3.

<p>Solubilized membrane extracts of SH-EP1-hα7-Ric-3 and SH-EP1-hα7 cell lines were probed with anti-Ric-3 polyclonal antibodies. Ric-3 antibody immunoreactivity at 41 kDa confirms the presence of Ric-3 in membrane extracts from SH-EP1-hα7-Ric-3 cells (A). There is no corresponding band in SH-EP1-hα7 membrane extracts (B). The anti-GAPDH antibody immunoreactivity was utilized as a loading control.</p

Experimental design.

<p>Five biological replicates of both SH-EP1-hα7-Ric-3 cells and SH-EP1-hα7 cells were independently processed and analyzed. Triton X-100 solubilized α7-nAChR protein complexes were isolated from SH-EP1-hα7-Ric-3 and SH-EP1-hα7 extracts using α-bgtx-affinity beads. Binding of α7-nAChRs to affinity beads was confirmed with ¹²⁵I-α-bgtx radioligand binding assays. Separately, α7-nAChR protein complexes isolated from SH-EP1-hα7-Ric-3 and SH-EP1-hα7 were eluted from affinity beads using 1 M carbachol. Eluted proteins were reduced and alkylated before being digested with trypsin in-solution. Digested peptides from each of the five samples prepared from SH-EP1-hα7-Ric-3 and SH-EP1-hα7 cells were analyzed with a Q Exactive mass spectrometer, spectra identified using the Mascot algorithm and results analyzed using ProteoIQ. Identified α7-nAChR associated proteins from SH-EP1-hα7-Ric-3 and SH-EP1-hα7 cells were compared. Associations only identified with Ric-3 co-expression in SH-EP1-hα7-Ric-3 cells were determined to be Ric-3-mediated changes in the α7-nAChR interactome.</p

EC₅₀ values for 4BP-TQS with the fixed concentration of ACh/nicotine for the rescued mutants.

<p>EC₅₀ values for 4BP-TQS with the fixed concentration of ACh/nicotine for the rescued mutants.</p

PNU120596 concentration-response for the rescued nonfunctional mutants with a fixed ACh or nicotine concentration.

<p><b>A</b>. Representative current traces induced by increasing concentration of PNU-120596 in the presence of 200 μM ACh. <b>B</b>. Normalized and averaged (each group had at least 6 oocytes) current responses to ACh from A. Lines are nonlinear least squares fits of the normalized averages of the responses to the Hill equation. The derived EC₅₀ values from individual fits are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137588#pone.0137588.t002" target="_blank">Table 2</a>. <b>C</b>. Representative current traces induced by increasing concentration of PNU-120596 in the presence of 200 μM nicotine. <b>D</b>. Normalized and averaged current responses (each group had at least 6 oocytes) to ACh from C. The derived EC₅₀ values from individual fits are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137588#pone.0137588.t002" target="_blank">Table 2</a>.</p

Concentration-responses of W55G mutant to ACh and nicotine.

<p>A. Concentration responses of the wild type and W55G to ACh. Top: raw current traces; bottom: normalized and averaged currents. Lines are least-squares fit of the data to the Hill equation. The resulting EC₅₀ for ACh in the wild type receptor was 210.5±24.3 μM, and the EC₅₀ for W55G mutant was 1375.3±130.5 μM (N = 5). B. Concentration responses of the wild type and W55G to nicotine. Top: raw current traces; bottom: normalized and averaged currents. Lines are least-squares fit of the data to the Hill equation. The resulting EC₅₀ values for nicotine were 43.6±4.8 μM and 530.40±12.91 μM for the wild type and mutant receptor respectively (N = 5).</p

Agonist-responses for the nonfunctional mutants in the presence of a PAM or agonist-PAM.

<p>A. Co-application of 31.6 μM PNU-120596 with 200 μM ACh or nicotine rescued the receptor functions for some of the nonfunctional mutants. The same amount of cRNA was injected for each group, and recordings were performed after 3 days in 9–19 oocytes for each group). The bar graph represents the average currents rescued by PNU-120596. In case of the wild type, the current represents the rescued current from desensitization. B, Direct activation of nonfunctional mutants by 4BP-TQS. The same amount of cRNA was injected for each group, and recordings were performed after 3 days in 10–17 oocytes in each group). C, Co-application of 4BP-TQS with ACh or nicotine rescued more mutants. The same amount of cRNA was injected for each group, and recordings were performed after 3 days in 9–19 oocytes in each group. Asterisk (****) represents that the difference between the wild type and each mutant is statistically significant (P<0.0001) in Tukey multiple comparison test of one-way ANOVA. ♦♦, ♦♦♦, or ♦♦♦♦ represent the difference between blank and each mutant with statistical significance (P<0.01, P<0.001, or P<0.0001).</p

Different interactions of ACh and nicotine with the AChBP binding residues.

<p>A. The binding pocket between chain A and chain B of the AChBP co-crystalized with ACh (PBDID: 3WIP); B. The binding pocket between chain A and chain B of the AChBP co-crystalized with nicotine (PBDID: 1UW6). The residues of Cys191 and Trp55 in the human α7nAChR are labeled next to their homologous residues in the AChBP. Arrows indicate different interactions.</p