RegIIA: An α4/7-conotoxin from the venom of Conus regius that potently blocks α3β4 nAChRs

Franco, Aldo; Kompella, Shiva N.; Akondi, Kalyana Bharati; Melaun, Christian; Daly, Norelle L.; Luetje, Charles W.; Alewood, Paul F.; Craik, David J.; Adams, David J.; Marí, Frank

doi:10.1016/j.bcp.2011.11.006

Cited by 50 publications

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“…␣4/7-Conotoxin RegIIA from Conus regius has been reported to potently inhibit ␣3␤4 besides ␣3␤2 and ␣7 nAChRs (15). Interestingly, a non-natural analogue of RegIIA in which two residues in loop 2 were exchanged by alanines exhibited an enhanced selectivity for the ␣3␤4 subtype compared with the wild-type toxin (16).…”

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confidence: 99%

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

Cuny

Kompella

Tae

et al. 2016

Journal of Biological Chemistry

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Edited by Paul Fraser␣-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The ␣3␤4 nAChR, a predominant subtype in the peripheral nervous system, has been implicated in various pathophysiological conditions. As many ␣-conotoxins have multiple pharmacological targets, compounds specifically targeting individual nAChR subtypes are needed. In this study, we performed mutational analyses to evaluate the key structural components of human ␤2 and ␤4 nAChR subunits that determine ␣-conotoxin selectivity for ␣3␤4 nAChR. ␣-Conotoxin RegIIA was used to evaluate the impact of non-conserved human ␤2 and ␤4 residues on peptide affinity. Two mutations, ␣3␤2[T59K] and ␣3␤2[S113R], strongly enhanced RegIIA affinity compared with wild-type ␣3␤2, as seen by substantially increased inhibitory potency and slower off-rate kinetics. Opposite point mutations in ␣3␤4 had the contrary effect, emphasizing the importance of loop D residue 59 and loop E residue 113 as determinants for RegIIA affinity. Molecular dynamics simulation revealed the side chains of ␤4 Lys 59 and ␤4 Arg 113 formed hydrogen bonds with RegIIA loop 2 atoms, whereas the ␤2 Thr 59 and ␤2 Ser 113 side chains were not long enough to form such interactions. Residue ␤4 Arg 113 has been identified for the first time as a crucial component facilitating antagonist binding. Another ␣-conotoxin, AuIB, exhibited low activity at human ␣3␤2 and ␣3␤4 nAChRs. Molecular dynamics simulation indicated the key interactions with the ␤ subunit are different to RegIIA. Taken together, these data elucidate the interactions with specific individual ␤ subunit residues that critically determine affinity and pharmacological activity of ␣-conotoxins RegIIA and AuIB at human nAChRs.

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confidence: 99%

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

Cuny

Kompella

Tae

et al. 2016

Journal of Biological Chemistry

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“…Surprisingly, an initial screening with 100 nM RegIIA at ha3b2 demonstrated only minor (∼12%) reduction of AChevoked current amplitude, whereas the same concentration almost completely blocked the homologous rat a3b2 nAChR subtype ( Fig. 1A) (Franco et al, 2012). However, at rat and human a3b4 nAChR subtypes, 100 nM RegIIA exhibited similar inhibitory effects on ACh-evoked currents (Fig.…”

Section: Methodsmentioning

confidence: 83%

“…Plasmid DNAs encoding human a3, b2 and b4 nAChR subunits were obtained from OriGene Technologies Inc. (Rockville, MD) and the fulllength cDNAs were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091). Rat a3, b2 and b4 nAChR cDNA clones described previously (Franco et al, 2012) were also subcloned into pT7TS. Individual amino acids in the N-terminal extracellular domain (ECD) that precedes transmembrane segment I of rat a3 and b2 nAChRs were mutated to the corresponding human residues using the Geneart Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

“…over 10 independent simulations and two a3 (1) Results a-Conotoxin RegIIA Exhibits Differential Selectivity at the Rat and Human a3b2 but Not a3b4 nAChR Subtypes. RegIIA has been shown to selectively inhibit ra3b2, ra3b4 and ha7 nAChR subtypes (Franco et al, 2012). To examine if RegIIA exhibits species-specific activity differences, inhibition of ACh-evoked currents at human nAChR subtypes expressed in Xenopus oocytes was investigated.…”

Section: Methodsmentioning

confidence: 99%

“…a-Conotoxins are classified as selective antagonists of muscle and neuronal nAChRs and are promising pharmacological probes and potential therapeutics (Olivera et al, 2008;Muttenthaler et al, 2011;Lebbe et al, 2014). a4/7-Conotoxin RegIIA, isolated from the venom of Conus regius, has previously been characterized as a potent inhibitor of rat a3b2, a3b4, and human a7 nAChRs at nanomolar concentrations (Franco et al, 2012). Furthermore, a recent study using alanine-scanning mutagenesis identified a novel [N11A,N12A]RegIIA analog that selectively targets the rat a3b4 nAChR subtype (Kompella et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Molecular Basis for Differential Sensitivity of α-Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors

et al. 2015

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a-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The a3b2 and a3b4 nAChR subtypes are expressed in the central and peripheral nervous systems and play a critical role in various pathophysiological conditions ranging from nicotine addiction to the development and progression of lung cancer. Here we used the a4/7-conotoxin RegIIA, a disulfidebonded peptide from the venom of Conus regius, and its analog [N11A,N12A]RegIIA to probe the specific pharmacological properties of rat and human nAChR subtypes. nAChR subtypes were heterologously expressed in Xenopus oocytes and two-electrode voltage clamp recordings used to investigate the effects of the peptides on nAChR activity. RegIIA potently inhibited currents evoked by acetylcholine (ACh) at rat a3b2 (IC 50 5 10.7 nM), whereas a 70-fold lower potency was observed at human a3b2 nAChR (IC 50 5 704.1 nM). Conversely, there were no speciesspecific differences in sensitivity to RegIIA at the a3b4 nAChR. Receptor mutagenesis and molecular dynamics studies revealed that this difference can be attributed primarily to a single amino acid change: Glu198 on the rat a3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species-and subunit-specific receptor-antagonist interaction.

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Mechanism of interactions between α-conotoxin RegIIA and carbohydrates at the human α3β4 nicotinic acetylcholine receptor

Zheng

Tae

Xue

et al. 2021

Mar Life Sci Technol

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Conotoxins are marine peptide toxins from marine cone snails. The α-conotoxin RegIIA can selectively act on human (h) α3β4 nicotinic acetylcholine receptor (nAChR), and is an important lead for drug development. The high-resolution cryo-electron microscopy structure of the α3β4 nAChR demonstrates several carbohydrates are located near the orthosteric binding sites, which may affect α-conotoxin binding. Oligosaccharide chains can modify the physical and chemical properties of proteins by changing the conformation, hydrophobicity, quality and size of the protein. The purpose of this study is to explore the effect of oligosaccharide chains on the binding modes and activities of RegIIA and its derivatives at hα3β4 nAChRs. Through computational simulations, we designed and synthesized RegIIA mutants at position 14 to explore the importance of residue H14 to the activity of the peptide. Molecular dynamics simulations suggest that the oligosaccharide chains affect the binding of RegIIA at the hα3β4 nAChR through direct interactions with H14 and by affecting the C-loop conformation of the binding sites. Electrophysiology studies on H14 analogues suggest that in addition to forming direct interactions with the carbohydrates, the residue might play an important role in maintaining the conformation of the peptide. Overall, this study further clarifies the structure–activity relationship of α-conotoxin RegIIA at the hα3β4 nAChR and, also provides important experimental and theoretical basis for the development of new peptide drugs.

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RegIIA: An α4/7-conotoxin from the venom of Conus regius that potently blocks α3β4 nAChRs

Cited by 50 publications

References 58 publications

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

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

Molecular Basis for Differential Sensitivity of α-Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors

Mechanism of interactions between α-conotoxin RegIIA and carbohydrates at the human α3β4 nicotinic acetylcholine receptor

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