Action potentials in nerve and muscle fibers are mediated by voltage-gated sodium channels (VGSCs). There are several isoforms of these channels, and most are exquisitely sensitive to tetrodotoxin (TTX), the classic blocker of VGSCs. One such isoform is found exclusively in skeletal muscle, and it is targeted by the founding members in the mu-conopeptide family. Recently, new members of mu-conopeptides (provisionally called mu/nu-conopeptides) have been discovered that irreversibly block TTX-resistant VGSCs in sensory and sympathetic neurons of frog. VGSC-antagonists with this specificity are of medical interest since two TTX-resistant VGSC isoforms cloned from mammals are found largely in nociceptive sensory neurons. Thus, these mu/nu-conopeptides may serve as lead ligands in the development of improved analgesics. There are four specific aims: (1) Examine the activity and selectivity of mu/nu-conopeptides by electrophysiological assays of VGSCs in: (a) sensory and autonomic neurons, (b) motor axons, (c) skeletal muscle, and (d) Xenopus oocytes expressing cloned subtypes of VGSCs. (2) Map the functional distribution of TTX-resistant (mu/nu-conopeptide-sensitive) VGSCs in sensory nerves to examine possible differential expression at sensory endings versus the bulk, or conductive, portion of sensory fibers. This will be done by extracellular recording and focal drug application. (3) Examine the functional tructures of the mu/nu-conopeptides by in-vitro mutagenesis, construction of chimeras of various mu/nuand mu-conopeptides, and covalent labeling with moieties that can also serve as reporter groups for use in the next aim. (4) Map the subcellular distribution of labeled mu/nu-conopeptide binding sites along sensory axons and termini by microscopy. This histological study will complement the electrophysiological experiments in Aim 2.
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