RESUMO
The mammalian voltage-dependent KCNQ channels are responsible for distinct types of native potassium currents and are associated with several human diseases. We cloned a novel Drosophila KCNQ channel (dKCNQ) based on its sequence homology to the mammalian genes. When expressed in Chinese hamster ovary cells, dKCNQ gives rise to a slowly activating and slowly deactivating current that activates in the subthreshold voltage range. Like the M-current produced by mammalian KCNQ channels, dKCNQ current is sensitive to the KCNQ-specific blocker linopirdine and is suppressed by activation of a muscarinic receptor. dKCNQ is also similar to the mammalian channels in that it binds calmodulin (CaM), and CaM binding is necessary to produce functional currents. In situ hybridization analysis demonstrates that dKCNQ mRNA is present in brain cortical neurons, the cardia (proventriculus), and the nurse cells and oocytes of the ovary. We generated mutant flies with deletions in the genomic sequence of dKCNQ. Embryos produced by homozygous deletion females exhibit disorganized nuclei and fail to hatch, suggesting strongly that a maternal contribution of dKCNQ protein and/or mRNA is essential for early embryonic development.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Canais de Potássio KCNQ/fisiologia , Animais , Células CHO , Cricetinae , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Canais de Potássio KCNQ/antagonistas & inibidores , Canais de Potássio KCNQ/genética , Masculino , Camundongos , Bloqueadores dos Canais de Potássio/farmacologia , RNA Mensageiro/fisiologiaRESUMO
We have cloned and characterized mouse and human variants of MONaKA, a novel protein that interacts with and modulates the plasma membrane Na,K-ATPase. MONaKA was cloned based on its sequence homology to the Drosophila Slowpoke channel-binding protein dSlob, but mouse and human MONaKA do not bind to mammalian Slowpoke channels. At least two splice variants of MONaKA exist; the splicing is conserved perfectly between mouse and human, suggesting that it serves some important function. Both splice variants of MONaKA are expressed widely throughout the CNS and peripheral nervous system, with different splice variant expression ratios in neurons and glia. A yeast two-hybrid screen with MONaKA as bait revealed that it binds tightly to the beta1 and beta3 subunits of the Na,K-ATPase. The association between MONaKA and Na,K-ATPase beta subunits was confirmed further by coimmunoprecipitation from transfected cells, mouse brain, and cultured mouse astrocytes. A glutathione S-transferase-MONaKA fusion protein inhibits Na,K-ATPase activity from whole brain or cultured astrocytes. Furthermore, transfection of MONaKA inhibits 86Rb+ uptake via the Na,K-ATPase in intact cells. These results are consistent with the hypothesis that MONaKA modulates brain Na,K-ATPase and may thereby participate in the regulation of electrical excitability and synaptic transmission.
Assuntos
Membrana Celular/enzimologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Processamento Alternativo , Animais , Membrana Celular/genética , Células Cultivadas , Clonagem Molecular/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Vias Neurais/enzimologia , Vias Neurais/fisiologia , Proteínas Serina-Treonina Quinases , Ratos , ATPase Trocadora de Sódio-Potássio/genética , Sinapses/enzimologia , Sinapses/fisiologiaRESUMO
As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.