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

α-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The α3β4 nAChR, a predominant subtype in the peripheral nervous system, has been implicated in various pathophysiological conditions. As many α-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 β2 and β4 nAChR subunits that determine α-conotoxin selectivity for α3β4 nAChR. α-Conotoxin RegIIA was used to evaluate the impact of non-conserved human β2 and β4 residues on peptide affinity. Two mutations, α3β2[T59K] and α3β2[S113R], strongly enhanced RegIIA affinity compared with wild-type α3β2, as seen by substantially increased inhibitory potency and slower off-rate kinetics. Opposite point mutations in α3β4 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 β4 Lys59 and β4 Arg113 formed hydrogen bonds with RegIIA loop 2 atoms, whereas the β2 Thr59 and β2 Ser113 side chains were not long enough to form such interactions. Residue β4 Arg113 has been identified for the first time as a crucial component facilitating antagonist binding. Another α-conotoxin, AuIB, exhibited low activity at human α3β2 and α3β4 nAChRs. Molecular dynamics simulation indicated the key interactions with the β subunit are different to RegIIA. Taken together, these data elucidate the interactions with specific individual β subunit residues that critically determine affinity and pharmacological activity of α-conotoxins RegIIA and AuIB at human nAChRs

Alanine scan ofα-conotoxin regIIA reveals a selective α3β4 nicotinic acetylcholine receptor antagonist

Activation of the alpha 3 beta 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 alpha 3 beta 4 nAChR antagonists, alpha-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new beta 4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by alpha 3 beta 4, alpha 3 beta 2, and alpha 7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the alpha 3 beta 4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the alpha 3 beta 4 than the alpha 3 beta 2 nAChR subtype. We also report synthesis of [N11A, N12A] RegIIA, a selective alpha 3 beta 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 alpha 3 beta 4 and alpha 3 beta 2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of alpha 3 beta 2 (alpha 3-Tyr(92), Ser(149), Tyr(189), Cys(192), and Tyr(196); beta 2-Trp(57), Arg(81), and Phe(119)) may form the molecular basis for the selectivity shift

Polyaromatic hydrocarbons in pollution: a heart-breaking matter

Air pollution is associated with detrimental effects on human health, including decreased cardiovascular function. However, the causative mechanisms behind these effects have yet to be fully elucidated. Here we review the current epidemiological, clinical and experimental evidence linking pollution with cardiovascular dysfunction. Our focus is on particulate matter (PM) and the associated low molecular weight polycyclic aromatic hydrocarbons (PAHs) as key mediators of cardiotoxicity. We begin by reviewing the growing epidemiological evidence linking air pollution to cardiovascular dysfunction in humans. We next address the pollution‐based cardiotoxic mechanisms first identified in fish following the release of large quantities of PAHs into the marine environment from point oil spills (e.g. Deepwater Horizon). We finish by discussing the current state of mechanistic knowledge linking PM and PAH exposure to mammalian cardiovascular patho‐physiologies such as atherosclerosis, cardiac hypertrophy, arrhythmias, contractile dysfunction and the underlying alterations in gene regulation. Our aim is to show conservation of toxicant pathways and cellular targets across vertebrate hearts to allow a broad framework of the global problem of cardiotoxic pollution to be established. AhR; Aryl hydrocarbon receptor. Dark lines indicate topics discussed in this review. Grey lines indicate topics reviewed elsewhere.publishedVersio

Molecular basis for differential sensitivity of α-conotoxin RegIIA at rat and human neuronal nicotinic acetylcholine receptors

α-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The α3β2 and α3β4 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 α4/7-conotoxin RegIIA, a disulfide-bonded 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 α3β2 (IC50 = 10.7 nM), whereas a 70-fold lower potency was observed at human α3β2 nAChR (IC50 = 704.1 nM). Conversely, there were no species-specific differences in sensitivity to RegIIA at the α3β4 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 α3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species- and subunit-specific receptor-antagonist interaction

Alanine scan ofα-conotoxin regiia reveals a selective α3α4 nicotinic acetylcholine receptor antagonist

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)-evoked currents mediated by α3β4, α3β2, and α7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the α3β4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the α3β4 than the α3β2 nAChR subtype. We also report synthesis of [N11A,N12A]RegIIA, a selective α3β4 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 α3β4 and α3β2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of α3β2 (α3-Tyr92, Ser149, Tyr189, Cys192, and Tyr196; β2-Trp57, Arg81, and Phe119) may form the molecular basis for the selectivity shift

Determination of the alpha-conotoxin Vc1.1 binding site on the alpha 9 alpha 10 nicotinic acetylcholine receptor

a-Conotoxin Vc1.1 specifically and potently inhibits the nicotinic acetylcholine receptor subtype alpha 9 alpha 10 (alpha 9 alpha 10 nAChR) and is a potential novel treatment for neuropathic pain. Here, we used a combination of computational modeling and electrophysiology experiments to determine the Vc1.1 binding site on the alpha 9 alpha 10 nAChR. Interactions of Vc1.1 with two probable binding sites, alpha 9 alpha 10 and alpha 10 alpha 9, were modeled. Mutational energies calculated by assuming specific interactions in the alpha 10 alpha 9 binding site correlated better with electrophysiological recordings than those assuming interactions with the alpha 9 alpha 10 binding site. Two novel Vc1.1 analogues, [N9F]Vc1.1 and [N9W]Vc1.1, were predicted to have large differences in affinity between the two binding sites. Data from functional studies were consistent with computational predictions that assumed preferred binding of Vc1.1 to the alpha 10 alpha 9 pocket. Moreover, our modeling study suggested that a single hydrogen bond formed between Vc1.1 and position 59 of the alpha 10 alpha 9 pocket confers specificity to rat versus human alpha 9 alpha 10 nAChRs