RESUMO
Xenopus laevis oocytes have become a pre-eminent tool for studying cloned ion channels, primarily because they intrinsically express low levels of most types of ion channels. However, when these cells are used for single channel studies, it is essential to determine whether or not oocytes contain even low levels of endogenous ion channels with properties similar to the channel being investigated. We show here that X. laevis oocytes express endogenous large-conductance Ca2(+)-activated K+ channels with properties similar to mammalian isoforms of this channel. The endogenous channels exhibit a voltage-dependence of 12-14 mV per e-fold change in open probability (po), can be activated by micromolar Ca2+ concentrations, and have a single channel conductance of approximately 200 pS in symmetrical 110 mM K+ solutions. Patch clamp experiments indicate that this endogenous channel is present at low densities (approximately 1 channel/3000 microns2). If endogenous channel subunits can form functional tetramers with other exogenous potassium channel subunits, then they will give rise to the expression of a heterogeneous channel population. Therefore, studies involving the heterologous expression of large-conductance Ca2(+)-activated K+ channels in Xenopus laevis oocytes require careful analysis and interpretation.
Assuntos
Canais de Potássio Cálcio-Ativados , Canais de Potássio/análise , Animais , Canais de Potássio Ativados por Cálcio de Condutância Alta , Oócitos/química , Técnicas de Patch-Clamp , Xenopus laevisRESUMO
We have cloned and expressed nine Ca(2+)-activated K+ channel isoforms from human brain. The open reading frames encode proteins ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse. All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 (hslo). RNA splicing occurs at four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1-hbr9). hslo mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in Xenopus oocytes produces robust voltage and Ca(2+)-activated K+ currents. Splice variants differ significantly in their Ca2+ sensitivity, suggesting a broad functional role for these channels in the regulation of neuronal excitability.
Assuntos
Canais de Potássio/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Cromossomos Humanos Par 10 , Clonagem Molecular , Primers do DNA/química , Expressão Gênica , Genes , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Dados de Sequência Molecular , Oócitos , Canais de Potássio/classificação , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Xenopus laevisRESUMO
Two different developmental patterns of stimulation of phosphoinositide (PI) metabolism by excitatory amino acid (EAA) receptors were observed during the postnatal maturation of various brain regions. A 'burst' in PI metabolism was seen at postnatal day 6 (PND6) in olfactory bulb and cerebellum and at PND9 in hippocampus. In cortex and thalamus/hypothalamus high levels of PI metabolism were observed initially, and then began to decline at PND15 and PND18, respectively. NMDA inhibition of PI metabolism was generally found to parallel the EAA activation but the persistence of inhibition varied in the different brain regions.
