Structure, Dynamics, and Selectivity of the Sodium Channel Blocker μ-Conotoxin SIIIA

Abstract

µ-SIIIA, a novel µ-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-sensitive sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remained unknown. We have determined the structure of µ-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, µ-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11–16, while residues 3–5 in the N-terminal region form a helix-like turn resembling 310 helix. The Trp12 and His16 side chains are in close proximity, as in the related conotoxin µ-SmIIIA, but Asn2 is further away. Dynamics measurements show that the N-terminus and Ser9 have larger magnitude motions on the sub-ns timescale, while the C-terminus is more rigid. Cys4, Trp12 and Cys13 undergo significant conformational exchange on µs - ms timescales. µ-SIIIA is a potent, nearly irreversible blocker of NaV1.2, but also blocks NaV1.4 and NaV1.6 with submicromolar potency. The selectivity profile of µ-SIIIA, including poor activity against the cardiac sodium channel, NaV1.5, is similar to that of the closely related µ-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal NaV1.2. The structural and functional characterization described in this paper of an analgesic µ-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.

μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve

“…Data are quantified in Table 1 and Table S2. rNa V 1.2, 1.3 and hNa V 1.7 (25); and KIIIA and SIIIA on rNa V 1.1, 1.2, 1.3, and 1.7 and mNa V 1.6 (15,29). For the most part, the reported affinities of the μ-conopeptides agree with each other and the affinities in Table 1.…”

Integrated Oxidative Folding of Cysteine/Selenocysteine Containing Peptides: Improving Chemical Synthesis of Conotoxins

“…The solution was shaken for 30 min at 0 8C, centrifuged at 7000 RPM for 5 min, filtered through a 0. [19] Electrophysiological assays were carried out as described in Zhang et al [26] Received: December 13, 2008 Published online: February 10, 2009 . Keywords: conotoxin · cysteine · NMR spectroscopy · oxidative folding · selenium …”

Discovery and characterization of NaV modulatory venom peptides

“…Recurring features include an Arg crucial for activity on the loop 2 region of the peptide, C-terminal amidation, and a hydroxylated Pro that seems to aid proper folding but is not always present [338].…”

“…Since those first discoveries, numerous µ-conotoxins have been discovered through genomic analysis or activity-guided isolation from crude venom and have demonstrated a wide range of activity and selectivity profiles for Na V channels [342][343][344]. Solution structures and mutagenesis studies of a few select µ-conotoxins have begun to reveal key residues, loop regions, and even backbone lengths necessary to impart selectivity between different Na V channel isoforms [337,338,342,345]. The µ-conotoxins have been a focus for the development of novel analgesics targeting single-target Na V isoforms that contribute to nociception.…”

“…kinoshitai) and µ-SIIIA (C. striatus) have been shown to have analgesic properties in mouse inflammatory pain assays [338,346]. The µ-conotoxins small size, high affinity and diverse selectivity of inhibition for TTXs and TTXr Na V channels make them strong prospective therapeutic agents.…”