Dataset Information

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

ACCESS DATA

Publications

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

Similar Datasets

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.

Project description:Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 µM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation.

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.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets