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Alanine scan of ?-conotoxin RegIIA reveals a selective ?3?4 nicotinic acetylcholine receptor antagonist.

ABSTRACT: Activation of the ?3?4 nicotinic acetylcholine receptor (nAChR) subtype has recently been implicated in the pathophysiology of various conditions, including development and progression of lung cancer and in nicotine addiction. As selective ?3?4 nAChR antagonists, ?-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new ?4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by ?3?4, ?3?2, and ?7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the ?3?4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the ?3?4 than the ?3?2 nAChR subtype. We also report synthesis of [N11A,N12A]RegIIA, a selective ?3?4 nAChR antagonist (IC50 of 370 nM) that could potentially be used in the treatment of lung cancer and nicotine addiction. Molecular dynamics simulations of RegIIA and [N11A,N12A]RegIIA bound to ?3?4 and ?3?2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of ?3?2 (?3-Tyr(92), Ser(149), Tyr(189), Cys(192), and Tyr(196); ?2-Trp(57), Arg(81), and Phe(119)) may form the molecular basis for the selectivity shift.

SUBMITTER: Kompella SN

PROVIDER: S-EPMC4294472 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Alanine scan of α-conotoxin RegIIA reveals a selective α3β4 nicotinic acetylcholine receptor antagonist.

Kompella Shiva N SN Hung Andrew A Clark Richard J RJ Marí Frank F Adams David J DJ

The Journal of biological chemistry 20141119 2

Activation of the α3β4 nicotinic acetylcholine receptor (nAChR) subtype has recently been implicated in the pathophysiology of various conditions, including development and progression of lung cancer and in nicotine addiction. As selective α3β4 nAChR antagonists, α-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new α4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evok ...[more]

PMID: 25411242

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Project description:α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure-activity relationship (SAR) of EI revealed that the "Arg1-Asn2-Hyp3" residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1-3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

Dataset Information

Alanine scan of ?-conotoxin RegIIA reveals a selective ?3?4 nicotinic acetylcholine receptor antagonist.

Publications

Alanine scan of α-conotoxin RegIIA reveals a selective α3β4 nicotinic acetylcholine receptor antagonist.

Similar Datasets

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