Ethanol directly depresses AMPA and NMDA glutamate currents in spinal cord motor neurons independent of actions on GABAA or glycine receptors.
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Ethanol is a general anesthetic agent as defined by abolition of movement in response to noxious stimulation. This anesthetic endpoint is due to spinal anesthetic actions. This study was designed to test the hypothesis that ethanol acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors. Whole cell recordings were made in visually identified motor neurons in spinal cord slices from 14- to 23-day-old rats. Currents were evoked by stimulating a dorsal root fragment or by brief pulses of glutamate. Ethanol at general anesthetic concentrations (50-200 mM) depressed both responses. Ethanol also depressed glutamate-evoked responses in the presence of tetrodotoxin (300 nM), showing that its actions are postsynaptic. Block of inhibitory gamma-aminobutyric acidA and glycine receptors by bicuculline (50 microM) and strychnine (5 microM), respectively, did not significantly reduce the effects of ethanol on glutamate currents. Ethanol also depressed glutamate-evoked currents when the inhibitory receptors were blocked and either D, L-2-amino-5-phosphonopentanoic acid (40 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 microM) were applied to block N-methyl-D-aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, respectively. The results show that ethanol exerts direct depressant effects on both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate glutamate currents in motor neurons. Enhancement of gamma-aminobutyric acidA and glycine inhibition is not required for this effect. Direct depression of glutamatergic excitatory transmission by a postsynaptic action on motor neurons thus may contribute to general anesthesia as defined by immobility in response to a noxious stimulus. (+info)
Genes encoding three new members of the leukocyte antigen 6 superfamily and a novel member of Ig superfamily, together with genes encoding the regulatory nuclear chloride ion channel protein (hRNCC) and an N omega-N omega-dimethylarginine dimethylaminohydrolase homologue, are found in a 30-kb segment of the MHC class III region.
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Many of the genes in the class III region of the human MHC encode proteins involved in the immune and inflammatory responses. We have sequenced a 30-kb segment of the MHC class III region lying between the heat shock protein 70 and TNF genes as part of a program aimed at identifying genes that could be involved in autoimmune disease susceptibility. The sequence analysis has revealed the localization of seven genes, whose precise position and order is cen-G7-G6-G6A-G6B-G6C-G6D-G6E-tel, five of which are fully encoded in the sequence, allowing their genomic structures to be defined. Three of them (G6C, G6D, and G6E) encode putative proteins that belong to the Ly-6 superfamily, known to be GPI-anchored proteins attached to the cell surface. Members of the family are specifically expressed and are important in leukocyte maturation. A fourth gene, G6B, encodes a novel member of the Ig superfamily containing a single Ig V-like domain and a cytoplasmic tail with several signal transduction features. The G6 gene encodes a regulatory nuclear chloride ion channel protein, while the G6A gene encodes a putative homologue of the enzyme N omega,N omega-dimethylarginine dimethylaminohydrolase, which is thought to be involved in regulating nitric oxide synthesis. In addition, three microsatellite markers, 9N-1, 82-2, and D6S273 are contained within the sequence, the last two of which have been reported to be strongly associated with the autoimmune disease ankylosing spondylitis. (+info)
Structural and ionic determinants of 5-nitro-2-(3-phenylprophyl-amino)-benzoic acid block of the CFTR chloride channel.
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1. The goals of this study were to identify the structural components required for arylaminobenzoate block of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and to determine the involvement of two positively charged amino acid residues, found within the channel, in drug binding. 2. Wild-type and mutant CFTR chloride channels were expressed in Xenopus oocytes and CFTR currents measured using the two microelectrode voltage clamp. Block of the wild-type CFTR current by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) occurred in a voltage-dependent manner with preferential inhibition of the inward currents (Kd = 166 microM at -90 mV). 3. Removal of the phenyl ring from the aliphatic chain of NPPB, with the compound 2-butylamino-5-nitrobenzoic acid, caused only a small change in CFTR inhibition (Kd = 243 microM), while addition of an extra phenyl ring at this position (5-nitro-2-(3,3-diphenylpropylamino)-benzoic acid) increased drug potency (Kd = 58 microM). In contrast, removal of the benzoate ring (2-amino-4-phenylbutyric acid) or the 5-nitro group (2-(3-phenylpropylamino)-benzoic acid) of NPPB severely limited drug block of the wild-type channel. 4. NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner. However, the Kds for NPPB block were increased to 371 and 1573 microM, for the K335E and R347E mutants, respectively. 5. NPPB block of the inward wild-type CFTR current was reduced in the presence of 10 mM of the permeant anion SCN-. 6. These studies present the first step in the development of high affinity probes to the CFTR channel. (+info)
Ion channels in presynaptic nerve terminals and control of transmitter release.
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The primary function of the presynaptic nerve terminal is to release transmitter quanta and thus activate the postsynaptic target cell. In almost every step leading to the release of transmitter quanta, there is a substantial involvement of ion channels. In this review, the multitude of ion channels in the presynaptic terminal are surveyed. There are at least 12 different major categories of ion channels representing several tens of different ion channel types; the number of different ion channel molecules at presynaptic nerve terminals is many hundreds. We describe the different ion channel molecules at the surface membrane and inside the nerve terminal in the context of their possible role in the process of transmitter release. Frequently, a number of different ion channel molecules, with the same basic function, are present at the same nerve terminal. This is especially evident in the cases of calcium channels and potassium channels. This abundance of ion channels allows for a physiological and pharmacological fine tuning of the process of transmitter release and thus of synaptic transmission. (+info)
Endocrine control of Na+,K+-ATPase and chloride cell development in brown trout (Salmo trutta): interaction of insulin-like growth factor-I with prolactin and growth hormone.
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A 2-factorial (3x3) injection experiment was used to investigate the effect and interaction between different hormones on the initial phase of seawater (SW) acclimation in brown trout (Salmo trutta). Each fish was given 4 injections on alternate days in freshwater (FW). Factor 1 was either saline, 2 micrograms ovine prolactin (oPRL)/g, or 2 micrograms ovine growth hormone (oGH)/g. Factor 2 was either 0, 0. 01, or 0.1 mirograms recombinant human insulin-like growth factor-I (rhIGF-I)/g. In each of the 9 treatment groups, half of the fish were subjected to a 48-h SW-challenge test, and the remaining fish were sham-transferred to FW one day after the last injection. Hypo-osmoregulatory performance was increased by GH and impaired by PRL treatment as judged by changes in plasma osmolality, [Na+], [Cl-], total [Mg] and muscle water content (MWC) after SW transfer. IGF-I reduced plasma osmolality after transfer to SW but had no effect on plasma total [Mg] or MWC. The effects of the two factors on plasma osmolality, [Na+], [Cl-], and MWC were additive. In sham-transferred fish, GH and IGF-I, alone and in combination, stimulated Na+,K+-ATPase alpha-subunit mRNA (alpha-mRNA) content in the gill. This was paralleled by an overall increase in gill Na+, K+-ATPase activity in fish treated with 0.01 micrograms IGF-I/g. Simultaneous administration of PRL completely inhibited the increase in gill alpha-mRNA observed in the IGF-I-injected groups. Combination of GH and IGF-I did not further affect the alpha-mRNA level relative to the single hormone-injected groups. There was an overall decrease in Na+,K+-ATPase activity in pyloric caeca and middle intestine by the low dose and both doses of IGF-I respectively. No effect was observed in the posterior intestine. PRL and GH treatments did not affect enzyme activity in any intestinal segment. Both doses of IGF-I increased Na+,K+-ATPase-immunoreactive (NKIR) cell density in gill primary filaments. PRL and GH had no effect on primary filament NKIR cell density. GH and both doses of IGF-I reduced secondary lamellar NKIR cell density, whereas PRL had no effect. The main conclusion is that IGF-I and GH induce an overall redistribution of NKIR cells away from the secondary lamella onto the primary filament of FWacclimated trout. This is associated with an overall increased alpha-mRNA level in the gill, which may reflect an increased expression within individual NKIR cells in the primary filament. PRL completely abolished the IGF-I stimulation of alpha-mRNA levels, suggesting a desensitisation of the gill tissue to IGF-I, which may explain the overall anti-SW adaptive effect of PRL. (+info)
Elimination of the slow gating of ClC-0 chloride channel by a point mutation.
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The inactivation of the ClC-0 chloride channel is very temperature sensitive and is greatly facilitated by the binding of a zinc ion (Zn2+) from the extracellular side, leading to a Zn2+-induced current inhibition. To further explore the relation of Zn2+ inhibition and the ClC-0 inactivation, we mutated all 12 cysteine amino acids in the channel and assayed the effect of Zn2+ on these mutants. With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition. Upon mutating C212 to serine or alanine, the inactivation of the channel in macroscopic current recordings disappears and the channel does not show detectable inactivation events at the single-channel level. At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced. The other two cysteine mutants, C213G and C480S, as well as a previously identified mutant, S123T, also affect the inactivation of the channel to some degree, but the temperature-dependent inactivation process is still present, likewise the high sensitivity of the Zn2+ inhibition. These results further support the assertion that the inhibition of Zn2+ on ClC-0 is indeed due to an effect on the inactivation of the channel. The absence of inactivation in C212S mutants may provide a better defined system to study the fast gating and the ion permeation of ClC-0. (+info)
GABA-Induced Cl- current in cultured embryonic human dorsal root ganglion neurons.
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gamma-Aminobutyric acid (GABA)-activated channels in embryonic (5-8 wk old) human dorsal root ganglion (DRG) neurons in dissociated culture were characterized by whole cell and single-channel techniques. All DRG neurons when held at negative holding membrane potentials displayed inward current to micromolar concentrations of GABA applied by pressure pulses from closely positioned micropipettes. The current was directly proportional to the concentration of GABA (EC50, 111 microM; Hill coefficient, 1.7). DRG neurons also responded to micromolar concentrations of pentobarbital and alphaxalone but not to cis-4-aminocrotonic acid (CACA), glycine, or taurine. Baclofen (100 microM) affected neither the holding currents nor K+ conductance (when patch pipettes were filled with 130 mM KCl) caused by depolarizing pulses. Whole cell GABA-currents were blocked by bicuculline, picrotoxin, and t-butylbicyclophosphorothionate (TBPS; all at 100 microM). The reversal potential of whole cell GABA-currents was close to the theoretical Cl- equilibrium potential, shifting with changes in intracellular Cl- concentration in a manner expected for Cl--selective channels. The whole cell I-V curve for GABA-induced currents demonstrated slight outward rectification with nearly symmetrical outside and inside Cl- concentrations. Spectral analysis of GABA-induced membrane current fluctuations showed that the kinetic components were best fitted by a triple Lorentzian function. The apparent elementary conductance for GABA-activated Cl- channels determined from the power spectra was 22.6 pS. Single-channel recordings from cell-attached patches with pipettes containing 10 microM GABA indicated that GABA-activated channels have a main and a subconductance level with values of 30 and 19 pS, respectively. Mean open and closed times of the channel were characterized by two or three exponential decay functions, suggesting two or three open channel states and two closed states. Single channels showed a lack of rectification. The actions of GABA on cultured human embryonic DRG neurons are mediated through the activation of GABAA receptors with properties corresponding to those found in the CNS of human and other mammalian species but differing from those of cultured human adult DRG neurons. (+info)
Alphaxalone activates a Cl- conductance independent of GABAA receptors in cultured embryonic human dorsal root ganglion neurons.
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Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5alpha-pregnane-3alpha-ol-11,20-dione) effects on GABAA receptors and on Cl- currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl- currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1-5.0 microM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 +/- 0.15). The alphaxalone-induced whole cell current was carried largely by Cl- ions. Its reversal potential was close to the theoretical Cl- equilibrium potential, changing with a shift in the external Cl- concentration as predicted by the Nernst equation for Cl- ions. And because the alphaxalone-current was not suppressed by the competitive GABAA receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 microM), it did not appear to result from activation of GABAA receptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl- concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 microM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl- channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 microM alphaxalone revealed a single conductance state with a chord conductance of approximately 29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl- channels whose electrophysiological and pharmacological properties are distinct from those of Cl- channels associated with GABAA receptors. (+info)