Stoichiometry of N-methyl-D-aspartate receptors within the suprachiasmatic nucleus.

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.

Functional analysis of the weaver mutant GIRK2 K+ channel and rescue of weaver granule cells.

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.

Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma 2L subunit.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Kir4.1 potassium channel subunit is crucial for oligodendrocyte development and in vivo myelination.

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.

GABAA receptor beta 1, beta 2, and beta 3 subunits: comparisons in DBA/2J and C57BL/6J mice.

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.

Intrinsic gating properties of a cloned G protein-activated inward rectifier K+ channel.

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.

Sodium/calcium exchanger in heart muscle: molecular biology, cellular function, and its special role in excitation-contraction coupling.

The Na/Ca exchanger has been examined with respect to its molecular biology, its cellular function, and its role in excitation-contraction coupling. The Na/Ca exchanger plays a central part in excitation-contraction coupling, setting the level of sarcoplasmic reticular calcium and contributing to the triggering of sarcoplasmic reticular calcium release. Functional biophysical studies with isolated single cells and caged calcium provide evidence that the Na/Ca exchanger works as a two step sequential transporter. In the heart there are about 250 exchangers.mu-2, operating at a turnover rate of up to about 2500.s-1, with the exchanger carrying -2.56 charges under normal conditions. The Na/Ca exchanger has been recently cloned from diverse mammalian species and several tissues and is largely conserved. It is clear, however, that the function of the Na/Ca exchanger is different in the different tissues. Thus work is in progress in several laboratories, including ours, to determine how the Na/Ca exchanger achieves its tissue specific function. Several modulatory motifs have been seen in studies of the exchanger that may explain some of the tissue specific differences. Interestingly the modulation of the Na/Ca exchanger (for example, by protons, sodium, calcium, ATP, calmodulin) seems to arise from interactions with the intracellular loop.

Functional expression of the human cardiac Na+/Ca2+ exchanger in Sf9 cells: rapid and specific Ni2+ transport.

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.

Potassium buffering in the central nervous system.

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.

The alpha 1, alpha 2, and alpha 3 subunits of GABAA receptors: comparison in seizure-prone and -resistant mice and during development.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, opens chloride channels through actions on GABAA receptors. We now report base and amino acid sequences of the alpha 1, alpha 2, and alpha 3 subunits from GABAA receptors of audiogenic seizure-prone (DBA/2J) and -resistant (C57BL/6J) inbred strains of mice. Inbreeding had fixed different alleles of the alpha 1 subunit in the two strains, giving five base differences in the cDNAs. None of these affected amino acid sequence, but one did create a NsiI restriction site potentially useful in mapping genomic DNA. No base or amino acid sequence differences between the strains were detected for the other two subunits. Northern blots revealed no apparent strain differences in message levels for these three subunits in whole brains of the mice at 3 weeks of age, the peak of seizure susceptibility in DBA/2J, but did reveal distinct regional and developmental patterns of expression among the subunits in mouse brain.

Alternative splicing of the Na(+)-Ca2+ exchanger gene, NCX1.

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.

Characterization of recombinant GABAA receptors produced in transfected cells from murine alpha 1, beta 1, and gamma 2 subunit cDNAs.

In order to explore the structural basis of GABAA receptor function, we have expressed murine alpha 1, beta 1, and gamma 2 subunit cDNAs by transient transfection of human 293 cells. Expression of GABAA receptors was measured by ligand binding assay and by electrophysiological analysis. As in other species, expression of the alpha 1 and beta 1 subunits produced a receptor that was insensitive to modulation by benzodiazepines as measured by electrophysiological analysis; however, a small number of flunitrazepam binding sites were detectable. The coexpression of the gamma 2 subunit was found to be essential for this modulation, and also resulted in a dramatic (14-fold) increase in the number of binding sites for flunitrazepam. On the coexpression of all 3 subunit cDNAs, a receptor was produced that exhibited a similar number of binding sites for flunitrazepam and muscimol.

Strain comparisons and developmental profile of the delta subunit of the murine GABAA receptor.

GABAA receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of gamma-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABAA receptor delta subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunit's postnatal developmental profile. We did not detect any unique features in the delta subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the delta subunit's first putative transmembrane region.

The GABA(A) receptor gamma1-subunit in seizure prone (DBA/2) and resistant (C57BL/6) mice.

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.

Evaluación toxicológica y de metales (Cu, Pb, Ni, Zn y Cd) en el sedimento del reservorio Paiva Castro en Mairiporã-SP, Brasil / Toxicological and metal evaluation (Cu, Pb, Ni, Zn and Cd) in the sediment of Paiva Castro reservoir, in Mairiporã-SP, Brasil

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.

Inwardly rectifying potassium channel Kir4.1 is responsible for the native inward potassium conductance of satellite glial cells in sensory ganglia.

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.

Na/Ca exchanger isoforms expressed in kidney.

The cardiac (versus retinal rod) Na/Ca exchanger gene has been cloned, sequenced and shown by RNA analysis to be present in diverse tissues. Analysis of published sequences shows that a single isoform is found in heart tissue from many species (NACA1 isoform). We provide evidence here by ribonuclease (RNase) protection assays and by reverse transcriptase-polymerase chain reaction (PCR) amplification with sequence analysis that a new isoform encoding the Na/Ca exchanger is present in renal tissue. This isoform (NACA3) reveals a 7-amino acid deletion in the tested region compared with the NACA2 isoform described by Reilly and Shugrue [Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F1105-F1109, 1992] and is the dominant exchanger transcript in kidney. Analysis of the sequence of all isoforms indicates that the differences in the isoforms reside in the large intracellular loop region of the protein. Alternative splicing of a single Na/Ca exchanger message may be responsible for these tissue-specific transcripts.

Mutually exclusive and cassette exons underlie alternatively spliced isoforms of the Na/Ca exchanger.

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.

Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by G beta gamma subunits and function as heteromultimers.

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.