R A Harris

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.—Harris, R. A., Mihic, S. J., Dildy‐Mayfield, J. E., Machu, T. K. Actions of anesthetics on ligandgated ion channels: role of receptor subunit composition. FASEBJ. 9, 1454‐1462 (1995)

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 α1β1 or α1β1γ21 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.

Alcohol intoxication: ion channels and genetics.

Acute in vitro exposure to ethanol and other intoxicant‐anesthetics activates γ‐aminobutyric acid (GABA)‐stimulated chloride channels and inhibits voltage‐dependent calcium and sodium channels of isolated brain membranes. The question of whether these neurochemical actions are responsible for intoxication in vivo has been addressed using animal populations displaying genetic differences in sensitivity to alcohol and benzodiazepine intoxication. These genetic approaches include inbred strains, selected lines, recombinant inbred strains, and heterogeneous stocks. Genetic differences in ion channel function provide strong evidence for a role of the GABA‐stimulated chloride channel in ethanol and benzodiazepine intoxication; the role of calcium and sodium channels is less clear.— Harris, R. A.; Allan, A. M. Alcohol intoxication: ion channels and genetics. FASEB J. 3: 1689‐1695; 1989.

Platelet-derived Growth Factor Induces a Long-term Inhibition of N-Methyl-D-aspartate Receptor Function

Platelet-derived growth factor (PDGF) is a multifunctional protein that plays important roles in many tissues, including the mammalian central nervous system. PDGF and PDGF receptors (PDGFRs) are expressed in virtually every region of the central nervous system where they are involved in the development, survival, growth, and differentiation of both neuronal and glial cells. We now report that a brief activation of PDGFRs produced a long-lasting inhibition of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents in CA1 pyramidal neurons in rat hippocampal slices. PDGF also inhibited NMDA receptors (NMDA-Rs) in cultured hippocampal neurons by a mechanism that involves a decrease in single channel open probability. Non-NMDA receptor function was not affected by PDGF in hippocampal neurons. Experiments with mutant PDGFRs and chelation of intracellular Ca2+ in Xenopus oocytes indicate that this inhibition depends on a phospholipase C-γ-induced elevation of intracellular Ca2+ levels. The PDGF-induced inhibition of NMDA-Rs is produced by a mechanism different than the well characterized phenomenon of Ca2+-dependent NMDA-R run down because the effect of PDGF was blocked by the phosphatase inhibitor, calyculin A, and was not affected by the microtubule polymerizing agent, phalloidin. Because elevations of PDGF levels are associated with neurological trauma or disease, we propose that PDGF can exert neuroprotective effects by inhibiting NMDA-R-dependent excitotoxicity.

Enflurane inhibits NMDA, AMPA, and kainate-induced currents in Xenopus oocytes expressing mouse and human brain mRNA.

Effects of enflurane, an inhalational anesthetic, on NMDA, AMPA, and kainate‐gated currents were examined in Xenopus laevis oocytes expressing mouse or human brain mRNA. In oocytes expressing mouse mRNA, enflurane at an anesthetic concentration (1.8 mM) inhibited the NMDA‐, AMPA‐, and kainate‐induced currents by 29–40%, 30–33%, and 20–27%, respectively, suggesting that all three glutamate ionotropic receptors are susceptible to suppression by inhalational anesthetics. Furthermore, inhibition by enflurane was independent of the concentrations of the agonists (NMDA, AMPA, and kainate) or the NMDA‐coagonist (glycine). This suggests that enflurane inhibition does not result from a competitive interaction at glutamate or glycine binding sites. Enflurane also suppressed the oscillation and apparent desensitization of NMDA currents, suggesting an inhibition of Ca2+ influx through the NMDA channel. In oocytes expressing human brain mRNA, only kainate produced observable currents. Kainate currents of human channels were smaller in size than those of the mouse; however, the kainate concentration‐response curve and percent inhibition (27–29%) by enflurane were similar for mice and humans. The results suggest that human and mouse kainate receptors have similar pharmacological characteristics.— Lin, L.‐H., Chen, L. L., Harris, R. A. Enflurane inhibits NMDA, AMPA, and kainateinduced currents in Xenopus oocytes expressing mouse and human brain mRNA. FASEB J. 7: 479‐485; 1993.

Cerebellar GABAB receptors modulate function of GABAA receptors.

Interactions between GABAA and GABAB receptors were studied using muscimol‐stimulated uptake of 36Cl− by membrane vesicles from mouse cerebellum. Baclofen inhibited muscimol‐stimulated 36Cl− uptake and this action was more pronounced with longer flux times (30 vs. 3 s) and after predesensitization of GABAA receptors. Baclofen also inhibited 36Cl− flux by cortical membranes but was more effective with cerebellar preparations. The action of baclofen was stereoselective, calcium‐dependent, and blocked by the GABAB receptor antagonist 2‐OH‐saclofen. It was mimicked by GTP‐γ‐S but not by GDP‐β‐S, which suggests that baclofen may be acting via a G protein. The action of baclofen was inhibited by U73122, an inhibitor of phospholipase C. However, the potassium channel blockers tetraethylammonium or Ba2+ did not affect the action of baclofen. The results show that activation of GABAB receptors can inhibit the function of GABAA receptors and suggest that this action involves either a nondesensitizing subtype of GABAA receptor or the rate of recycling of desensitized to nondesensitized receptors. We speculate that this action of baclofen results from activation of phospholipase C and phosphorylation of a subtype of GABAA receptor by protein kinase C.—Hahner, L.; McQuilkin, S.; Harris, R. A. Cerebellar GABAB receptors modulate function of GABAA receptors. FASEB J. 5: 2466–2472; 1991.

Rapid Communication Activation of Protein Kinase C Inhibits Kainate-Induced Currents in Oocytes Expressing Glutamate Receptor Subunits

Abstract: 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 GluR 1+3 receptors. PMA inhibited the desensitization, and this effect could be observed before PMAs 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

Acute and Chronic Ethanol Exposure Alters the Function of Hippocampal Kainate Receptors Expressed in Xenopus Oocytes

Abstract: 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 μM kainate responses by 45% compared to 15% inhibition of 400 μM 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.