Sites of volatile anesthetic action on kainate (Glutamate receptor 6) receptors.

Molecular mechanisms of anesthetic action on neurotransmitter receptors are poorly understood. The major excitatory neurotransmitter in the central nervous system is glutamate, and recent studies found that volatile anesthetics inhibit the function of the alpha-amino-3-hydroxyisoxazolepropionic acid subtype of glutamate receptors (e.g. glutamate receptor 3 (GluR3)), but enhance kainate (GluR6) receptor function. We used this dissimilar pharmacology to identify sites of anesthetic action on the kainate GluR6 receptor by constructing chimeric GluR3/GluR6 receptors. Results with chimeric receptors implicated a transmembrane region (TM4) of GluR6 in the action of halothane. Site-directed mutagenesis subsequently showed that a specific amino acid, glycine 819 in TM4, is important for enhancement of receptor function by halothane (0. 2-2 mM). Mutations of Gly-819 also markedly decreased the response to isoflurane (0.2-2 mM), enflurane (0.2-2 mM), and 1-chloro-1,2, 2-trifluorocyclobutane (0.2-2 mM). The nonanesthetics 1, 2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane had no effect on the functions of either wild-type GluR6 or receptors mutated at Gly-819. Ethanol and pentobarbital inhibited the function of both wild-type and mutant receptors. These results suggest that a specific amino acid, Gly-819, is critical for the action of volatile anesthetics, but not of ethanol or pentobarbital, on the GluR6 receptor.

Selective actions of a detergent on ligand-gated ion channels expressed in Xenopus oocytes.

Cytoclean a commercially available detergent, has selective actions on ligand-gated ion channels. Cytoclean (0.0005-0.01% v/v) potentiated 50 microM glycine responses in oocytes expressing alpha-2 glycine receptors by 23 +/- 7% to 342 +/- 43%. Cytoclean is composed of five components dissolved in water, but only one reagent, Bio-Soft D-62, modulated responses of oocytes expressing alpha-2 glycine receptors. Bio-Soft D-62 (0.00005-0.001% w/v), potentiated 50 microM glycine responses by 13 +/- 1% to 474 +/- 50%. Bio-Soft D-62 is composed of linear alkylbenzene sulfonate (> 95% C12 chain). The effects of Cytoclean or Bio-Soft D-62 were examined on alpha-1 beta-2 and alpha-1 beta-2 gamma-2L gamma-aminobutyric acidA, gamma-aminobutyric acid rho 1, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid, kainate and 5-hydroxytryptamine3 receptors expressed in Xenopus laevis oocytes. Enhancement of gamma-aminobutyric acidA receptor function ranged from approximately 21% to 458% with Cytoclean (0.0001-0.01%), respectively. Bio-Soft D-62 (0.001%) inhibited GABA rho 1 receptor function by approximately 72%. Cytoclean had no effect on 5-hydroxytryptamine3 or GluR6 function, but Cytoclean (0.005% and 0.01%) inhibited GluR3-mediated currents by approximately 21% and approximately 41%, respectively. These results suggest that trace amounts of Cytoclean, such as amounts adhering to glassware, may modulate ion channel function and potentially confound experimental results.

Actions of long chain alcohols on GABAA and glutamate receptors: relation to in vivo effects.

1. The effects of n-alcohols on GABAA and glutamate receptor systems were examined, and in vitro effectiveness was compared with in vivo effects in mice and tadpoles. We expressed GABAA, NMDA, AMPA, or kainate receptors in Xenopus oocytes and examined the actions of n-alcohols on receptor function using two-electrode voltage clamp recording. 2. The function of GABAA receptors composed of alpha 1 beta 1 or alpha 1 beta 1 gamma 2L subunits was potentiated by all of the n-alcohols studied (butanol-dodecanol). 3. In contrast to GABAA receptors, glutamate receptors expressed from mouse cortical mRNA or from cRNAs encoding AMPA (GluR3)- or kainate (GluR6)-selective subunits were much less sensitive to longer chain alcohols. In general, octanol and decanol were either without effect or high concentrations were required to produce inhibition. 4. In contrast to the lack of behavioural effects by long chain alcohols reported previously, decanol produced loss of righting reflex in short- and long-sleep mice, indicating that the in vivo effects of decanol may be due in part to actions at GABAA receptors. Furthermore, butanol, hexanol, octanol, and decanol produce similar potentiation of GABAA receptor function at concentrations required to cause loss of righting reflex in tadpoles, an in vivo model where alcohol distribution is not a compromising factor. 5. Thus, the in vivo effects of long chain alcohols are not likely to be due to their actions on NMDA, AMPA, or kainate receptors, but may be due instead to potentiation of GABAA receptor function.

Anesthetics produce subunit-selective actions on glutamate receptors.

We assessed the involvement of specific glutamate receptors in the action of anesthetics. In addition to the clinical anesthetics enflurane, isoflurane and halothane, we tested novel halogenated compounds, which are anesthetic or nonanesthetic in vivo, on glutamate receptor (GluR) subunits. these volatile compounds as well as pentobarbital and phenobarbital were tested on kainate-induced currents in Xenopus oocytes expressing GluR1, GluR3, GluR2+3 or GluR6 subunits. The anesthetic 1-chloro-1,2,2-triflurocyclobutane (F3) weakly inhibited kainate responses in oocytes expressing GluR3 receptors but not oocytes expressing GluR1 or GluR2+3 receptors. Surprisingly, F3 potentiated kainate responses in oocytes expressing the kainate-selective receptor GluR6. The nonanesthetics 2,3-chlorooctafluorobutane (F8) and 1,2-dichlorohexafluorocyclobutane (F6) did not affect GluR3 or GluR6 kainate responses. Isoflurane weakly inhibited although enflurane and halothane modestly inhibited kainate responses in oocytes expressing GluR1, GluR3 or GluR2+3 receptors. As with F3, isoflurane, enflurane and halothane potentiated kainate-induced currents of GluR6 receptors. The respective inhibitory and potentiating effects of halothane on GluR3 and GluR6 receptors were enhanced by increasing duration of halothane exposure. In contrast to the opposing action of volatile anesthetics, pentobarbital and phenobarbital inhibited GluR3 and GluR6 kainate responses and had a much greater effect on GluR6 receptors. These results provide novel evidence that anesthetics produce selective actions on glutamate receptors, suggesting that subunit composition may determine the role of glutamate receptors in anesthesia.

Actions of anesthetics on ligand-gated ion channels: role of receptor subunit composition.

Molecular cloning of cDNAs coding for ligand-gated ion channel subunits makes it possible to study the pharmacology of recombinant receptors with defined subunit compositions. Many laboratories have used these techniques recently to study actions of agents that produce general anesthesia. We review the effects of volatile and intravenous anesthetics on recombinant GABAA, glycine, AMPA, kainate, NMDA, and 5HT3 receptors. Evidence for and against specific ligand-gated ion channel subunits as targets responsible for anesthesia or the side effects of anesthetic agents is discussed for each type of receptor. Subunit specific actions of some of the agents suggest that construction and testing of certain chimeric receptor subunits may be useful for defining the amino acid sequences responsible for anesthetic actions.

Ethanol inhibits kainate responses of glutamate receptors expressed in Xenopus oocytes: role of calcium and protein kinase C.

Recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors expressed in oocytes are inhibited by ethanol and the sensitivity to ethanol depends on the kainate concentration and the subunit(s) expressed. For example, GluR3 kainate channels are more sensitive to inhibition by ethanol than GluR6 channels in the presence of maximally effective kainate concentrations. To determine if the ethanol inhibition was influenced by the cation permeability (Na+ vs Na+ and Ca2+) of the channels expressed, we compared ethanol inhibition of Ca(2+)-permeable glutamate receptors (GluRs) in oocytes perfused with normal- and high-Ca2+ buffers. The ethanol inhibition was much greater when Ca2+ was the only permeant cation. When Ba2+ was substituted for Ca2+, the ethanol inhibition was reduced, although it was still greater than with normal buffer. The enhanced ethanol inhibition of kainate-stimulated Ca2+ currents was reduced in oocytes injected with the Ca2+ chelator BAPTA, suggesting a role for intracellular Ca2+ in mediating enhanced ethanol sensitivity of kainate channels. The enhanced ethanol inhibition of Ca2+ currents was not due to a direct ethanol inhibition of Ca(2+)-stimulated Cl- currents in the oocyte because ethanol produced no effect on Ca(2+)-stimulated Cl-currents induced by injection of myo-inositol-1,4,5-trisphosphate. Because Ca2+ activates protein kinase C (PKC) and because we found that the PKC activator phorbol 12-myristate 13-acetate inhibits kainate responses (Dildy-Mayfield and Harris, 1994), we examined the role of PKC in mediating the enhanced ethanol inhibition of kainate responses produced by increased Ca2+. Inhibition of PKC by injection of the PKC inhibitor peptide or calphostin C prevented the enhanced ethanol inhibition of kainate-induced Ca2+ responses without altering ethanol inhibition in normal buffer. Thus, ethanol inhibition of kainate channels may involve two mechanisms, one that is independent of PKC and a second type that is due to activation of PKC under conditions of elevated Ca2+, resulting in enhanced inhibition of kainate responses.

Ethanol inhibits the function of 5-hydroxytryptamine type 1c and muscarinic M1 G protein-linked receptors in Xenopus oocytes expressing brain mRNA: role of protein kinase C.

Effects of ethanol on the function of Ca(2+)-activated Cl- channels activated by G protein-coupled serotonin (5-hydroxytryptamine, (5-HT)1c) and muscarinic M1 cholinergic receptors were studied in Xenopus oocytes expressing mouse whole-brain mRNA. Ethanol (25-200 mM) inhibited currents evoked by both 5-HT and acetylcholine (ACh), in a concentration-dependent manner. The maximal effect was obtained with 150 mM ethanol, which produced 65 and 49% inhibition of 5-HT and ACh responses, respectively. In the presence of 100 mM ethanol, the EC50 values for both 5-HT and ACh were increased about 4-fold. In contrast, in oocytes expressing rat cerebellar mRNA, metabotropic glutamate receptor responses were much less sensitive to ethanol. To examine potential postreceptor sites for ethanol inhibition, guanosine-5'-O-(3-thio)triphosphate and myo-inositol-1,4,5-trisphosphate were injected intracellularly. Ethanol (100 mM) did not significantly inhibit the currents produced by either guanosine-5'-O-(3-thio)triphosphate or myo-inositol-1,4,5-trisphosphate. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate markedly inhibited 5-HT-induced responses. Both the PKC inhibitor peptide and staurosporine prevented ethanol inhibition of 5-HT-induced responses. Moreover, ethanol, similarly to phorbol-12-myristate-13-acetate and opposite to PKC inhibitors, enhanced the rate of Ca(2+)-activated Cl- current desensitization induced by repeated applications of 5-HT. These results indicate that certain types of receptor-G protein interactions are more susceptible than others to uncoupling by ethanol and that ethanol inhibition of 5-HT1c receptors requires PKC-mediated phosphorylation. We suggest that ethanol may activate PKC, which phosphorylates the receptors, resulting in inhibition of the responses.

Activation of protein kinase C inhibits kainate-induced currents in oocytes expressing glutamate receptor subunits.

The effect of protein kinase C (PKC) activation on maximal kainate (KA)-induced currents was studied in Xenopus oocytes expressing the glutamate receptor (GluR) subunits GluR3, GluR1 + 3, GluR2 + 3, and GluR6. The PKC activator phorbol 12-myristate 13-acetate (PMA) inhibited peak KA responses in a time-dependent manner. The magnitude of inhibition was greatest in GluR6-expressing oocytes. Desensitizing KA currents characterized by a peak, transient current followed by a slower, desensitizing current were observed in oocytes expressing GluR3 and GluR1 + 3 receptors. PMA inhibited the desensitization, and this effect could be observed before PMA's inhibition of peak current amplitude. PMA-mediated inhibition of both desensitization and peak current amplitude was prevented by intracellular injection of the protein kinase C (PKC) inhibitor peptide. These results suggest that the function of GluRs is regulated by PKC-dependent phosphorylation.

GABAA and glutamate receptor subunit mRNAs in cortex of mice chemically kindled with FG 7142.

Messenger RNA (mRNA) for several subunits of the GABAA receptor was measured in the cortex of mice chemically kindled with FG 7142. At 10 days after the final FG 7142 injection, beta 2 and gamma 2S subunit mRNA were significantly increased. At 31 days, alpha 1, alpha 3, beta 2, and gamma 2L mRNA were elevated. In contrast, levels of mRNA for four subunits of the glutamate receptor in the cortex of FG 7142-kindled mice killed at 31 days were not significantly increased. Previous investigations have shown a reduction in GABA-gated chloride channel function and density in mice kindled with FG 7142, and the increases in subunit mRNA found in the present studies may be a response to these decreases. These results indicate that chemical kindling produces long-lasting changes in expression of genes coding for specific neurotransmitter receptor subunits.

Comparison of ethanol sensitivity of rat brain kainate, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone proprionic acid and N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

The effect of ethanol (EtOH) on kainate (KA), DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone proprionic acid and N-methyl-D-aspartate (NMDA) receptor-operated channels was examined electrophysiologically in Xenopus laevis oocytes expressing mRNA from rat hippocampus and cerebellum. EtOH (50, 100 mM) inhibited KA-induced currents but did not alter the EC50 for KA (approximately 78 microM). For a series of n-alcohols, potency for inhibition of KA responses was related to chain length. 6,7-dinitroquinoxaline-2,3-dione inhibited maximum KA responses with an IC50 of approximately 1 microM; EtOH (50, 100 mM) did not alter the IC50 for 6,7-dinitroquinoxaline-2,3-dione but did not produce further inhibition of KA-induced currents. Despite the apparent noncompetitive inhibition produced by EtOH on KA receptor-mediated responses, the EtOH inhibition increased as the KA concentration decreased in hippocampal and cerebellar mRNA expressing oocytes. This differential inhibition was not due to the different current amplitudes stimulated by low vs. high KA concentrations. In contrast, oocytes expressing NMDA channels demonstrated a constant percent inhibition by EtOH in the presence of 25 to 200 microM NMDA. Altering the extracellular Ca++ concentration did not affect the ability of EtOH to inhibit NMDA responses. Maximal NMDA-stimulated currents were inhibited by 100 mM EtOH to a lesser extent (31%) in oocytes injected with rat cerebellar mRNA than oocytes expressing rat hippocampal mRNA (47%), suggesting brain regional differences in NMDA channel inhibition by EtOH.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute and chronic ethanol exposure alters the function of hippocampal kainate receptors expressed in Xenopus oocytes.

The effects of acute and extended ethanol exposure on N-methyl-D-aspartate- and kainate-induced currents were examined electrophysiologically in Xenopus oocytes expressing rat hippocampal mRNA. Ethanol inhibited responses stimulated by low and high concentrations of N-methyl-D-aspartate to a similar degree. However, responses produced by low or high concentrations of kainate were differentially inhibited by ethanol. Low kainate concentration responses were much more sensitive to ethanol than high kainate concentrations (e.g., 50 mM ethanol inhibited 12.5 microM kainate responses by 45% compared to 15% inhibition of 400 microM kainate responses). In oocytes cultured in 100 mM ethanol for 1-5 days, the ethanol inhibition of maximum N-methyl-D-aspartate and kainate responses was not different from that in non-ethanol-exposed oocytes. Ethanol treatment, however, selectively decreased the ethanol sensitivity of low kainate concentration responses. Currents stimulated by N-methyl-D-aspartate or kainate were not different between control and ethanol-treated oocytes, indicating that ethanol exposure did not interfere with channel expression. The selective actions of acute and extended ethanol exposure on low kainate responses may indicate selective actions of ethanol on subtypes of kainate receptors expressed in oocytes.

Ethanol and voltage- or receptor-mediated increases in cytosolic Ca2+ in brain cells.

Dissociated brain cells were isolated from newborn rat pups and loaded with fura-2. Different mechanisms for stimulating increased free intracellular Ca2+ concentrations [( Ca2+]i) were examined in the absence and presence of ethanol. KCl, carbachol, and kainate concentration-dependently increased [Ca2+]i. Quisqualate also elevated [Ca2+]i but did not produce clear concentration-dependent increases. KCl, carbachol, and quisqualate responses reached peak levels within 10-30 s and then desensitized within 90 s. However, kainate-stimulated increases in [Ca2+]i plateaued and did not decline after 90 s. Of these different [Ca2+]i-mediated processes, only 60 mM KCl stimulation was significantly inhibited by 100 mM ethanol, while lower KCl concentrations were not affected. Carbachol-induced release of intracellular Ca2+ and activation of non-NMDA (i.e., kainate, quisqualate) excitatory amino acid receptor-operated cation channels were also not significantly inhibited by 100 mM ethanol. Thus, in acutely dissociated brain cells from newborn rats, only Ca2+ influx via voltage- and, as reported previously, NMDA-operated Ca2+ channels were sensitive to ethanol inhibition.

Mechanism of inhibition of N-methyl-D-aspartate-stimulated increases in free intracellular Ca2+ concentration by ethanol.

Dissociated brain cells were isolated from newborn rat pups and loaded with fura-2. These cells were sensitive to low N-methyl-D-aspartate (NMDA) concentrations with EC50 values for NMDA-induced intracellular Ca2+ concentration ([Ca2+]i) increases of approximately 7-16 microM measured in the absence of Mg2+. NMDA-stimulated [Ca2+]i increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mM KCl prior to NMDA addition. Under these predepolarized conditions, 100 mM ethanol inhibited 25 microM NMDA responses by approximately 50%, which was similar to the ethanol inhibition observed in buffer without added Mg2+. Ethanol did not alter [Ca2+]i prior to NMDA addition. In the absence of Mg2+, 50 and 100 mM ethanol did not significantly alter the EC50 value for NMDA, but did inhibit NMDA-induced increases in [Ca2+]i in a concentration-dependent manner at 4, 16, 64, and 256 microM NMDA. Whereas NMDA-induced increases in [Ca2+]i were dependent on extracellular Ca2+ and were inhibited by Mg2+, the ability of 100 mM ethanol to inhibit 25 microM NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100 microM) enhanced 25 microM NMDA-induced increases in [Ca2+]i by approximately 50%. Glycine (1-100 microM) prevented the 100 mM ethanol inhibition of NMDA-stimulated [Ca2+]i observed in the absence of exogenous glycine. MK-801 (25-400 nM) inhibited 25 microM NMDA-stimulated rises in [Ca2+]i in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)