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• W2022181012 abstract "Non-native disulfide isomers of α-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of “globular” and “ribbon” isomers of α-conotoxin AuIB have been characterized on α3β4 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to ∼50%. The maximal inhibition was stoichiometry-dependent because altering α3:β4 RNA injection ratios either increased AuIB(ribbon) efficacy (10α:1β) or completely abolished blockade (1α:10β). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity α3β4 nAChRs with corresponding EC50 values of 22.6 and 176.9 μm, respectively. Increasing the agonist concentration antagonized the inhibition of LS α3β4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS α3β4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS α3β4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both α3β4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS α3β4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS α3β4 nAChRs competitively, whereas AuIB(globular) inhibits α3β4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism. Non-native disulfide isomers of α-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of “globular” and “ribbon” isomers of α-conotoxin AuIB have been characterized on α3β4 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to ∼50%. The maximal inhibition was stoichiometry-dependent because altering α3:β4 RNA injection ratios either increased AuIB(ribbon) efficacy (10α:1β) or completely abolished blockade (1α:10β). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity α3β4 nAChRs with corresponding EC50 values of 22.6 and 176.9 μm, respectively. Increasing the agonist concentration antagonized the inhibition of LS α3β4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS α3β4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS α3β4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both α3β4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS α3β4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS α3β4 nAChRs competitively, whereas AuIB(globular) inhibits α3β4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism. Conotoxins are short disulfide-rich bioactive peptides that have been originally isolated from venoms of carnivorous mollusk cone snails, belonging to the genus Conus. α-Conotoxins are among the largest class of conotoxins found in the venom of most cone snail species (1.Azam L. McIntosh J.M. Acta Pharmacol. Sin. 2009; 30: 771-783Crossref PubMed Scopus (141) Google Scholar). This class of conotoxins targets various subtypes of nicotinic acetylcholine receptors (nAChRs) 2The abbreviations used are: nAChRnicotinic acetylcholine receptorAChBPacetylcholine-binding proteinHFhydrogen fluorideHShigh sensitivity α3β4 nAChRLSlow sensitivity α3β4 nAChRRP-HPLCreversed phase-high performance liquid chromatographyPDBProtein data bank. and is distinguished by four cysteines arranged in a CC-C-C pattern. nicotinic acetylcholine receptor acetylcholine-binding protein hydrogen fluoride high sensitivity α3β4 nAChR low sensitivity α3β4 nAChR reversed phase-high performance liquid chromatography Protein data bank. α-Conotoxins have attracted considerable attention as some of them, such as Vc1.1 and RgIA, have been shown to possess analgesic activity in rodent behavioral models of neuropathic pain (2.Callaghan B. Haythornthwaite A. Berecki G. Clark R.J. Craik D.J. Adams D.J. J. Neurosci. 2008; 28: 10943-10951Crossref PubMed Scopus (151) Google Scholar, 3.Luo S. Kulak J.M. Cartier G.E. Jacobsen R.B. Yoshikami D. Olivera B.M. McIntosh J.M. J. Neurosci. 1998; 18: 8571-8579Crossref PubMed Google Scholar). Interestingly, AuIB has recently been shown to be analgesic in vivo despite the fact that it acts on the α3β4 nAChR subtype different from the α9α10 nAChR targeted by Vc1.1 and RgIA. 3M. J. Christie, personal communication. Vc1.1 and RgIA have been shown to suppress N-type Ca2+ channel currents in dorsal root ganglion (DRG) neurons of neonatal and adult rats and wild type and α9 knock-out mice via activation of GABAB G protein-coupled receptors (2.Callaghan B. Haythornthwaite A. Berecki G. Clark R.J. Craik D.J. Adams D.J. J. Neurosci. 2008; 28: 10943-10951Crossref PubMed Scopus (151) Google Scholar). Similarly, AuIB inhibits N-type Ca2+ channels in rat DRG neurons analogous to Vc1.1 and RgIA and its effect can be blocked with selective GABAB receptor antagonists. 4B. Callaghan and D. J. Adams, unpublished observations. GABAB-mediated inhibition of N-type Ca2+ channels is proposed as an analgesic mechanism for α-conotoxins Vc1.1, RgIA, and AuIB (2.Callaghan B. Haythornthwaite A. Berecki G. Clark R.J. Craik D.J. Adams D.J. J. Neurosci. 2008; 28: 10943-10951Crossref PubMed Scopus (151) Google Scholar), which can reconcile obvious differences in their nAChR subtype selectivity. α-Conotoxin AuIB has been characterized on oocyte-expressed nAChRs and shown to be selective primarily for the α3β4 nAChR subtype (3.Luo S. Kulak J.M. Cartier G.E. Jacobsen R.B. Yoshikami D. Olivera B.M. McIntosh J.M. J. Neurosci. 1998; 18: 8571-8579Crossref PubMed Google Scholar). The α3β4 nAChR subtype is a predominant subtype in autonomic ganglia, adrenal medulla, and in subpopulations of central nervous system neurons such as medial habenula and dorsal medulla (4.Millar N.S. Gotti C. Neuropharmacology. 2009; 56: 237-246Crossref PubMed Scopus (325) Google Scholar). AuIB is a 15-residue conotoxin with an unusual 4/6 intercysteine spacing. Native AuIB peptide found in the venom, like the vast majority of other α-conotoxins, has 2–8, 3–15 (Cys1-Cys3, Cys2-Cys4) cystine globular connectivity (5.Dutton J.L. Bansal P.S. Hogg R.C. Adams D.J. Alewood P.F. Craik D.J. J. Biol. Chem. 2002; 277: 48849-48857Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). When AuIB is synthesized chemically, a disulfide bond isomer having 2–15, 3–8 (Cys1-Cys4, Cys2-Cys3) connectivity is co-produced as a by-product, which is the ribbon isoform of AuIB (Fig. 1A) (5.Dutton J.L. Bansal P.S. Hogg R.C. Adams D.J. Alewood P.F. Craik D.J. J. Biol. Chem. 2002; 277: 48849-48857Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Surprisingly, the ribbon isomer of AuIB has been shown previously to be ∼10-fold more potent at nAChRs of rat parasympathetic ganglion neurons compared with the globular (native) peptide isoform (5.Dutton J.L. Bansal P.S. Hogg R.C. Adams D.J. Alewood P.F. Craik D.J. J. Biol. Chem. 2002; 277: 48849-48857Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). However, when AuIB(ribbon) was probed on rat nAChRs heterologously expressed in Xenopus oocytes it was reported to be less active than the globular isomer (6.Nicke A. Samochocki M. Loughnan M.L. Bansal P.S. Maelicke A. Lewis R.J. FEBS Lett. 2003; 554: 219-223Crossref PubMed Scopus (48) Google Scholar). The difference in activity of the two AuIB isomers on native versus recombinant nAChRs remains to be elucidated. Taken together, there is an incomplete understanding of diversity of mechanisms of action of α-conotoxin AuIB and its isoforms. Here, we further explore the inhibitory mechanisms of globular and ribbon isomers of AuIB on rat α3β4 nAChRs expressed in Xenopus oocytes. Manipulations of the α3:β4 subunit ratios show that α3β4 nAChRs expressed in oocytes are present in different subunit stoichiometries and inhibition by AuIB(ribbon), but not AuIB(globular), is limited to one of the receptor stoichiometries. Inhibition by AuIB(ribbon) is consistent with a competitive antagonism, whereas AuIB(globular) inhibits α3β4 nAChRs via a non-competitive mechanism. The peptide was assembled on a 4-methylbenzhydrylamine resin by manual tert-butoxycarbonyl solid-phase peptide synthesis using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate-mediated in situ neutralization protocol with N,N′-dimethylformamide as solvent (7.Schnölzer M. Alewood P. Jones A. Alewood D. Kent S.B.H. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (938) Google Scholar). HF deprotection and cleavage was performed by treatment of the dried peptide resin (300 mg) with 10 ml of HF/p-cresol/p-thio-cresol (18:1:1, v/v/v) for 2 h at 0 °C. Following evaporation of the HF, the peptide was precipitated and washed with cold ether, filtered, and re-dissolved in 30 ml of 50% acetonitrile, 1% trifluoroacetic acid, and lyophilized. Oxidative folding was carried out in 0.1 m NH4HCO3 (pH 8.2, concentration = 0.1 μm) resulting in formation of the globular and ribbon isomers of AuIB (Fig. 1B). Oxidation was monitored by RP-HPLC, liquid chromatography mass spectrometry, and mass spectrometry, and the peptide isomers were isolated using preparative C18 RP-HPLC. CD spectroscopy was performed on a Jasco J-810 spectropolarimeter. Spectra were recorded at room temperature under nitrogen atmosphere. Peptides were dissolved in 20 mm phosphate buffer, containing 30% trifluoroethanol at pH 7. The peptide concentration was determined by quantitative RP-HPLC. The peptides were transferred into a 0.01-cm path length demountable cell and data were recorded over 5 scans, from 260 to 185 nm at 10 nm/min, with a resolution of 1 nm and a response time of 0.25 s. CD data in ellipticity was converted to mean residue ellipticity ([θ]R) using the equation: [θ]R = θ/(10 × C × Np × l), where θ is the ellipticity in millidegrees, C is the peptide molar concentration (M), l is the cell path length (cm), and Np is the number of peptide residues. CD was used to confirm the globular and ribbon AuIB structure. AuIB(globular) showed CD spectra with α-helical content as opposed to that of AuIB(ribbon), which has less secondary structure (Fig. 1C). Plasmid DNAs encoding rat α3 and β4 nAChR subunits were obtained from J. Patrick (Baylor College of Medicine, Houston, TX). After multiplication plasmid DNA was linearized with appropriate restriction enzymes and cRNA was synthesized in vitro using a SP6 in vitro transcription kit (mMessage mMachine; Ambion, Foster City, CA). RNA for α3 and β4 subunits was synthesized in parallel on the same day using identical procedures to maximize consistency between subunits in concentration and purity. RNA concentration was controlled spectrophotometrically for each new aliquot of RNA and before injections. Total amount of RNA injected per oocyte was ∼5 ng in ∼50 nl/volume. Stage V to VI oocytes obtained from Xenopus laevis were subsequently incubated for 2–8 days at 18 °C before electrophysiological recordings as described previously (6.Nicke A. Samochocki M. Loughnan M.L. Bansal P.S. Maelicke A. Lewis R.J. FEBS Lett. 2003; 554: 219-223Crossref PubMed Scopus (48) Google Scholar). Oocytes were transferred to the recording chamber (∼50 μl volume) and perfused at 3–5 ml/min with ND96 solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES, pH 7.5) by a gravity fed perfusion system. nAChR-mediated currents were evoked by pipetting 100 μl of ACh-containing solutions into the bath with the perfusion stopped. Oocytes were preincubated with the peptide for ∼5 min prior to ACh application and subsequently ACh was co-applied together with the peptide. Two-electrode voltage clamp recordings from Xenopus oocytes were made at room temperature (21–23 °C) using a GeneClamp 500B amplifier (Molecular Devices Corp., Sunnyvale, CA) at a holding potential of −80 mV. ACh-induced currents were recorded and their peak amplitudes were measured with pClamp 9 software (Molecular Devices Corp.). ACh-evoked currents recorded following exposure to peptides were normalized to an average of 2–6 control ACh-induced currents. Data from several experiments were pooled and each data point represents the average of 3–8 cells ± S.E. Estimates of toxins potencies (IC50, nH) were obtained by fitting data points to the equation: % response = 100/{1 + ([toxin]/IC50)nH} with SigmaPlot8 (Systat Software Inc., San Jose, CA). The molecular model of rat α3 subunit was generated using α1 nAChR subunit (Protein Data Bank (PDB) code 2QC1 (8.Dellisanti C.D. Yao Y. Stroud J.C. Wang Z.Z. Chen L. Nat. Neurosci. 2007; 10: 953-962Crossref PubMed Scopus (357) Google Scholar)). The rat β4 subunit homology model was generated using the β chain of Torpedo acetylcholine receptor (PDB ID 2BG9 (9.Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1419) Google Scholar)), as several attempts by using high resolution AChBP crystal structures were not satisfactory. The open and closed C-loop conformations of α3 subunit were resembled using co-crystal structures of AChBP in complex with different molecular sizes of ligands (Ac-AChBP-TxIA(A10L) (PDB code 2UZ6 (10.Dutertre S. Ulens C. Büttner R. Fish A. van Elk R. Kendel Y. Hopping G. Alewood P.F. Schroeder C. Nicke A. Smit A.B. Sixma T.K. Lewis R.J. EMBO J. 2007; 26: 3858-3867Crossref PubMed Scopus (146) Google Scholar)), Ac-AChBP-methyllycaconitine (PDB code 2BYR (11.Hansen S.B. Sulzenbacher G. Huxford T. Marchot P. Taylor P. Bourne Y. EMBO J. 2005; 24: 3635-3646Crossref PubMed Scopus (575) Google Scholar)), Ac-AChBP-nicotine (PDB code 1UW6 (12.Celie P.H. van Rossum-Fikkert S.E. van Dijk W.J. Brejc K. Smit A.B. Sixma T.K. Neuron. 2004; 41: 907-914Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar)), and Ls-AChBP-HEPES (PDB code 1I9B (13.Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1580) Google Scholar)). Templates and interfaces were assembled using Aplysia californica acetylcholine-binding protein (PDB code 2BR8 (14.Celie P.H. Kasheverov I.E. Mordvintsev D.Y. Hogg R.C. van Nierop P. van Elk R. van Rossum-Fikkert S.E. Zhmak M.N. Bertrand D. Tsetlin V. Sixma T.K. Smit A.B. Nat. Struct. Mol. Biol. 2005; 12: 582-588Crossref PubMed Scopus (294) Google Scholar)) as the template, with programs Modeler 9 version 2 (15.Fiser A. Sali A. Methods Enzymol. 2003; 374: 461-491Crossref PubMed Scopus (1355) Google Scholar) and Sculptor, and/or by docking simulations using the program HEX 5.1 (16.Ritchie D.W. Kemp G.J.L. Proteins Struct. Funct. Genet. 2000; 39: 178-194Crossref PubMed Scopus (488) Google Scholar). All sequence alignments were generated using ClustalW (17.Larkin M.A. Blackshields G. Brown N.P. Chenna R. McGettigan P.A. McWilliam H. Valentin F. Wallace I.M. Wilm A. Lopez R. Thompson J.D. Gibson T.J. Higgins D.G. Bioinformatics. 2007; 23: 2947-2948Crossref PubMed Scopus (22660) Google Scholar) and were further manually adjusted based on secondary structure alignment. The subunit α3 and β4 and the pentamers (α3)3(β4)2 and (α3)2(β4)3 were validated individually using online server Verify3D (18.Bowie J.U. Lüthy R. Eisenberg D. Science. 1991; 253: 164-170Crossref PubMed Scopus (2417) Google Scholar) and Ramachandran plot available from ProFunc data base (19.Laskowski R.A. Watson J.D. Thornton J.M. Nucleic Acids Res. 2005; 33: W89-W93Crossref PubMed Scopus (510) Google Scholar). The solvent accessible area of each interface was calculated using online server CASTp (20.Dundas J. Ouyang Z. Tseng J. Binkowski A. Turpaz Y. Liang J. Nucleic Acids Res. 2006; 34: W116-W118Crossref PubMed Scopus (1416) Google Scholar). In addition, we further validated our homology models by performing a global docking simulation with ACh. The outcome showed that ACh only docked to the classical agonist binding site in the α(+)β(−) interface and was unaffected by subunit stoichiometry (supplemental Fig. S1). Each of the top 20 lowest energy NMR structures of globular (PDB code 1MXN (5.Dutton J.L. Bansal P.S. Hogg R.C. Adams D.J. Alewood P.F. Craik D.J. J. Biol. Chem. 2002; 277: 48849-48857Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar)) and ribbon (PDB code 1MXP (5.Dutton J.L. Bansal P.S. Hogg R.C. Adams D.J. Alewood P.F. Craik D.J. J. Biol. Chem. 2002; 277: 48849-48857Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar)) AuIB were docked to all (α3)3(β4)2 and (α3)2(β4)3 pentamer models (assembled by both approaches with different C-loop conformations) individually using the program HEX 5.1, followed by energy minimization. The solutions that disagreed with the known constraints, such as binding at the transmembrane region or inside the ion permeation pathway were excluded. All docking simulation with (α3)3(β4)2 and (α3)2(β4)3 pentamer models were successful, except for docking of ribbon AuIB to (α3)3(β4)2. Global docking (without energy minimization) of ribbon AuIB to (α3)3(β4)2 pentamer showed that ribbon AuIB docked in the C-loop binding site. However, no solution could be obtained after energy minimization. Therefore, we tried the α(+)β(−) dimer extracted directly from the (α3)3(β4)2 pentamer models, an approach used previously for docking simulations at AChBP-derived models of nAChRs. The docking solutions after energy minimization showed that ribbon AuIB docked at the same location as the global docking in the (α3)3(β4)2 pentamer. For consistency, dimer extracted from (α3)3(β4)2 and (α3)2(β4)3 pentamer models were also used for docking simulation with globular and ribbon AuIB. Outcomes of docking simulations of globular and ribbon AuIB to dimer (α3)3(β4)2 and (α3)2(β4)3 models are consistent with results obtained using pentameric models. The top 20 energetically favorable docking solutions of each combination were selected and are presented in Fig. 8. Concentration-response curves were obtained for the inhibition of rat α3β4 nAChRs expressed in Xenopus oocytes by globular and ribbon isomers of AuIB α-conotoxin (Fig. 2). Surprisingly, the two isomers of AuIB demonstrated a marked difference in their efficacy for inhibition of α3β4 nAChRs. AuIB(globular) at a concentration of 10 μm completely inhibited α3β4 nAChRs (95.3 ± 0.9%, n = 6; p < 0.03), whereas AuIB(ribbon) did not exceed 50% block even at a concentration of 30 μm (Fig. 2, A and B). The concentration-response relationships for AuIB(globular) and AuIB(ribbon) gave IC50 values of 2.48 and 0.77 μm, respectively (Table 1). We hypothesized that the difference in inhibitory action of the two AuIB isomers may be due to specific action of the AuIB(ribbon) on one of the two likely ((α3)3(β4)2 versus (α3)2(β4)3) α3β4 nAChR stoichiometries. The concentration-response curves shown in Fig. 2B were obtained on oocytes injected with equal amounts of α3 and β4 RNA (1:1 injection ratio). Theoretically, a 1:1 injection ratio may yield equal occurrence of the (α3)2(β4)3 and (α3)3(β4)2 stoichiometries, which would result in maximal inhibition of 50% of the whole cell ACh-evoked current amplitude if blockade of α3β4 nAChRs by AuIB(ribbon) is stoichiometry dependent.TABLE 1Comparison of concentration-response curves parameters of AuIB analogues obtained in oocytes injected with different α3 and β4 nAChR subunit ratiosα3:β4 ratioAuIB(globular)AuIB(ribbon)IC50nHIC50nHμmμm10:11.11.380.860.941:12.481.370.77aMaximal efficacy ∼50%. Data were acquired with 50 μm ACh used as agonist.0.971:103.01.0NAbNA, not active.a Maximal efficacy ∼50%. Data were acquired with 50 μm ACh used as agonist.b NA, not active. Open table in a new tab To probe the stoichiometry sensitivity of α3β4 nAChR inhibition by AuIB isomers, experiments were carried out on oocytes injected with different amounts of RNA encoding α3/β4 subunits. Concentration-response curves for inhibition of ACh-evoked currents by AuIB isomers in oocytes injected at 10:1 versus 1:10 α3:β4 ratios are shown on Fig. 3A. The efficacy of inhibition (maximal block) by AuIB(ribbon) at α3-dominant (10α:1β) nAChRs increased to 73.2 ± 3.0% (n = 8, p < 0.001) at 10 μm (Fig. 2A) but the IC50 of 0.86 μm remained approximately the same as for 1:1 injected oocytes (IC50 = 0.77 μm, Table 1). In contrast, in β4-dominant (1α:10β) nAChRs, the ribbon isoform of AuIB exhibited a dramatic decrease in inhibitory effect averaging only 14.2 ± 1.1% maximal block at 30 μm AuIB(ribbon) (Fig. 3A, panel ii). In comparison, maximal block by AuIB(globular) was unaffected by varying the α3:β4 ratios at both α3-dominant (10:1 ratio) nAChRs and β4-dominant (1:10 ratio) nAChRs (Fig. 3A, panels i and ii). We note that nAChR inhibition by AuIB(ribbon) in oocytes injected with the 10:1 ratio of mRNAs was incomplete (Fig. 3B, panel ii). This incomplete block likely reflects expression of a population of (α3)2(β4)3 receptors insensitive to the ribbon isomer (Fig. 3A, panel ii). Comparison of the concentration-response relationships obtained from oocytes with different α3β4 nAChR subunit ratios indicates distinct modes of action of AuIB(ribbon) versus AuIB(globular) (Fig. 3). Shifting α3:β4 ratios did not change the maximal inhibition as well as the other parameters of the AuIB(globular) concentration-response curve (Fig. 3B, panel i). Half-maximal inhibitory concentrations (IC50) and Hill slopes (nH) obtained for AuIB(globular) inhibition of ACh-evoked currents were 1.1 (1.38), 2.48 (1.37), and 3.0 μm (1.0) for 10:1, 1:1, and 1:10 ratios, respectively (Table 1). In contrast, when the subunit ratio changed from 10:1 to 1:10, the concentration-response curve of AuIB(ribbon) flattened out to exhibit no significant inhibition at β-dominated oocytes (Fig. 3B, panel ii). These results suggest an intrinsic difference in the mechanisms of action of the two isomers of α-conotoxin AuIB. Varying the α3:β4 subunit ratio in oocytes in either direction had a marked effect on the ACh-induced current amplitude. Application of 50 μm ACh elicited the largest currents in oocytes injected at a 1:10 ratio, and the smallest current amplitudes in oocytes injected with a 10:1 α3:β4 ratio. Injecting all three different ratios of RNA in oocytes resulted in a significant difference between the average ACh-evoked current amplitudes in the three groups (Fig. 4A). The average amplitude of ACh-evoked currents in the 10:1 ratio injected oocytes was 1.66 ± 0.14 μA and 13.74 ± 2.26 μA in 1:1, and 23.33 ± 3.48 μA in 1:10 (n = 17–21). The current amplitudes in the 10:1 and 1:10 ratio injected oocytes were significantly different from the current amplitudes obtained for the 1:1 ratio injected oocytes (p < 0.001, 10:1; p < 0.05 1:10, unpaired t test) (Fig. 4A). In a series of experiments on 10:1 ratio injected oocytes, 2.5 mm ACh elicited current amplitudes similar to those observed for 1:10 injected oocytes (data not shown) indicating that the smaller current observed in response to 50 μm ACh in 10:1 oocytes was not due to reduced nAChR expression. We hypothesized that the sensitivity to ACh (EC50) may account for different current amplitudes observed for the three different subunit ratios. To test this possibility, ACh sensitivity of oocytes injected with different α3:β4 subunit ratios was examined. The ACh concentration-response curves obtained for 10:1 and 1:10 ratio groups were strikingly different with an EC50 of 176.9 ± 23.9 μm (n = 4–7) for 10:1 ratio injected oocytes and 22.56 ± 1.41 μm (n = 3–8) for the 1:10 oocyte group (Fig. 4B). The Hill slope was also different between the two ratio groups (2.1 ± 0.5 for 10:1 and 1.00 ± 0.1 for 1:10) suggesting that the 10:1 α3β4 nAChR may bind 2 ACh molecules, whereas the 1:10 α3β4 nAChR binds a single agonist molecule (Fig. 4B). The β-dominated nAChRs (1:10) are high sensitivity receptors and α-dominated nAChRs (10:1) are low sensitivity α3β4 nicotinic receptors. The ACh concentration-response curve for the 1:1 injected oocytes was fit with an EC50 of 92.8 ± 6.6 μm and nH = 1.3 ± 0.1 (n = 3–8), which was between the values obtained for 1:10 and 10:1 ratios (Fig. 4B). The parameters obtained from the ACh concentration-response curves for the three subunit ratios are summarized in Table 2. These findings indicate that the different stoichiometries of α3β4 nAChRs expressed in oocytes underlie the differences in EC50, Hill slope, and ACh-evoked current amplitudes observed for the three groups.TABLE 2Agonist sensitivity of different α3β4 nAChR stoichiometriesα3:β4 ratioEC50 AChnHμm10:1176.9 ± 23.872.14 ± 0.581:192.88 ± 6.611.32 ± 0.131:1022.56 ± 1.411.00 ± 0.06 Open table in a new tab The ACh concentration-response curves determined for the three different α3:β4 ratios indicate that 50 μm ACh used in our initial experiments is below the EC50 for the 10:1 ratio and close to saturation for the 1:10 ratio injected oocytes (Fig. 5). The difference in the EC50 may account for the differential inhibitory effect of AuIB(ribbon) tested on all three injection groups. The ACh dependence of the inhibition of 10:1 ratio α3β4 nAChRs by both AuIB isomers was investigated for three different ACh concentrations: 50, 175 (∼EC50), and 500 μm. Concentration-response curves for AuIB isomers obtained at different ACh concentrations are shown in Fig. 5. The inhibition of α3β4 nAChRs by AuIB(globular) was not strongly dependent on ACh concentration: IC50 values of 1.1, 0.9, and 2.3 μm were obtained for 50, 175, and 500 μm ACh, respectively (Table 3). Overall, increasing the agonist concentration produced a small rightward shift of the concentration-response curve only at saturating ACh concentration (500 μm) without affecting maximal inhibition achieved. In contrast, the efficacy of α3β4 nAChRs inhibition by AuIB(ribbon) decreased with increasing ACh concentration (Fig. 5B). 10 μm AuIB(ribbon) inhibited α3β4 nAChRs by 73.2 ± 3.0% using 50 μm ACh, 34.2 ± 2.1% at 175 μm, and 22.2 ± 3.1% at 500 μm ACh (Fig. 5B). Inhibition of α3β4 nAChRs at a 10:1 ratio by AuIB(ribbon) is sensitive to the ACh concentration, whereas AuIB(globular) is relatively insensitive to the agonist concentration suggesting a competitive mechanism of action for the ribbon isomer of AuIB and non-competitive for the globular isomer.TABLE 3Concentration-response curve parameters determined for AuIB(globular) and -(ribbon) inhibition at different α3:β4 subunit ratios and ACh concentrations (n ≥ 3)AuIB isomer10:11:10[ACh]IC50nH[ACh]IC50nHμmμmGlobular50 μm1.11.385 μm1.240.85175 μm0.861.3750 μm3.01.0500 μm2.361.685 mm5.81.63Ribbon50 μm0.860.943 μmNAaNA, not active. ND, not determined.175 μmNDb50 μmNA500 μmNDa NA, not active.ND, not determined. Open table in a new tab The effect of ACh concentration on the inhibition of β4-dominant oocytes (1:10 ratio) by AuIB isomers was examined. AuIB(globular) inhibition of β4-dominant nAChRs was tested for three different ACh concentrations: 5 μm, 50 μm (∼EC50), and 5 mm. The concentration-response curves obtained for the globular isomer exhibited a rightward shift at low toxin concentrations; however, AuIB(globular) retained full efficacy even at a saturating ACh concentration (5 mm). The maximal inhibition produced by 30 μm AuIB(globular) was 97.8 ± 0.2% at 5 μm ACh, 94.4 ± 1.1% at 50 μm ACh, and 96.1 ± 1.3% at 5 mm ACh (Fig. 6A). Half-maximal inhibitory AuIB(globular) concentrations were 1.24, 3.0, and 5.8 μm for 5 μm, 50 μm, and 5 mm ACh, respectively (Table 3). Because AuIB(ribbon) inhibition is competitive and ACh concentration dependent in LS nAChRs, there is a possibility that in HS inhibition is masked due to lower ACh EC50 for HS; however, AuIB(ribbon) was not active in 1:10 ratio injected oocytes even at low ACh concentrations (3 μm) (Fig. 6B, inset). These data suggest that the lack of activity of AuIB(ribbon) at 1:10 nAChRs may be due to dependence of the inhibition on α3β4 nAChR stoichiometry, probably, because of a modified ACh binding site at 1:10 α3β4 nAChRs. Effects of different ACh concentrations on concentration-response curve parameters for the AuIB isomers obtained in 10:1 and 1:10 oocytes are summarized in Table 3. AuIB(globular) inhibition of α3β4 nAChRs, in contrast to AuIB(ribbon), is not significantly impaired by subunit stoichiometry or agonist concentration. The mechanism of nAChR inhibition by AuIB(globular) is consistent with non-competitive receptor antagonism. To further examine mechanism(s) of action of AuIB(globular), ACh concentration-response curves were determined in the presence of different concentrations of AuIB(globular) at 1:10 ratio injected oocytes where the ribbon isomer is inactive. Increasing concentrations of AuIB(globular) did not induce a parallel shift of the ACh concentration-response curve (see Fig. 7A) and even saturating ACh concentrations did not impair inhibition of HS α3β4 nAChRs by AuIB(globular) when it" @default.
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• W2022181012 title "α-Conotoxin AuIB Isomers Exhibit Distinct Inhibitory Mechanisms and Differential Sensitivity to Stoichiometry of α3β4 Nicotinic Acetylcholine Receptors" @default.
• W2022181012 cites W1595347157 @default.
• W2022181012 cites W1637250296 @default.
• W2022181012 cites W1788430178 @default.
• W2022181012 cites W1964673525 @default.
• W2022181012 cites W1986084361 @default.
• W2022181012 cites W2004164502 @default.
• W2022181012 cites W2006398084 @default.
• W2022181012 cites W2010215361 @default.
• W2022181012 cites W2031173955 @default.
• W2022181012 cites W2037966476 @default.
• W2022181012 cites W2039817644 @default.
• W2022181012 cites W2039936652 @default.
• W2022181012 cites W2040325257 @default.
• W2022181012 cites W2041720100 @default.
• W2022181012 cites W2041812461 @default.
• W2022181012 cites W2050593080 @default.
• W2022181012 cites W2053846150 @default.
• W2022181012 cites W2059235702 @default.
• W2022181012 cites W2074568561 @default.
• W2022181012 cites W2077776336 @default.
• W2022181012 cites W2081065619 @default.
• W2022181012 cites W2083881933 @default.
• W2022181012 cites W2086784961 @default.
• W2022181012 cites W2091181223 @default.
• W2022181012 cites W2091465745 @default.
• W2022181012 cites W2092364608 @default.
• W2022181012 cites W2097292426 @default.
• W2022181012 cites W2097345469 @default.
• W2022181012 cites W2099277573 @default.
• W2022181012 cites W2100270845 @default.
• W2022181012 cites W2100494857 @default.
• W2022181012 cites W2109732014 @default.
• W2022181012 cites W2133553818 @default.
• W2022181012 cites W2137015675 @default.
• W2022181012 cites W2143014450 @default.
• W2022181012 cites W2146837416 @default.
• W2022181012 cites W2147835957 @default.
• W2022181012 cites W2148168931 @default.
• W2022181012 cites W2148677994 @default.
• W2022181012 cites W2156819887 @default.
• W2022181012 cites W2160223767 @default.
• W2022181012 cites W2337946333 @default.
• W2022181012 cites W4246356731 @default.
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