ABSTRACT
GABAA receptor function was studied in outside-out patches from guinea pig hippocampal neurons using a drug application system with an exchange time of under 1.5 ms. Application of GABA to these patches induced a Cl- conductance that desensitized with prolonged exposure. Increasing GABA concentrations induced larger conductance increases that were associated with more complex patterns of desensitization. Smaller GABA responses desensitized with monophasic kinetics, whereas large responses displayed bi- and triphasic kinetics. Desensitization of the response to 1 mM GABA was triphasic in about 70% of the patches (tau = 15.4, 207, and 1370 ms) and biphasic in about 30% of the patches (tau = 44 and 725 ms). All phases of desensitization reversed at the Cl- equilibrium potential. Over the concentration range from 3 microM to 3 mM, both the rate and the extent of desensitization increased; however, complete desensitization was rarely observed. The increase in desensitization rate was due to an increase in the relative contribution of the faster phases with increasing GABA. The time constants of the three phases were independent of concentration. The different phases are not mediated by separate receptor populations, because double pulse experiments demonstrated interconversion among the fastest phase and the two slower phases. We demonstrate the plausibility of a model in which multiphasic desensitization is a consequence of the faster association rate at higher GABA concentrations.
Subject(s)
Receptors, GABA-A/metabolism , Animals , Biophysical Phenomena , Biophysics , Chloride Channels/drug effects , Chloride Channels/metabolism , Dose-Response Relationship, Drug , Drug Resistance , Electric Conductivity , Guinea Pigs , Hippocampus/metabolism , In Vitro Techniques , Kinetics , Models, Neurological , Neurons/metabolism , Potassium Chloride/pharmacology , Receptors, GABA-A/drug effects , Sodium Chloride/pharmacology , gamma-Aminobutyric Acid/administration & dosage , gamma-Aminobutyric Acid/pharmacologyABSTRACT
We have studied the effects of divalent cations on the gamma-aminobutyric acid (GABA) response of voltage-clamped spinal cord neurons, using the whole-cell recording configuration. Zn, Cd, Ni, and Mn (but not Ba, Ca, or Mg) inhibit GABA-induced whole-cell currents when applied extracellularly. Although Zn is an effective inhibitor when applied extracellularly, it is ineffective when applied intracellularly. Inhibition by these cations is mediated by a common saturable recognition site that is distinct from the recognition sites for GABA, benzodiazepines, barbiturates, picrotoxin, or steroids. The maximal inhibition, or efficacy of inhibition, of GABA-induced currents is greater for Zn than for Cd, Ni, or Mn. The order of potency is Cd greater than Zn much greater than Ni much greater than Mn. Inhibition by Zn is partially surmountable by GABA, consistent with a decrease in both the maximum response and the affinity for GABA. The dose-response curve for inhibition of the GABA response by Zn is shifted to the right at a high GABA concentration but is unaffected by the presence of chlordiazepoxide, pentobarbital, or 5 beta-pregnan-3 alpha-ol-20-one. The results are consistent with a model in which a Zn-sensitive modulatory site exerts negative allosteric control over GABA receptor function.
Subject(s)
Zinc/pharmacology , gamma-Aminobutyric Acid/pharmacology , Allosteric Regulation , Animals , Cadmium/pharmacology , Cells, Cultured , Chick Embryo , Dose-Response Relationship, Drug , Nickel/pharmacology , Receptors, GABA-A/drug effectsABSTRACT
The effects of acute ethanol exposure of chick spinal cord neurons were studied in tissue culture, using whole-cell voltage-clamp techniques. Results indicate that ethanol produces a persistent increase in the sensitivity of spinal neurons to GABA and glycine, with no change in input resistance or resting membrane potential. Glutamate responses, in contrast, are unaffected by ethanol.
Subject(s)
Chlorides/physiology , Ethanol/pharmacology , Glycine/pharmacology , Spinal Cord/physiology , gamma-Aminobutyric Acid/pharmacology , Animals , Azides/pharmacology , Benzodiazepines/pharmacology , Cells, Cultured , Chick Embryo , Glutamates/pharmacology , Glutamic Acid , Membrane Potentials/drug effects , Receptors, GABA-A/drug effects , Receptors, GABA-A/physiology , Receptors, Glycine , Receptors, Neurotransmitter/drug effects , Receptors, Neurotransmitter/physiology , Spinal Cord/cytology , Spinal Cord/drug effectsABSTRACT
A brief survey of computer algorithms that have been developed to generate predictions of the secondary structures of RNA molecules is presented. Two particular methods are described in some detail. The first utilizes a thermodynamic energy minimization algorithm that takes into account the likelihood that short-range folding tends to be favored over long-range interactions. The second utilizes an interactive computer graphic modelling algorithm that enables the user to consider thermodynamic criteria as well as structural data obtained by nuclease susceptibility, chemical reactivity and phylogenetic studies. Examples of structures for prokaryotic 16S and 23S ribosomal RNAs, several eukaryotic 5S ribosomal RNAs and rabbit beta-globin messenger RNA are presented as case studies in order to describe the two techniques. Anm argument is made for integrating the two approaches presented in this paper, enabling the user to generate proposed structures using thermodynamic criteria, allowing interactive refinement of these structures through the application of experimentally derived data.
