Zhang Q , Du Y , Zhang J , Xu X , Xue F , Guo C , Huang Y , Lukas RJ , Chang Y .

R01 GM085237 NIGMS NIH HHS , R01GM085237 NIGMS NIH HHS

	Fig 2. Current responses of the wild type and mutant receptors to 3.16mM ACh or nicotine with non-injected oocyte controls.A. 13 mutants and wild type control along with un-injected oocyte control (for nonfunctional mutants). The same amount of cRNAs for the wild type or mutant receptors was injected. On the 3rd post-injection day, the oocytes were tested with ACh (with 1 μM atropine) or nicotine. The name of each condition is indicated at the bottom of each bar. Each group had 8–18 oocytes from two sets of experiments. Asterisk (*, **, or ****) represents that the difference between the wild type and each mutant is statistically significant (P<0.05, P<0.01, or P<0.0001) in Tukey multiple comparison test of one-way ANOVA. ♦, ♦♦ or ♦♦♦♦ represent the statistical difference with P<0.05, P<0.01, or P<0.0001 between blank and each mutant. B. W55G mutant and its wild type control in a separate experiment (10 oocytes each group). ****: P<0.0001 with 2-sided grouped t-test.
	Fig 3. ACh concentration-response for most of the functional mutants.A. Representative current traces induced by ACh for the wild type and mutant receptors with the concentrations indicated. B. Averaged and normalized concentration-responses of charge (6–8 oocytes each group). Lines are nonlinear least squares fits of the normalized averages of the responses to the Hill equation. C. Bar graph of the pEC50 values (negative logEC50s) derived from B. ***: P<0.001 when compared to the WT value.
	Fig 4. Concentration-responses of W55G mutant to ACh and nicotine.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).
	Fig 5. Agonist-responses for the nonfunctional mutants in the presence of a PAM or agonist-PAM.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).
	Fig 6. PNU120596 concentration-response for the rescued nonfunctional mutants with a fixed ACh or nicotine concentration.A. Representative current traces induced by increasing concentration of PNU-120596 in the presence of 200 μM ACh. 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 Table 2. C. Representative current traces induced by increasing concentration of PNU-120596 in the presence of 200 μM nicotine. D. 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 Table 2.
	Fig 7. Concentration response of 4BP-TQS direct activation of the wild type control and Y93C, C191Y, and Y211C mutants.A, Representative current traces induced by increasing concentration of 4BP-TQS. B. Normalized and averaged current responses to 4BP-TQS from A. Lines are nonlinear least squares fits of the normalized averages of the responses to the Hill equation. The resulting EC50 values were 5.54±0.31, 8.21±1.03, 13.73±1.73, and 4.23±0.30 μM for the wild type control and Y93C, C191Y, and Y211C mutants respectively (n = 6 for each group).
	Fig 8. 4BP-TQS concentration-response for the rescued nonfunctional mutants with a fixed ACh or nicotine concentration.A. Representative current traces induced by increasing concentration of 4BP-TQS in the presence of 200 μM ACh. B. Normalized and averaged (each group had 5–6 oocytes) current responses 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 Table 3. C. Representative current traces induced by increasing concentration of 4BP-TQS in the presence of 200 μM nicotine. D. Normalized and averaged current responses (each group had 5–6 oocytes) from C. The derived EC50 values from individual fits are listed in Table 3.
	Fig 9. Coexpression of wild type and nonfunctional mutant.The same amount of cRNAs encoding the wild type or wild type plus mutant (in 1:1 ratio) were injected into Xenopus oocytes, and recorded with two electrode voltage clamp for ACh-induced current after 3 days of injection. A. Peak current induced by 3.16mM ACh for all groups on the 3 post-injection day. "****": P<0.0001. Each group had at least 16 oocytes. B. Averaged concentration response relationships of the wild type and wild type plus mutant as indicated. Each group is the average of six oocyte data. C. EC50 values were derived by fitting individual concentration-response curves in B. pEC50 values (negative logEC50) are used to plot the data. ("**", “***”, “****”: p<0.01, P<0.001, or P<0.0001). D. Linear regression analysis for the relationship between Hill slope and LogEC50.
	Fig 10. Different interactions of ACh and nicotine with the AChBP binding residues.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.
	Fig 1. Location of 14 mutations in the α7 nAChR receptor.A: The residues with the mutations in the binding region (loops A (TYR93), and C (CYS191, LYS192, ASP197) of the principal side of a binding pocket, and D (TRP55) of the complementary side of the neighboring binding pocket); B, The residues with the mutations in the coupling region (loop 2 (ASN47), loop 9 (ASN171 and GLU173), pre-M1 (ARG205 and ARG206) and M1 (TYR211 and GLY212).

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