Abongwa M , Buxton SK , Courtot E , Charvet CL , Neveu C , McCoy CJ , Verma S , Robertson AP , Martin RJ .

R01 AI047194 NIAID NIH HHS , R21 AI092185 NIAID NIH HHS

	Figure 1. (A) Amino acid sequence alignment of Asu‐ACR‐16 and human‐α7 nAChR subunits. The signal peptide (bright green box), ACh‐binding loops A–F (pink boxes), cys‐loop (yellow box) and transmembrane regions TM1–TM4 (turquoise boxes) are indicated. The vicinal cysteines (black‐edged box) that characterize an α‐subunit are present in the C‐binding loop. The blue‐edged box between TM2 and TM3 represents the region where PNU120596 acts on α7 nAChRs. Green arrows are residues important for positive allosteric modulation of α7 receptors by ivermectin. Grey (and grey outline) arrows are residues important for permeability of α7 receptors to Ca2 +. Black (and black outline) arrows are residues affecting α7 receptor desensitization. Residues in C‐binding loop of α7 nAChRs that bind α‐BTX are highlighted in grey. (B) Distance tree showing relationships of ACR‐16 homologues in parasitic nematode species with AChR subunit sequences from C.elegans. A neighbour joining tree was generated with deduced amino acid sequence from AChR subunits representative from the ACR‐16, ACR‐8, UNC‐38, UNC‐29 and DEG‐3 group as defined by Mongan et al., (1998). Three letter prefixes in AChR subunit names: Ace, Asu, Cel, Hco, Llo, Nam, Sra and Tca, refer to A. ceylanicum, A. suum, C. elegans, H. contortus, L. loa, N. americanus, S. ratti and T. canis respectively. ACR‐16 orthologues are highlighted in red. Numbers at each branch indicate percentage bootstrap values (>80%) corresponding to 1000 replicates. The scale bar represents substitutions per site. The Cel‐lgc‐30 subunit sequence was used as an outgroup.
	Figure 2. Localization of Asu‐ACR‐16 in different body tissues of the A.suum worm using RT‐PCR and single‐cell RT‐PCR (n = 5). (A) RT‐PCR analysis of Asu‐acr‐16 (lanes 2, 4, 6, 8, 10) and gapdh control (lanes 3, 5, 8, 9, 11) in gut (g), oviduct (o), pharynx (p), somatic muscle strip (m) and head region (h). The PCR products size for acr‐16 and gapdh is 468 and 411 bp respectively. (B) Single‐cell RT‐PCR of Asu‐ACR‐16 in pharyngeal muscle (2, 4, 6, 8, 10) and in somatic muscle (1., 3., 5., 7., 9.). RT‐PCR of gapdh control in pharyngeal muscle (3, 5, 7, 9, 11) and in somatic muscle (2., 4., 6., 8., 10.). 1, FastRuler High Range DNA ladder; ntc1, no‐template controls for acr‐16; ntc2, no‐template controls for gapdh.
	Figure 3. Effects of the ancillary proteins, RIC‐3, UNC‐50 and UNC‐74, from different nematode species, on Asu‐ACR‐16 expression. (A) Sample traces represented as inward currents produced in response to 100 μM ACh. (B) Bar chart (mean ± SEM) showing (left to right) current (in nA) generated in response to 100 μM ACh produced. Control (ctrl): Asu‐acr‐16 alone (n = 21). Black bar: Asu‐acr‐16 plus Hco ‐ric‐3, unc‐50 and unc‐74 (n = 15). Vertical line fill: Asu‐acr‐16 plus Asu ric‐3, unc‐50 and unc‐74 (n = 15). Horizontal line fill: Asu‐acr‐16 plus Hco ‐ric‐3 (n = 17). Checkered fill: Asu‐acr‐16 plus Xle ‐ric‐3 (n = 20). No fill: Asu‐acr‐16 plus Asu‐ric‐3 (n = 23). Asu‐acr‐16 on its own did not respond to ACh, and the largest current size was obtained when Asu‐acr‐16 was co‐injected with Asu‐ric‐3. * P < 0.05; significantly different as indicated; Tukey's multiple comparison tests.
	Figure 4. Effects of nAChR agonists and anthelmintics on Asu‐ACR‐16. Sample traces and bar chart (mean ± SEM) showing rank order potency series for nAChR agonists: nicotine (nic), cytisine (cyt), 3‐bromocytisine (3‐bc), epibatidine (epi), DMPP, choline (cho), betaine (bet), lobeline (lobe) and A844606; and cholinergic anthelmintics: oxantel (oxa), morantel (mor), levamisole (lev), methyridine (met), thenium (the), bephenium (beph), tribendimidine (tbd) and pyrantel (pyr); on Asu‐ACR‐16. Overall, the rank order potency series for agonists and anthelmintics on Asu‐ACR‐16 when normalized to the control 100 μM ACh current was as follows: 100 μM nic (n = 21) ~ 100 μM cyt (n = 21) ~ 100 μM 3‐bc (n = 15) ~ 100 μM epi (n = 15) > 100 μM DMPP (n = 21) > 100 μM oxa (n = 36 ) >>> 100 μM cho (n = 15) = 100 μM bet (n = 15) = 100 μM lobe (n = 15) = 100 μM A844606 (n = 15) = mor (n = 21) = 100 μM lev (n = 21) = 100 μM met (n = 15) = 100 μM the (n = 15) = 100 μM beph (n = 15) = 30 μM tbd (n = 15) = 100 μM pyr (n = 15). * P < 0.05; significantly different as indicated; Tukey's multiple comparison tests.
	Figure 5. Asu‐ACR‐16 desensitization rate constant fit. Bar chart (mean ± SEM) showing Asu‐ACR‐16 desensitization in response to ACh, 3‐bromocytisine (3‐bc), cytisine (cyt), nicotine (nic) and epibatidine (epi). The rank order for Asu‐ACR‐16 time constants of desensitization was as follows: 100 μM ACh (12.6 ± 2.1 s, n = 6) ~ 100 μM 3‐bc (11.2 ± 2.8 s, n = 6) ~ 100 μM cyt (7.3 ± 0.7 s, n = 6) ~ 100 μM nic (6.6 ± 1.0 s, n = 4) ~ 100 μM epi (6.2 ± 0.8 s, n = 6). Insert: Sample trace with red line signifying desensitization fit. * P < 0.05; significantly different as indicated; Tukey's multiple comparison tests.
	Figure 6. Nicotine (nic) and ACh concentration–response relationships for Asu‐ACR‐16 in the absence of antagonists. (A) Sample traces for ACh (top trace) and nicotine (lower trace) dose–response relationships for Asu‐ACR‐16. (B) ACh and nic dose–response curves for Asu‐ACR‐16. EC50 values (mean ± SEM, μM) were 5.9 ± 0.1 for ACh, Hill slope (n H) = 3.9 ± 0.3, n = 6, and 4.5 ± 0.2 for nic, n H = 3.4 ± 0.2, n = 6.
	Figure 7. (A) Effects of selected nAChR antagonists on Asu‐ACR‐16‐mediated ACh responses. Bar chart showing effects of selected nAChR antagonists on Asu‐ACR‐16. Results were expressed as mean ( ± SEM) % inhibition of currents elicited by 100 μM ACh, n = 6, for all antagonists. dTC, mecamylamine (mec) and MLA completely blocked Asu‐ACR‐16‐mediated ACh responses, while paraherquamide (para), derquantel (der), hexamethonium (hexa) and DHβE only produced a partial block of Asu‐ACR‐16‐mediated ACh responses and α‐BTX produced an almost insignificant block of Asu‐ACR‐16‐mediated ACh responses. Rank order potency series for the nAChR antagonists each tested at a concentration of 10 μM was as follows: mecamylamine (n = 6) = MLA (n = 6) ≈ dTC (n = 6) > paraherquamide (n = 6) ~ derquantel (n = 6) ~ hexamethonium (hexa) (n = 6) ~ DHβE (n = 6) > α‐BTX (n = 6). * P < 0.05; significantly different as indicated; Tukey's multiple comparison tests. We used ANOVA and Bartlett's test for variance inhomogeneity and found no significant difference and Tukey's multiple comparison tests. (B) Dose–response relationships for Asu‐ACR‐16 in the presence of antagonists. (B1) Sample traces for ACh concentration–response relationships for Asu‐ACR‐16 in the presence of 1 μM derquantel and 1 μM DHβE. (B2) ACh concentration–response plots for Asu‐ACR‐16 in the presence of 1 μM derquantel and 1 μM DHβE. Derquantel caused a reduction in the maximum response, but no change in EC50, whereas DHβE caused both a reduction in the maximum response and a right shift in the EC50.
	Figure 8. Effects of PAMs of human‐α7 on Asu‐ACR‐16‐mediated ACh responses. Bar charts showing blocking actions of human‐α7 PAMs; (A) 10 μM ivermectin (n = 6), (B) 3 μM genistein (n = 6) and (C) 3 μM PNU120596 (n = 6), on the responses of Asu‐ACR‐16 to ACh. The type 1 PAMs, ivermectin and genistein, as well as the type 2 PAM, PNU120596, caused a reduction in Asu‐ACR‐16 responses to 10, 30 and 100 μM ACh, with the reduction being more pronounced at 10 than at 30 or 100 μM ACh.

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