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1.
Acta toxicol. argent ; 26(1): 1-11, mayo 2018. ilus, tab
Article in Spanish | LILACS | ID: biblio-973611

ABSTRACT

El hombre ha cambiado el ambiente para sostener la demanda global de recursos naturales como el agua. La gestión de los cuerpos hídricos tiene que ser constante, con el propósito preventivo y correctivo, dependiendo del estado de antropización de cada sistema. El objetivo de este artículo fue analizar la toxicidad del sedimento y los metales Mn, Zn, Pb, Ni, Cd y Cu, en el reservorio Paiva Castro, que abastece la Region Metropolitana de São Paulo. Se realizaron 2 muestreos (Mayo 2011, estación seca y Enero de 2012, estación húmeda) y se analizaron 5 puntos próximos a la captación de agua por la Companhia de Saneamento Básico do Estado de São Paulo. Se realizaron ensayos de toxicidad aguda y crónica en sedimento, a través de ensayos biológicos con el cladócero Daphnia similis y el insecto Chironomus xanthus. El tratamiento de datos se realizó con el test de Fisher (mortalidad). El nivel de asociación entre las variables en sedimento y en los test ecotoxicológicos fueron evaluados por test no-paramétricos, a través del coeficiente de correlación de Spearman's. Los resultados del presente trabajo señalaron bajas concentracones de metales en el sedimento del área de estudio y ausencia de toxicidad en los organismos ensayados. Se puede concluir que área estudiada del reservorio Paiva Castro se encuentra poco impactada por los metales, sin efectos directos sobre la calidad de vida los organismos bentónicos: D. similis y C. xanthus.


Man had changed the natural environment in an attempt trying to supply the global demand for resources. The management of the hydric bodies has to be constant, with preventive and corrective purpose, depending on the eutrophization state of each one. The objective of this article was to analyze the sediment toxicity and the metals Mn, Zn, Pb, Ni, Cd and Cu, in the Paiva Castro reservoir, that supply the Metropolitan Region of São Paulo. It was made 2 collections (May 2011, dry season and January 2012, wet season). It was analyzed 5 points next to the water captation station by the Companhia de Saneamento Básico do Estado de São Paulo. The sediment was analyzed as for acute and chronic toxicity through bioassays with the cladocerans Daphnia similis and the insect Chironomus xanthus. Data treatment was done with Fisher Exact Test (mortality). The association level between the variables in sediment and ecotoxicological tests was available in non-parametric tests, through the Spearman's correlation coefficient. Oriented on the results presented in this work, pointing low concentrations of heavy metals in the sediments presented in the collect local, and the absence of toxicity, we can say that at this reservoir, at least in the collect area, it's low impacted, not implicating in direct interferences in the quality of life of benthonic organisms.


Subject(s)
Zinc/toxicity , Cadmium/toxicity , Water Reservoirs/prevention & control , Sediments/analysis , Copper/toxicity , Lead/toxicity , Manganese/toxicity , Nickel/toxicity , Brazil , Metals, Heavy/toxicity , Toxicity Tests, Acute/analysis , Toxicity Tests, Acute/methods , Toxicity Tests, Chronic/analysis , Toxicity Tests, Chronic/methods
2.
J Neurophysiol ; 103(6): 3448-64, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20410362

ABSTRACT

The circadian pacemaker within the suprachiasmatic nucleus (SCN) confers daily rhythms to bodily functions. In nature, the circadian clock will adopt a 24-h period by synchronizing to the solar light/dark cycle. This light entrainment process is mediated, in part, at glutamatergic synapses formed between retinal ganglion afferents and SCN neurons. N-methyl-D-aspartate receptors (NMDARs) located on SCN neurons gate light-induced phase resetting. Despite their importance in circadian physiology, little is known about their functional stoichiometry. We investigated the NR2-subunit composition with whole cell recordings of SCN neurons within the murine hypothalamic brain slice using a combination of subtype-selective NMDAR antagonists and voltage-clamp protocols. We found that extracellular magnesium ([Mg](o)) strongly blocks SCN NMDARs exhibiting affinities and voltage sensitivities associated with NR2A and NR2B subunits. These NMDAR currents were inhibited strongly by NR2B-selective antagonists, Ro 25-6981 (3.5 microM, 55.0 +/- 9.0% block; mean +/- SE) and ifenprodil (10 microM, 55.8 +/- 3.0% block). The current remaining showed decreased [Mg](o) affinities reminiscent of NR2C and NR2D subunits but was highly sensitive to [Zn](o), a potent NR2A blocker, showing a approximately 44.2 +/- 1.1% maximal inhibition at saturating concentrations with an IC(50) of 7.8 +/- 1.1 nM. Considering the selectivity, efficacy, and potency of the drugs used in combination with [Mg](o)-block characteristics of the NMDAR, our data show that both diheteromeric NR2B NMDARs and triheteromeric NR2A NMDARs (paired with an NR2C or NR2D subunits) account for the vast majority of the NMDAR current within the SCN.


Subject(s)
Neurons/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Suprachiasmatic Nucleus/cytology , Animals , Dose-Response Relationship, Drug , Drug Interactions , Excitatory Amino Acid Antagonists/pharmacology , In Vitro Techniques , Magnesium/pharmacology , Mice , Mice, Inbred C57BL , Neurons/drug effects , Neurons/physiology , Patch-Clamp Techniques/methods , Phenols/pharmacology , Piperidines/pharmacology , Protein Subunits/metabolism , Receptors, N-Methyl-D-Aspartate/chemistry , Suprachiasmatic Nucleus/metabolism
3.
Neuroscience ; 166(2): 397-407, 2010 Mar 17.
Article in English | MEDLINE | ID: mdl-20074622

ABSTRACT

Satellite glial cells (SGCs) surround primary afferent neurons in sensory ganglia, and increasing evidence has implicated the K(+) channels of SGCs in affecting or regulating sensory ganglion excitability. The inwardly rectifying K(+) (Kir) channel Kir4.1 is highly expressed in several types of glial cells in the central nervous system (CNS) where it has been implicated in extracellular K(+) concentration buffering. Upon neuronal activity, the extracellular K(+) concentration increases, and if not corrected, causes neuronal depolarization and uncontrolled changes in neuronal excitability. Recently, it has been demonstrated that knockdown of Kir4.1 expression in trigeminal ganglia leads to neuronal hyperexcitability in this ganglia and heightened nociception. Thus, we investigated the contribution of Kir4.1 to the membrane K(+) conductance of SGCs in neonatal and adult mouse trigeminal and dorsal root ganglia. Whole cell patch clamp recordings were performed in conjunction with immunocytochemistry and quantitative transcript analysis in various mouse lines. We found that in wild-type mice, the inward K(+) conductance of SGCs is blocked almost completely with extracellular barium, cesium and desipramine, consistent with a conductance mediated by Kir channels. We then utilized mouse lines in which genetic ablation led to partial or complete loss of Kir4.1 expression to assess the role of this channel subunit in SGCs. The inward K(+) currents of SGCs in Kir4.1+/- mice were decreased by about half while these currents were almost completely absent in Kir4.1-/- mice. These findings in combination with previous reports support the notion that Kir4.1 is the principal Kir channel type in SGCs. Therefore Kir4.1 emerges as a key regulator of SGC function and possibly neuronal excitability in sensory ganglia.


Subject(s)
Ganglia, Sensory/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Potassium/metabolism , Satellite Cells, Perineuronal/metabolism , Analysis of Variance , Animals , Image Processing, Computer-Assisted , Immunohistochemistry , Ion Channel Gating/physiology , Ion Transport/physiology , Mice , Mice, Transgenic , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying/genetics , Reverse Transcriptase Polymerase Chain Reaction
4.
Neuroscience ; 129(4): 1045-56, 2004.
Article in English | MEDLINE | ID: mdl-15561419

ABSTRACT

Rapid changes in extracellular K+ concentration ([K+](o)) in the mammalian CNS are counteracted by simple passive diffusion as well as by cellular mechanisms of K+ clearance. Buffering of [K+](o) can occur via glial or neuronal uptake of K+ ions through transporters or K+-selective channels. The best studied mechanism for [K+](o) buffering in the brain is called K+ spatial buffering, wherein the glial syncytium disperses local extracellular K+ increases by transferring K+ ions from sites of elevated [K+](o) to those with lower [K+](o). In recent years, K+ spatial buffering has been implicated or directly demonstrated by a variety of experimental approaches including electrophysiological and optical methods. A specialized form of spatial buffering named K+ siphoning takes place in the vertebrate retina, where glial Muller cells express inwardly rectifying K+ channels (Kir channels) positioned in the membrane domains near to the vitreous humor and blood vessels. This highly compartmentalized distribution of Kir channels in retinal glia directs K+ ions from the synaptic layers to the vitreous humor and blood vessels. Here, we review the principal mechanisms of [K+](o) buffering in the CNS and recent molecular studies on the structure and functions of glial Kir channels. We also discuss intriguing new data that suggest a close physical and functional relationship between Kir and water channels in glial cells.


Subject(s)
Cell Membrane/physiology , Central Nervous System/physiology , Potassium Channels/physiology , Potassium/metabolism , Water-Electrolyte Balance/physiology , Animals , Aquaporins/metabolism , Buffers , Humans , Neuroglia/cytology , Neuroglia/physiology , Retina/cytology , Retina/physiology
5.
J Neurosci ; 21(15): 5429-38, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11466414

ABSTRACT

To understand the cellular and in vivo functions of specific K(+) channels in glia, we have studied mice with a null mutation in the weakly inwardly rectifying K(+) channel subunit Kir4.1. Kir4.1-/- mice display marked motor impairment, and the cellular basis is hypomyelination in the spinal cord, accompanied by severe spongiform vacuolation, axonal swellings, and degeneration. Immunostaining in the spinal cord of wild-type mice up to postnatal day 18 reveals that Kir4.1 is expressed in myelin-synthesizing oligodendrocytes, but probably not in neurons or glial fibrillary acidic protein-positive (GFAP-positive) astrocytes. Cultured oligodendrocytes from developing spinal cord of Kir4.1-/- mice lack most of the wild-type K(+) conductance, have depolarized membrane potentials, and display immature morphology. By contrast, cultured neurons from spinal cord of Kir4.1-/- mice have normal physiological characteristics. We conclude that Kir4.1 forms the major K(+) conductance of oligodendrocytes and is therefore crucial for myelination. The Kir4.1 knock-out mouse is one of the few CNS dysmyelinating or demyelinating phenotypes that does not involve a gene directly involved in the structure, synthesis, degradation, or immune response to myelin. Therefore, this mouse shows how an ion channel mutation could contribute to the polygenic demyelinating diseases.


Subject(s)
Demyelinating Diseases/physiopathology , Myelin Sheath/metabolism , Oligodendroglia/metabolism , Potassium Channels, Inwardly Rectifying , Potassium Channels/metabolism , Protein Subunits , Spinal Cord Diseases/physiopathology , Animals , Apoptosis , Axons/pathology , Axons/ultrastructure , Cells, Cultured , Demyelinating Diseases/complications , Demyelinating Diseases/pathology , Disease Models, Animal , Gene Targeting , Membrane Potentials , Mice , Mice, Knockout , Oligodendroglia/cytology , Patch-Clamp Techniques , Phenotype , Potassium/metabolism , Potassium Channels/genetics , Spinal Cord/metabolism , Spinal Cord/pathology , Spinal Cord Diseases/complications , Spinal Cord Diseases/pathology , Survival Rate , Tremor/etiology , Vacuoles/pathology , Vacuoles/ultrastructure
6.
Proc Natl Acad Sci U S A ; 98(5): 2786-91, 2001 Feb 27.
Article in English | MEDLINE | ID: mdl-11226318

ABSTRACT

Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.


Subject(s)
Anxiety/genetics , Dopamine/metabolism , Point Mutation , Receptors, Nicotinic/metabolism , Animals , Female , Heterozygote , Immunohistochemistry , Mice , Mice, Mutant Strains , Pregnancy , Rats , Receptors, Nicotinic/genetics
7.
J Neurosci ; 20(15): 5733-40, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10908613

ABSTRACT

The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K(+) conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K(+) in the retina. To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of the Kir4.1 gene (Kir4.1 -/-). Müller cells from Kir4.1 -/- mice were not labeled with an anti-Kir4.1 antibody, although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody. In contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes. In situ whole-cell patch-clamp recordings showed a 10-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells compared with Kir4.1 +/+ cells. The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K(+) fluxes through Müller cells, was totally absent in retinas from Kir4.1 -/- mice. The b-wave of the ERG, in contrast, was spared in the null mice. Overall, these results indicate that Kir4.1 is the principal K(+) channel subunit expressed in mouse Müller glial cells. The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K(+) in the mouse retina.


Subject(s)
Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Potassium Channels/metabolism , Retina/chemistry , Retina/physiology , Amino Acid Sequence , Animals , Antibodies , Astrocytes/chemistry , Astrocytes/physiology , Electric Impedance , Electroretinography , Gene Expression/physiology , Ion Channel Gating/genetics , Membrane Potentials/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Patch-Clamp Techniques , Phenotype , Potassium Channels/immunology , RNA, Messenger/analysis , Rabbits , Retina/cytology
8.
Cell Calcium ; 25(1): 9-17, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10191956

ABSTRACT

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.


Subject(s)
Nickel/metabolism , Sodium-Calcium Exchanger/biosynthesis , Animals , Biological Transport , Calcium/metabolism , Cell Line , Cells, Cultured , Humans , Myocardium/metabolism , Rats , Rats, Wistar , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Sodium-Calcium Exchanger/genetics , Spodoptera , Time Factors
9.
Brain Res Bull ; 45(4): 421-5, 1998.
Article in English | MEDLINE | ID: mdl-9527017

ABSTRACT

Gamma-aminobutyric acid (GABA)A receptors are the sites of action for many antiepileptic drugs such as benzodiazepines and barbiturates. We report the results of molecular cloning of the gamma1-subunit from seizure prone DBA/2J and resistant C57BL/6J inbred mice, and analyses of nucleotide sequences and expression of the gamma1-subunit messenger RNA (mRNA) in DBA/2 and C57BL/6 inbred mice. The mouse gamma1-subunit complementary DNA (cDNA) shares 98% similarity with that of the rat at the level of amino acid sequence. Northern blot hybridization indicates that the gamma1-subunit mRNA is expressed predominantly in areas other than the cerebral cortex and cerebellum and shows little change with postnatal development. No differences have been found for the subunit between DBA/2 and C57BL/6 mice either for nucleotide sequence or for level of expression of the subunit's mRNA in whole brain by Northern blots at 3 weeks of age.


Subject(s)
Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Seizures/genetics , Seizures/metabolism , Acoustic Stimulation , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Time Factors
10.
Proc Natl Acad Sci U S A ; 94(19): 10461-6, 1997 Sep 16.
Article in English | MEDLINE | ID: mdl-9294233

ABSTRACT

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of "regulators of G protein signaling" (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent "basal" GIRK current was suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of "slow" postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.


Subject(s)
GTP-Binding Proteins/metabolism , Ion Channel Gating , Potassium Channels/physiology , Proteins/physiology , RGS Proteins , Acetylcholine/administration & dosage , Acetylcholine/pharmacology , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Kinetics , Potassium Channels/metabolism , Rats , Synaptic Transmission/physiology , Xenopus laevis
11.
Am J Physiol ; 273(1 Pt 1): C257-65, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9252464

ABSTRACT

cDNAs for the Na+/Ca2+ exchanger from Drosophila melanogaster (Dmel/Nck) have been cloned by homology screening using the human heart Na+/Ca2+ exchanger cDNA. The overall deduced protein structure for Dmel/Nck is similar to that of mammalian Na+/Ca2+ exchanger genes NCX1 and NCX2, having six hydrophobic regions in the amino terminus separated from six at the carboxy-terminal end by a large intracellular loop. Sequence comparison of the Drosophila exchanger cDNAs with NCX1 and NCX2 Na+/Ca2+ exchangers are approximately 46% identical at the deduced amino acid level. Consensus phosphorylation sites for both protein kinase C and protein kinase A are present on the intracellular loop region of the Dmel/Nck. Alternative splicing for the Dmel/Nck gene is suggested in the same intracellular loop region as demonstrated for NCX1. Functionally, the Drosophila Na+/ Ca2+ exchanger expressed in oocytes differs from expressed mammalian NCX1 with regard to Ca2+ transport in Ca2+/ Ca2+ exchange and the effect of monovalent-dependent Ca2+/ Ca2+ exchange. The Dmel/Nck gene maps to chromosome 3 (93A-B) using in situ hybridization to polytene chromosomes, the same position as the Na(+)-K(+)-ATPase, a related transporter. We conclude that, although extracellular Na+ concentration-dependent Ca2+ transport is subserved by both human and Drosophila Na+/Ca2+ exchangers, there are clear and important differences in the transporters, which should be useful in deducing how the Na+/Ca2+ exchanger protein function depends on its structure.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Chromosome Mapping , DNA, Complementary/chemistry , Drosophila/metabolism , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/chemistry , Cell Membrane/physiology , Cloning, Molecular , Consensus Sequence , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA, Complementary/metabolism , Drosophila/genetics , Genes, Insect , Genomic Library , Humans , In Situ Hybridization , Mammals , Molecular Sequence Data , Oocytes/physiology , Phosphorylation , Protein Kinase C/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Sodium-Calcium Exchanger , Xenopus
12.
Proc Natl Acad Sci U S A ; 93(26): 15429-34, 1996 Dec 24.
Article in English | MEDLINE | ID: mdl-8986828

ABSTRACT

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K+ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by G beta gamma subunits, but instead shows constitutive activation, and (ii) is no longer a K(+)-selective channel but conducts Na+ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na+ concentration. In particular, manipulations that decrease intracellular Na+ produce a component of Na(+)-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na+ influx through the wvGIRK2 channel, leading to additional Na+ activation. We also show that the wvGIRK2 channel is permeable to Ca2+, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K+ channels.


Subject(s)
Point Mutation , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Potassium Channels/physiology , Acetylcholine/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Female , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Glucose/pharmacology , Meglumine/pharmacology , Membrane Potentials/drug effects , Mice , Mice, Neurologic Mutants , Oligonucleotides, Antisense/pharmacology , Oocytes/drug effects , Oocytes/physiology , Ouabain/pharmacology , Patch-Clamp Techniques , Polymerase Chain Reaction , Potassium Channels/biosynthesis , Sodium/pharmacology , Thionucleotides , Xenopus laevis
13.
Neuron ; 16(5): 941-52, 1996 May.
Article in English | MEDLINE | ID: mdl-8630252

ABSTRACT

In the neurological mutant mouse weaver, granule cell precursors proliferate normally in the external germinal layer of the cerebellar cortex, but fail to differentiate. Granule neurons purified from weaver cerebella have greatly reduced G protein-activated inwardly rectifying K+ currents; instead, they display a constitutive Na+ conductance. Expression of the weaver GIRK2 channel in oocytes confirms that the mutation leads to constitutive activation, loss of monovalent cation selectivity, and increased sensitivity to three channel blockers. Pharmacological blockade of the Na+ influx in weaver granule cells restores their ability to differentiate normally. Thus, Na+ flux through the weaver GIRK2 channel underlies the failure of granule cell development in situ.


Subject(s)
Cerebellar Cortex/physiology , Potassium Channels, Inwardly Rectifying , Potassium Channels/genetics , Potassium Channels/physiology , Animals , Base Sequence , Cell Differentiation , DNA Primers , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Gene Expression Regulation, Developmental , Guanosine Triphosphate/physiology , In Situ Hybridization , Mice , Mice, Neurologic Mutants , Molecular Sequence Data , Oocytes , Point Mutation , Potassium/physiology , Receptors, Muscarinic/physiology , Signal Transduction , Sodium/physiology , Transfection , Xenopus laevis
14.
Ann N Y Acad Sci ; 779: 46-57, 1996 Apr 15.
Article in English | MEDLINE | ID: mdl-8659862

ABSTRACT

We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.


Subject(s)
Alternative Splicing , Carrier Proteins/genetics , Amino Acid Sequence , Animals , Calcium/metabolism , Humans , Molecular Sequence Data , Sequence Homology , Sodium/metabolism , Sodium-Calcium Exchanger , Transfection
16.
J Physiol ; 490 ( Pt 3): 633-45, 1996 Feb 01.
Article in English | MEDLINE | ID: mdl-8683463

ABSTRACT

1. The structural determinants of a G protein-activated inwardly rectifying potassium channel, GIRK1 (KIR3.1), involved in voltage- and time-dependent gating properties were investigated by heterologous expression of chimeric constructs and point mutants in Xenopus oocytes. 2. Chimeras between GIRK1 and the weakly rectifying potassium channel, ROMK1 (KIR1.1), indicate that residues in the putative transmembrane segments TM1 and TM2 affect the steep inward rectification of GIRK1, while residues in the main pore-forming domain, the P-region segment, are critical for the manifestation of GIRK1 time-dependent activation. 3. Phenylalanine 137 in the P-region of GIRK1 is unique; in ROMK1, as in other inward rectifiers, there is a serine residue at this position. Mutation of the phenylalanine 137 to serine leads to expression of currents with nearly time-independent activation. 4. An acidic residue (aspartate) in TM2 partially controls the time- and voltage-dependent gating in IRK1 (KIR2.1). Mutation of the equivalent aspartate 173 to glutamine in GIRK1 did not abolish the time-dependent activation but did decrease the degree of inward rectification. 5. These results reveal an important role for the P-region in controlling the time-dependent gating of an inwardly rectifying potassium channel and suggest a close relationship between permeation and gating in this family of K+ channels.


Subject(s)
Chimera/physiology , GTP-Binding Proteins/physiology , Gene Expression/genetics , Potassium Channels/genetics , Animals , Cloning, Molecular , Molecular Structure , Oocytes , Patch-Clamp Techniques , Time Factors , Xenopus
17.
Proc Natl Acad Sci U S A ; 92(14): 6542-6, 1995 Jul 03.
Article in English | MEDLINE | ID: mdl-7604029

ABSTRACT

Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits G beta 1 and G gamma 2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by G beta gamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels.


Subject(s)
Brain/physiology , GTP-Binding Proteins/physiology , Neurons/physiology , Potassium Channels, Inwardly Rectifying , Potassium Channels/physiology , Acetylcholine/pharmacology , Animals , Brain/metabolism , Cloning, Molecular , Evoked Potentials , Female , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Heart/physiology , Macromolecular Substances , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Myocardium/metabolism , Oocytes/physiology , Patch-Clamp Techniques , Potassium/pharmacology , Potassium Channels/biosynthesis , RNA, Complementary/metabolism , Receptors, Muscarinic/physiology , Xenopus
18.
J Gen Physiol ; 106(1): 1-23, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7494135

ABSTRACT

The voltage-, time-, and K(+)-dependent properties of a G protein-activated inwardly rectifying K+ channel (GIRK1/KGA/Kir3.1) cloned from rat atrium were studied in Xenopus oocytes under two-electrode voltage clamp. During maintained G protein activation and in the presence of high external K+ (VK = 0 mV), voltage jumps from VK to negative membrane potentials activated inward GIRK1 K+ currents with three distinct time-resolved current components. GIRK1 current activation consisted of an instantaneous component that was followed by two components with time constants tau f approximately 50 ms and tau s approximately 400 ms. These activation time constants were weakly voltage dependent, increasing approximately twofold with maximal hyperpolarization from VK. Voltage-dependent GIRK1 availability, revealed by tail currents at -80 mV after long prepulses, was greatest at potentials negative to VK and declined to a plateau of approximately half the maximal level at positive voltages. Voltage-dependent GIRK1 availability shifted with VK and was half maximal at VK -20 mV; the equivalent gating charge was approximately 1.6 e-. The voltage-dependent gating parameters of GIRK1 did not significantly differ for G protein activation by three heterologously expressed signaling pathways: m2 muscarinic receptors, serotonin 1A receptors, or G protein beta 1 gamma 2 subunits. Voltage dependence was also unaffected by agonist concentration. These results indicate that the voltage-dependent gating properties of GIRK1 are not due to extrinsic factors such as agonist-receptor interactions and G protein-channel coupling, but instead are analogous to the intrinsic gating behaviors of other inwardly rectifying K+ channels.


Subject(s)
GTP-Binding Proteins/physiology , Ion Channel Gating/physiology , Potassium Channels, Inwardly Rectifying , Potassium Channels/physiology , Animals , Cell Membrane/chemistry , Cell Membrane/physiology , Cell Membrane/ultrastructure , Electrophysiology , Female , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Membrane Potentials/physiology , Oocytes/cytology , Oocytes/physiology , Oocytes/ultrastructure , Receptors, Muscarinic/analysis , Receptors, Muscarinic/physiology , Receptors, Serotonin/analysis , Receptors, Serotonin/physiology , Time Factors , Xenopus laevis
19.
Biochim Biophys Acta ; 1261(1): 134-42, 1995 Mar 14.
Article in English | MEDLINE | ID: mdl-7893750

ABSTRACT

GABAA receptors link binding of GABA (gamma-aminobutyric acid) to inhibitory chloride flux in the brain. They are the site of action of several important classes of drugs, and have been implicated in animal models of epilepsy and in the actions of alcohol. We compare the sequence and expression of the beta 1, beta 2 and beta 3 subunits of GABAA receptors in two inbred strains of mice, DBA/2J and C57BL/6J, which differ markedly in seizure susceptibility and in a variety of behaviors related to alcohol. Only the beta 3 subunit had strain differences in cDNA nucleotide sequence, which did not affect amino acid sequence but which did create restriction fragment length polymorphisms (RFLPs) potentially useful in gene mapping. We have also tested mouse beta 1 and beta 2 subunits for internal alternative splicing, detecting none.


Subject(s)
Mice, Inbred C57BL/genetics , Mice, Inbred DBA/genetics , Receptors, GABA-A/genetics , Alcohol Drinking/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Mutational Analysis , DNA, Complementary/genetics , Gene Library , Genes , Genetic Predisposition to Disease , Mice , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , Receptors, GABA-A/chemistry , Seizures/genetics , Species Specificity
20.
J Biol Chem ; 269(7): 5145-9, 1994 Feb 18.
Article in English | MEDLINE | ID: mdl-8106495

ABSTRACT

We have analyzed the gene structure that gives rise to tissue-specific isoforms of the Na/Ca exchanger. Five distinct isoforms of the Na/Ca exchanger from rabbit brain, kidney, and heart have been identified previously to which we now add a new brain isoform. Reverse-transcribed polymerase chain reaction, library screening, and sequence analysis of cDNA coding regions indicate that the only significant alteration of the Na/Ca exchanger cDNA in rabbit brain, kidney, and heart isoforms is located in the carboxyl end of the putative intracellular loop of the protein, a region recently linked to ionic and metabolic regulation of the Na/Ca exchanger. Additionally, we find that the Na/Ca exchanger isoforms found in lung and skeletal muscle may arise from among these same six isoforms. Examination of the gene structure of the Na/Ca exchanger in rabbit indicates how the single gene that encodes for the Na/Ca exchanger is alternatively spliced to give rise to the five rabbit isoforms. Specifically, sequence analysis of the intron-exon boundaries reveals the presence of two "mutually exclusive" exons in conjunction with four "cassette" exons in the region of the Na/Ca exchanger gene that codes for the carboxyl end of the predicted intracellular loop region. This unusual arrangement of exons in the Na/Ca exchanger gene could allow for the generation of up to 32 different Na/Ca exchanger mRNAs and accounts for the isoforms identified to date.


Subject(s)
Alternative Splicing , Brain/metabolism , Carrier Proteins/biosynthesis , Exons , RNA, Messenger/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Calcium/metabolism , Carrier Proteins/genetics , DNA/chemistry , DNA/metabolism , DNA Primers , DNA, Complementary/chemistry , DNA, Complementary/metabolism , Genetic Variation , Genomic Library , Introns , Kidney/metabolism , Molecular Sequence Data , Myocardium/metabolism , Organ Specificity , Polymerase Chain Reaction , Protein Biosynthesis , Rabbits , Sequence Homology, Amino Acid , Sodium/metabolism , Sodium-Calcium Exchanger
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