Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na ؉ and Ca 2؉ channels have been characterized, relatively few have been identified that interact with K ؉ channels. We describe a novel Conus peptide that interacts with the Shaker K ؉ channel, M-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although M-conotoxin RIIIK is structurally similar to the -conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K ؉ channels. Studies using Shaker K ؉ channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe 425 Basic research on voltage-gated ion channels advances on two broad fronts: first, the identification and characterization of the numerous molecular isoforms that comprise each voltage-gated ion channel family. A different stream of research focuses on a few model systems intensively to uncover basic mechanistic insights. For both of these contrasting facets of ion channel research, the small peptides made by predatory cone snails (conotoxins) (1, 2) have considerable potential. Thus, the -conotoxins, one family of conotoxins, have become standard reagents for discriminating among Ca 2ϩ channel subtypes (3), and characterizing the functional role of each subtype. The -conotoxins that block voltage-gated channels are used as probes for the outer vestibule of the channel pore. The subject of this report is a novel conotoxin that has promising properties to be an important reagent for structure/function studies of the Shaker K ϩ channel, arguably the most intensively studied of all voltage-gated ion channels. However, the characterization of the peptide also defines a new family of conotoxins, the M family, that should provide a set of new ligands specific for different K ϩ channel isoforms.The first Conus peptide shown to target a voltage-gated ion channel was -conotoxin GIIIA, which was discovered and characterized two decades ago (4 -8). A characteristic feature of all -conotoxins is the arrangement of disulfide cross-links in the primary sequence, the -conotoxin pattern can be recognized by the following pattern of Cys residues: -CC-C-C-CC-, now defined as a class III (or M-1) conotoxin scaffold (9). After the discovery of the -conotoxins, other groups of Conus peptides with three disulfide bonds (the -conotoxins, ␦-conotoxins, O-conotoxins, and the spasmodic peptides) were characterized, but these had a different arrangement of Cys residues. Only one other family of conopeptides with a class III disulfide framework has been characterized, the -conotoxins (10). The latter are noncompetitive antagonists of nicotinic acetylcholine receptors.In this report, a peptide from Conus radiatus venom ducts that has the same class III...
In the central nervous system, two subtypes of sodium-and chloride-coupled GABA transporter exist. One is sensitive to ACHC, the other to ~.alanine. They are thought to be of neuronal and $1iai origin, reslZ:ctively. GABA transport in membrane v~icles derived from asttoglial ceils was found to be sodium-and chloride-dependent, electroganic and much more sensitive to fl-alanine than to ACHC. Immunobloning with antibodies dir~ted against a variety of sequenc,s of the ACHC-sensitive transporter indicated that none of these epitopes was shared by the glial transporter.
Previous studies identified homologues to mammalian myelin genes expressed in the teleost central nervous system (CNS), including myelin basic protein (MBP), protein zero (P0), and a member of the proteolipid protein family, DM20. In addition, an uncharacterized 36-kDa (36K) protein is a major component of teleost myelin, but is not a major component of myelin in other species. In the present study, we sought to better understand myelin proteins and myelination in one teleost, zebrafish, by molecular characterization of the zebrafish 36K protein. Purified zebrafish CNS myelin was isolated and the amino acid sequences of peptides present in the 36-kDa band were determined by mass spectrometry. These sequences matched a previously uncharacterized EST in The Institute for Genome Research (TIGR) zebrafish database that is related to the short-chain dehydrogenase/reductase (SDR) protein family. In vitro expression of the zebrafish 36K cDNA in Neuro 2a cells resulted in a protein product that was recognized by a 36K polyclonal antibody. The zebrafish 36K mRNA and protein expression patterns were determined and correlated to other known myelin gene expression profiles. In addition, we determined by in situ hybridization that a human 36K homologue (FLJ13639) is expressed in oligodendrocytes and neurons in the adult human cortex. This study identified a major myelin protein in zebrafish, 36K, as a member of the SDR superfamily; an expression pattern similar to other myelin genes was demonstrated.
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