A novel α4/7‐conotoxin LvIA from Conus lividus that selectively blocks α3β2 vs. α6/α3β2β3 nicotinic acetylcholine receptors

Abstract

This study was performed to discover and characterize the first potent α3β2-subtype-selective nicotinic acetylcholine receptor (nAChR) ligand. A novel α4/7-conotoxin, α-CTxLvIA, was cloned from Conus lividus. Its pharmacological profile at Xenopus laevis oocyte-expressed rat nAChR subtypes was determined by 2-electrode voltage-clamp electrophysiology, and its 3-dimensional (3D) structure was determined by NMR spectroscopy. α-CTx LvIA is a 16-aa C-terminally-amidated peptide with 2-disulfide bridges. Using rat subunits expressed in Xenopus oocytes, we found the highest affinity of α-CTxLvIA was for α3β2 nAChRs (IC50 8.7 nM), where blockade was reversible within 2 min. IC50 values were >100 nM at α6/α3β2β3, α6/α3β4, and α3β4 nAChRs, and ≥3 μM at all other subtypes tested. α3β2 vs. α6β2 subtype selectivity was confirmed for human-subunit nAChRs with much greater preference (300-fold) for α3β2 over α6β2 nAChRs. This is the first α-CTx reported to show high selectivity for human α3β2 vs. α6β2 nAChRs. α-CTxLvIA adopts two similarly populated conformations water: one (assumed to be bioactive) is highly structured, whereas the other is mostly random coil in nature. Selectivity differences with the similarly potent, but less selective, α3β2 nAChR antagonist α-CTx PeIA probably reside within the three residues, which differ in loop 2, given their otherwise similar 3D structures. α4/7-CTx LvIA is a new, potent, selective α3β2 nAChR antagonist, which will enable detailed studies of α3β2 nAChR structure, function, and physiological roles.

α-Conotoxins Identify theα3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells

“…a-Ctx PnIA is a peptide similar in sequence to TxIB and is selective for rat a3b2 and a6b2 nAChRs over the a3b4 and a6/a3b4 subtypes (Luo et al, 1999;Hone et al, 2012a). a-Ctx LvIA is a newly discovered peptide that shows some selectivity for a3b2 over the a3b4 subtype (Luo et al, 2014). The amino acid sequences of these small peptides are all similar, but vary in key positions that are known to be critical for activity.…”

Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins

“…␣-Conotoxin LvIA remarkably revealed a considerable specificity for ␣3␤2 over ␣6/␣3␤2␤3 rat and human nAChRs, a rare feature not shared by other ␣-conotoxins such as RegIIA (32). As loops 1 of the two peptides are identical, these specificity differences must be conferred by the second loop in which three residues (11, 12, and 14) are different.…”

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

“…Peptide Synthesis-A two-step oxidation protocol was used to synthesize ␣-CTx LvIA as described previously (25). Because this protocol worked well, we did not attempt a simpler onestep oxidation approach.…”

“…Molecular Modeling-A molecular model of the interaction between LvIA and the ligand-binding domain of ␣3␤2 nAChR was built by homology using the NMR solution structure of LvIA (Protein Data Bank (PDB) identifier 2mdq) and the crystal structure of the complex between acetylcholine-binding protein (AChBP) and conotoxin PnIA variant (PDB identifier 2br8) as templates, as described previously (25). The molecular model was refined by a 30-ns explicit water molecular dynamics simulation carried out with the GROMACS 4.6.5 (26) package and the ff03 force field (27), using a procedure described previously (28,29).…”

“…␣-CTx LvIA from Conus lividus was recently characterized and has high affinity for ␣3␤2 nAChRs, with an IC 50 of 8.7 nM (25). LvIA is notable for its ability to selectively block ␣3␤2 versus ␣6/␣3␤2␤3 or ␣3␤4 nAChRs.…”