Abstract
In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I β-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.
Highlights
As ligand-gated ion channels, neuronal nicotinic acetylcholine receptors are widely spread in the central and peripheral system [1]. nAChRs modulate the release of neurotransmitters, such as dopamine, norepinephrine, acetylcholine and γ-amino butyric acid [2], and they are involved in a variety of pathophysiologies, including chronic pain syndromes, epilepsy, Parkinson’s and Alzheimer’s [3,4,5,6]
The nAChR α3β2 subtype is expressed in the dorsal root ganglia and spinal cord, and it is involved in pain sensation [9,10]. nAChR α9α10 has been shown to be expressed on the cochlear outer hair cells, where it mediates the cholinergic efferent transmission [11]
The results showed that the PE residues ahead of the N-terminal of Mr1.7 were important for modulating its selectivity, and the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes
Summary
As ligand-gated ion channels, neuronal nicotinic acetylcholine receptors (nAChRs) are widely spread in the central and peripheral system [1]. nAChRs modulate the release of neurotransmitters, such as dopamine, norepinephrine, acetylcholine and γ-amino butyric acid [2], and they are involved in a variety of pathophysiologies, including chronic pain syndromes, epilepsy, Parkinson’s and Alzheimer’s [3,4,5,6]. NAChRs modulate the release of neurotransmitters, such as dopamine, norepinephrine, acetylcholine and γ-amino butyric acid [2], and they are involved in a variety of pathophysiologies, including chronic pain syndromes, epilepsy, Parkinson’s and Alzheimer’s [3,4,5,6]. Eight α and three β subunits (α2–α7, α9, α10, β2–β4) of nAChRs have been identified from mammalian neuronal cells [7]. They can form homopentamers (such as α7) and heteropentamers (such as α3β2, α3β4, α2β4, α4β2, α6β2β3, α2β2, and α9α10 subtypes) [8]. The nAChR α3β2 subtype is expressed in the dorsal root ganglia and spinal cord, and it is involved in pain sensation [9,10]. Vc1.1 and RgIA selectively target nAChR α9α10, and MII selectively targets nAChR α3β2 [12,13]
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