RESUMEN
Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel beta subunits (Kv beta). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv beta, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv beta.
Asunto(s)
Drosophila/genética , Genes de Insecto/genética , Familia de Multigenes/genética , Canales de Potasio/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , ADN Complementario/genética , Proteínas de Drosophila , Electrofisiología , Biblioteca de Genes , Datos de Secuencia Molecular , Oocitos , Oxidorreductasas/genética , Canales de Potasio/biosíntesis , Canales de Potasio/metabolismo , ARN , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Canales de Potasio de la Superfamilia Shaker , XenopusRESUMEN
We identified, by affinity chromatography, two putative binding proteins for the presynaptic snake venom toxin taipoxin. We have previously characterized one of these proteins [neuronal pentraxin (NP)] as a neuronally secreted protein with homology to acute-phase proteins. Here we report the identification of the second protein as a 49-kDa lumenal calcium binding protein that we have named taipoxin-associated calcium binding protein 49 (TCBP-49). This protein contains six EF-hand putative calcium binding domains and the carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL), identical to the yeast endoplasmic reticulum retention signal. Message for this protein is present in brain, liver, muscle, heart, kidney, and testis. Antibodies to this protein label reticular organelles of neurons and glia. This localization and the specific enrichment of native and recombinant TCBP-49 on columns of immobilized taipoxin raise the possibility that this protein interacts with internalized taipoxin, perhaps mediating its activation. The availability of pure TCBP-49 will allow direct tests of whether TCBP-49 alters the integrity of the oligomeric structure, phospholipase activity, or toxicity of taipoxin.
Asunto(s)
Proteínas de Unión al Calcio/aislamiento & purificación , Cromatografía de Afinidad , Venenos Elapídicos/metabolismo , Retículo Endoplásmico/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Química Encefálica , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Bromuro de Cianógeno , Datos de Secuencia Molecular , Neuroglía/química , Neuronas/química , Fragmentos de Péptidos/química , ARN Mensajero/análisis , RatasRESUMEN
We have identified, by affinity chromatography, a binding protein for the snake venom toxin taipoxin. The sequence of this 47 kDa protein is unique, is characteristic of a secreted protein, and has homology to the acute phase proteins serum amyloid P protein and C-reactive protein of the pentraxin family. We have named this protein neuronal pentraxin (NP), as Northern analysis and in situ hybridization demonstrate high message levels in neurons of cerebellum, hippocampus, and cerebral cortex. Because NP may be released synaptically and has homology to immune proteins potentially involved in uptake of lipidic, toxic, or other antigenic material, we suggest that NP may be involved in a general uptake of synaptic macromolecules.