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Key residues in the nicotinic acetylcholine receptor ?2 subunit contribute to ?-conotoxin LvIA binding.

ABSTRACT: ?-Conotoxin LvIA (?-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of ?3?2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the ?3?2 nAChR subtype from the ?6?2* (* indicates the other subunit) and ?3?4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the ?2 subunit versus those of the ?4 subunit on the binding of ?-CTx LvIA. Although two ?2 mutations, ?3?2[F119Q] and ?3?2[T59K], strongly enhanced the affinity of LvIA, the ?2 mutation ?3?2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the ?2-T59L mutant was combined with the ?3 subunit. There were no significant difference in inhibition of ?3?2[T59I], ?3?2[Q34A], and ?3?2[K79A] nAChRs when compared with wild-type ?3?2 nAChR. ?-CTx LvIA displayed slower off-rate kinetics at ?3?2[F119Q] and ?3?2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the ?2 subunit contributes to ?-CTx LvIA binding and selectivity. The results demonstrate that Val(111) is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework ?-conotoxins. Thr(59) and Phe(119) of the ?2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the ?4 subunit leads to increased affinity.

SUBMITTER: Zhangsun D

PROVIDER: S-EPMC4392282 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Key residues in the nicotinic acetylcholine receptor β2 subunit contribute to α-conotoxin LvIA binding.

Zhangsun Dongting D Zhu Xiaopeng X Wu Yong Y Hu Yuanyan Y Kaas Quentin Q Craik David J DJ McIntosh J Michael JM Luo Sulan S

The Journal of biological chemistry 20150220 15

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although ...[more]

PMID: 25713061

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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

Key residues in the nicotinic acetylcholine receptor ?2 subunit contribute to ?-conotoxin LvIA binding.

Publications

Key residues in the nicotinic acetylcholine receptor β2 subunit contribute to α-conotoxin LvIA binding.

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