Neuronal activity regulates phosphorylation-dependent surface delivery of G protein-activated inwardly rectifying potassium channels.
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ABSTRACT:
SUBMITTER: Chung HJ
PROVIDER: S-EPMC2613039 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundInwardly rectifying potassium channels are essential for normal potassium homeostasis, maintaining the cellular resting membrane potential, and regulating electrolyte transportation. Mutations in Kir channels have been known to cause debilitating diseases ranging from neurological abnormalities to renal and cardiac failures. Many efforts have been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and functions during embryonic development remain largely unknown.ResultsUsing zebrafish as a model, we identified and renamed 31 kir genes. We also analyzed Kir gene evolution by phylogenetic and syntenic analyses. Our data indicated that the four subtypes of the Kir genes might have already evolved out in chordates. These vertebrate Kir genes most likely resulted from both whole-genome duplications and tandem duplications. In addition, we examined zebrafish kir gene expression during early embryogenesis. Each subgroup's genes showed similar but distinct gene expression domains. The gene expression of ohnologous genes from teleost-specific whole-genome duplication indicated subfunctionalization. Varied temporal gene expression domains suggest that Kir channels may be needed for embryonic patterning or regulation.ConclusionsOur phylogenetic and developmental analyses of Kir channels shed light on their evolutionary history and potential functions during embryogenesis related to congenital diseases and human channelopathies.
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Project description:Activation of G protein-gated inwardly rectifying potassium (GIRK) channels leads to a hyperpolarization of the neuron's membrane potential, providing an important component of inhibition in the brain. In addition to the canonical G protein-activation pathway, GIRK channels are activated by small molecules but less is known about the underlying gating mechanisms. One drawback to previous studies has been the inability to control intrinsic and extrinsic factors. Here we used a reconstitution strategy with highly purified mammalian GIRK2 channels incorporated into liposomes and demonstrate that cholesterol or intoxicating concentrations of ethanol, i.e., >20?mM, each activate GIRK2 channels directly, in the absence of G proteins. Notably, both activators require the membrane phospholipid PIP2 but appear to interact independently with different regions of the channel. Elucidating the mechanisms underlying G protein-independent pathways of activating GIRK channels provides a unique strategy for developing new types of neuronal excitability modulators.
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Project description:BACKGROUND: Previous data from our laboratory has indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. METHODS: GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. RESULTS: GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with beta2-adrenergic antagonist ICI 118,551 (100 microM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the beta-adrenergic agonist isoproterenol (10 microM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 microM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4) mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4) mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells. CONCLUSION: We feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and beta-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results.
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