RESUMO
We have isolated, by degenerate PCR, a complementary DNA encoding a novel two pore domain potassium channel. This is the 7th functional member of the human tandem pore domain potassium channel family to be reported. It has an open reading frame of 1.125 kb and encodes a 374 amino acid protein which shows 62% identity to the human TASK-1 gene: identity to other human members of the family is 31-35% at the amino acid level. We believe this gene to be human TASK-3, the ortholog of the recently reported rat TASK-3 gene: amino acid identity between the two is 74%. 'Taqman' mRNA analysis demonstrated a very specific tissue distribution pattern, showing human TASK-3 mRNA to be localised largely in the cerebellum, in contrast rat TASK-3 was reported to be widely distributed. We have shown by radiation hybrid mapping that human TASK-3 can be assigned to chromosome 8q24.3. Human TASK-3 was demonstrated to endow Xenopus oocytes with a negative resting membrane potential through the presence of a large K(+) selective conductance. TASK-3 is inhibited by extracellular acidosis with a mid-point of inhibition around pH 6. 5, supporting the predictions from the sequence data that this is a third human TASK (TWIK-related acid sensitive K(+) channel) gene.
Assuntos
Cerebelo/metabolismo , Cromossomos Humanos Par 8 , Potenciais Evocados/fisiologia , Proteínas do Tecido Nervoso , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Variação Genética , Humanos , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Oócitos/fisiologia , Filogenia , Reação em Cadeia da Polimerase , Canais de Potássio/química , Canais de Potássio/fisiologia , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Neuromedins are a family of peptides best known for their contractile activity on smooth muscle preparations. The biological mechanism of action of neuromedin U remains unknown, despite the fact that the peptide was first isolated in 1985. Here we show that neuromedin U potently activates the orphan G protein-coupled receptor FM3, with subnanomolar potency, when FM3 is transiently expressed in human HEK-293 cells. Neuromedins B, C, K, and N are all inactive at this receptor. Quantitative reverse transcriptase-polymerase chain reaction analysis of neuromedin U expression in a range of human tissues showed that the peptide is highly expressed in the intestine, pituitary, and bone marrow, with lower levels of expression seen in stomach, adipose tissue, lymphocytes, spleen, and the cortex. Similar analysis of FM3 expression showed that the receptor is widely expressed in human tissue with highest levels seen in adipose tissue, intestine, spleen, and lymphocytes, suggesting that neuromedin U may have a wide range of presently undetermined physiological effects. The discovery that neuromedin U is an endogenous agonist for FM3 will significantly aid the study of the full physiological role of this peptide.