RESUMO
We have cloned a novel brain-specific inward rectifier K+ channel from a mouse brain cDNA library and designated it MB-IRK3. The mouse brain cDNA library was screened using a fragment of the mouse macrophage inward rectifier K+ channel (IRK1) cDNA as a probe. The amino acid sequence of MB-IRK3 shares 61% and 64% identity to MB-IRK1 and RB-IRK2, respectively. Xenopus oocytes injected with cRNA derived from this clone expressed a potassium current which showed inward-rectifying channel characteristics similar to MB-IRK1 and RB-IRK2 currents, but distinct from ROMK1 or GIRK1 current. However, the single channel conductance of MB-IRK3 was approximately 10 pS with 140 mM extracellular K+, which was distinct from that of MB-IRK1 (20 pS). MB-IRK3 mRNA expressed specifically in the forebrain, which clearly differed from MB-IRK1 and RB-IRK2 mRNAs. These results indicate that members of the IRK family with distinct electrophysiological properties express differentially and may play heterogenous functional roles in brain functions.
Assuntos
Química Encefálica , Clonagem Molecular , Expressão Gênica , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Bário/farmacologia , Sequência de Bases , Condutividade Elétrica , Eletrofisiologia , Feminino , Técnicas de Transferência de Genes , Potenciais da Membrana , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Canais de Potássio/química , Canais de Potássio/fisiologia , RNA Mensageiro/análise , Distribuição Tecidual , XenopusRESUMO
We have cloned a novel inward rectifier potassium channel from a rat brain cDNA library and designated it RB-IRK2. The rat brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. The amino acid sequence of RB-IRK2 shares 70%, 40% and 45% identity to mouse IRK1, rat ROMK1 and rat GIRK1, respectively. Xenopus oocytes injected with cRNA derived from RB-IRK2 expressed a potassium current which showed inward-rectifying channel characteristics similar to the IRK1 current, but distinct from the ROMK1 or the GIRK1 currents. However, the localization of RB-IRK2 mRNA in rat tissues, assessed by the Northern blot analysis, differed from that of mouse IRK1. These results indicate that the IRK family is composed of multiple genes, which express in different tissues and therefore may play heterogenous functional roles in various organs, including rat central nervous system.
Assuntos
Encéfalo/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Potenciais da Membrana , Camundongos , Dados de Sequência Molecular , Canais de Potássio/genética , Canais de Potássio/fisiologia , RNA Mensageiro/metabolismo , Ratos , XenopusRESUMO
We have cloned an inward-rectifier potassium channel from a mouse brain cDNA library, studied its distribution in the brain by in situ hybridization and determined the chromosomal localization of the gene. A mouse brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. Two duplicate clones of approximately 5.5 kb were obtained. Xenopus ococytes injected with cRNA derived from the clone expressed a potassium channel with inwardly rectifying channel characteristics. The amino acid sequence of the clone was identical to that of IRK1 recently cloned from a mouse macrophage cell line. In situ hybridization study showed the mouse brain IRK1 to be generally distributed throughout the brain, but in particular subsets of neurons at high levels. The gene was placed in the distal region of mouse chromosome 11, which contains several uncloned neurological mutations. These results provide the first demonstration of the cloning and distribution of an inward rectifier potassium channel from the nervous system.
