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Co-expression of Na(V)? subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking ?-conotoxins.

ABSTRACT: Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing ?-subunit (NaV 1) and one or two accessory ?-subunits (NaV ?s). Neurons in mammals can express one or more of seven isoforms of NaV 1 and one or more of four isoforms of NaV ?. The peptide ?-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in NaV 1. Hitherto, the effects of NaV ?-subunit co-expression on the activity of these toxins have not been comprehensively assessed.Four ?-conotoxins (?-TIIIA, ?-PIIIA, ?-SmIIIA and ?-KIIIA), TTX and STX were tested against NaV 1.1, 1.2, 1.6 or 1.7, each co-expressed in Xenopus laevis oocytes with one of NaV ?1, ?2, ?3 or ?4 and, for NaV 1.7, binary combinations of thereof.Co-expression of NaV ?-subunits modifies the block by ?-conotoxins: in general, NaV ?1 or ?3 co-expression tended to increase kon (in the most extreme instance by ninefold), whereas NaV ?2 or ?4 co-expression decreased kon (in the most extreme instance by 240-fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by NaV ?-subunit co-expression. Tests of NaV ?1?:??2 chimeras co-expressed with NaV 1.7 suggest that the extracellular portion of the NaV ? subunit is largely responsible for altering ?-conotoxin kinetics.These results are the first indication that NaV ? subunit co-expression can markedly influence ?-conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. ?-Conotoxins could, in principle, be used to pharmacologically probe the NaV ? subunit composition of endogenously expressed VGSCs.

SUBMITTER: Zhang MM

PROVIDER: S-EPMC3605869 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Co-expression of Na(V)β subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins.

Zhang Min-Min MM Wilson Michael J MJ Azam Layla L Gajewiak Joanna J Rivier Jean E JE Bulaj Grzegorz G Olivera Baldomero M BM Yoshikami Doju D

British journal of pharmacology 20130401 7

<h4>Background and purpose</h4>Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing α-subunit (NaV 1) and one or two accessory β-subunits (NaV βs). Neurons in mammals can express one or more of seven isoforms of NaV 1 and one or more of four isoforms of NaV β. The peptide μ-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in NaV 1. Hitherto, the effects of NaV β-subunit co-expression ...[more]

PMID: 23146020

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Project description:In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of ?-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related ?-conotoxins. The two major isomers of synthetic ?-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(V)1.2 (K(d) values of 5 and 230 nM, respectively). The solution structure for ?-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the ?-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an ?-helix spanning residues 7-12. In addition, the major folding isomers of ?-KIIIB, an N-terminally extended isoform of ?-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as ?-KIIIA, and both blocked Na(V)1.2 (K(d) values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic ?-KIIIA and ?-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of ?-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail's repertoire of active peptides.

Dataset Information

Co-expression of Na(V)? subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking ?-conotoxins.

Publications

Co-expression of Na(V)β subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins.

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