A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ

Abstract

A cone snail venom peptide, μO §-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligandbinding site. μO §-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJ SSG ). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) Na V 1.2. A mutant channel of rNa V 1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10 3 -fold less sensitive to GVIIJ SSG than was wild-type rNa V 1.2. In contrast, although rNa V 1.5 was >10 4 -fold less sensitive to GVIIJ SSG than Na V 1.2, an rNa V 1.5 mutant with a cysteine in the homologous location, rNa V 1.5[L869C], was >10 3 -fold more sensitive than wildtype rNa V 1.5. The susceptibility of rNa V 1.2 to GVIIJ SSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNa V β2 or rNa V β4, but not that of rNa V β1 or rNa V β3, protected rNa V 1.1 to -1.7 (excluding Na V 1.5) against block by GVIIJ SSG . Thus, GVIIJrelated peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which Na V β-and Na V α-subunits are present in native neurons.oltage-gated sodium channels (VGSCs) are responsible for the upstroke of action potentials in excitable tissues. Each VGSC is composed of a pore-and voltage sensor-bearing α-subunit and one or more auxiliary β-subunits. Mammals have nine α-subunit isoforms (Na V 1.1 to -1.9) and four β-subunit isoforms (Na V β1 to -β4) (1). An Na V 1 has about 2,000-aa residues arranged in four homologous domains, where each domain has six transmembrane spanning segments with an extracellular "pore" loop between segments 5 and 6 (1, 2); furthermore, each Na V 1 has about a dozen extracellular cysteine residues, all located in or near the pore loops. For the most part, not much is known about these cysteines (including whether they are disulfide bonded).Na V β-subunits can affect the function and cellular localization of Na V 1s (1, 3-5). Each Na V β-subunit has some 200-aa residues and consists of a single transmembrane segment with a large extracellular domain and a smaller intracellular domain (1). Na V β2-and Na V β4-subunits, unlike Na V β1-and Na V β3-subunits, are disulfide bonded to α-subunits (1, 6). A given neuron can have multiple isoforms of these subunits whose identities are challenging to appraise pharmacologically (7).Toxins that target VGSCs have been invaluable for probing the structure and function of these channels. Venoms are a rich source of such toxins. For example, in Conus snails, four families of neuroactive peptides have been characterized that target VGSCs:...

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks

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Insights into the origins of fish hunting in venomous cone snails from studies of Conus tessulatus

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