GABA-activated Chloride Currents Research Articles

Interaction of Lindane Isomers with Chloride Currents in Insect Membranes: Steric Requirements for Channel Modulation and Block

Electrophysiological methods were used to characterize the modulatory effects of hexachlorocyclohexane isomers on GABA-activated chloride currents in dissociated locust neurones. Conductance changes associated with hyperpolarising responses (or outward voltage-clamped currents), evoked by brief GABA pulses, were inhibited by the γ-isomer at 0.01–1 μM.The antagonistic action was noncompetitive and voltage-independent: the concentration required for half-maximal block was <0.01 μM.The rate of equilibration with the membrane/receptor was inversely proportional to the concentration applied and was not dependent upon agonist-induced channel gating: effects were only partially reversible with extensive washing. In contrast the noninsecticidal β-isomer was a low-affinity (≥10 μM) positive modulator of the inhibitory chloride channels. The modulatory action was voltage-dependent (more pronounced for inward currents at hyperpolarised potentials), did not reflect changes in chloride equilibrium potential, and was completely reversible. Nicotinic acetylcholine receptors were consistently antagonised by the β-isomer at 10 μM.The above results were interpreted by computer-assisted modelling and are discussed in the context of a pharmacophore model for picrotoxinin and convulsant insecticides. The γ-isomer overlays well with picrotoxinin and a convulsant butyrolactone whereas the β-isomer and anticonvulsant butyrolactones conform to a different template. Liganding mechanisms and comparative/species differences in chloride channel pharmacology are discussed in the context of potential for development of invertebrate-selective pesticides.

Anticonvulsant drug effects on spontaneous thalamocortical rhythms in vitro: valproic acid, clonazepam, and alpha-methyl-alpha-phenylsuccinimide.

Spontaneous thalamocortical epileptiform activity was elicited in rodent thalamocortical slices by a medium containing no added Mg2+. Multiple varieties of activity were generated in these slices, including simple thalamocortical burst complex (sTBC) activity that resembled the spike-wave discharges of generalized absence epilepsy, and complex thalamocortical burst complex (cTBC) activity that resembled generalized tonic-clonic seizure discharges. In a further pharmacological characterization of this activity, the effects of the broad-spectrum anticonvulsants valproic acid, alpha-methyl-alpha-phenylsuccinimide (the active metabolite of methsuximide) and clonazepam were studied. All three drugs were found to be effective in controlling both sTBC and cTBC activity when applied in clinically relevant concentration ranges. The effectiveness of valproic acid against spontaneous rhythms in vitro was not due to augmentation of GABAergic inhibition. No effect of valproic acid on GABA-activated chloride currents was evident in patch-clamp recordings of acutely isolated thalamic or cortical neurons. The equivalent general clinical and experimental spectrum of action of broadly effective anticonvulsants provided an additional correlation between the clinical efficacy of anticonvulsant drugs and their effects against epileptiform discharges in rodent thalamocortical slices. This further validates spontaneous generalized low-Mg2+ thalamocortical activity as a potentially valuable in vitro model of the primary generalized epilepsies, in which the cellular mechanisms underlying generation and control of these seizure discharges can be studied.

Differential effects of hexachlorocyclohexane isomers on the GABA receptor-chloride channel complex in rat dorsal root ganglion neurons

The GABA A receptor-chloride channel complex has recently been demonstrated by patch clamp experiments to be the target of cyclodiene insecticides. We have now examined the effects of four isomers of hexachlorocyclohexane (HCH), α-, β-, γ- and δ-HCH, on the GABA A receptor-chloride channel complex of rat dorsal root ganglion neurons using patch clamp techniques. When co-applied with 10 μM GABA, 1 μM γ-HCH slightly enhanced and then suppressed the GABA-induced chloride current. The desensitization of the current was greatly accelerated by γ-HCH in a dose-dependent manner. The acceleration of desensitization and the suppression of sustained component of current by γ-HCH occurred at lower concentration ranges than those for the suppression of peak current. When 10 μM δ-HCH was co-applied with 10 μM GABA, current was greatly enhanced and then suppressed, and the level of enhancement was much higher than that of γ-HCH. α- and β-HCH had little or no effect on the GABA-induced chloride current. The differential actions of these isomers on GABA-activated chloride currents account for variable symptoms of poisoning in insects and mammals.

The Antispastic Triazole MDL 27531 Selectively Enhanced GABA-Activated Chloride Current in Spinal Cord Motorneurons

The triazole MDL 27531, 4-methyl-3-methylsulfonyl-5-phenyl-4H-1,2,4-triazole was identified as an antispastic agent selective for protection against strychnine-induced seizures and hyperreflexia. These compounds did not displace the binding of [3H]muscimol, [3H]flunitrazepam or [35S]TBPS to the GABAA receptor. The present experiments were performed to better understand the mechanism of action of this compound. Whole cell and single channel patch clamp recordings were obtained from GABAA-receptor activated chloride channels using cultured murine spinal cord motorneurons or rat hippocampal pyramidal neurons. GABA (2-5 μM) or GABA plus MDL 27531 (100nM or 1μM) was applied from blunt micropipettes using positive pressure. In spinal motorneurons, the application of GABA plus MDL 27531 (100nM) potentiated GABA-activated chloride currents relative to GABA alone. However, GABA plus MDL 27531 (1μM) did not change or slightly reduced GABA-activated chloride currents. No increase in chloride current was observed with GABA plus MDL 27531 on hippocampal pyramidal neurons. These data indicate that MDL 27531 modulates GABA-activated chloride current selectively. This may be due to selective modulation of subunit configurations of the GABAA receptor. The effect observed with higher concentrations of MDL 27531 may reflect either the modulation of different subunit configurations of the GABAA receptor or interactive effects with additional binding sites. Preliminary studies indicate that MDL 27531 does not modulate glycine-activated chloride currents. Further studies are planned using cloned subunit configurations of the GABAA receptor complex.

Action of Phenylpyrazole Insecticides at the GABA-Gated Chloride Channel

The GABA-gated chloride channel is the proposed target for phenylpyrazole insecticides based on studies comparing four potent insecticidal compounds with two herbicidal but noninsecticidal analogs. The four insecticides used are 1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-1H-pyrazoles with the following heterocyclic substituents: 3-cyano-4-[(trifluoromethyl)sulfinyl]-5-amino (fipronil, 1), 4-[(trifluoromethyl)thiol (2), 4-[(trifluoromethyl)sulfinyl] (3), 4-[(trifluoromethyl)sulfonyl] (4). Pyrazole substituents of the two herbicidal analogs examined are 5-amino-4-nitro (5), 5-(2-chloropropanamide)-4-nitro (6). Piperonyl butoxide strongly antagonizes the toxicity of thioether 2 to houseflies, does not alter the potency of sulfoxide 3 and sulfone 4, and strongly synergizes sulfoxide 1, indicating the importance of oxidative activation and detoxification to insecticidal activity. Insecticides 1-4, but not herbicides 5 and 6, yield poisoning signs in house flies and mice similar or identical to those produced by trioxabicyclooctanes such as 1-[(4-ethynyl)phenyl]-4-n-propy-2,6,7-trioxabicyclo[2.2.2]octane (EBOB), which is known to block the γ-aminobutyric acid (GABA)-gated chloride channel. A dieldrin-resistant house fly strain with a low-affinity EBOB binding site is also tolerant to 1 and 2. The first four phenylpyrazoles are more potent than the latter two as inhibitors of [3H]EBOB binding with 50% inhibition levels of 2-20 nM in house fly head membranes and 1700-10,100 nM in mouse brain membranes. Compounds 1 and 2 are much more potent than 5 and 6 in inhibiting ivermectin-induced neurotransmitter release from cricket central nervous system synaptosomes. Compound 1 is also more effective than 2 and 5 at 20 μM in blocking GABA-dependent chloride uptake in mouse brain microvesicles; in this assay 6 is as effective as 1 although they act by different mechanisms in vivo in mice based on the symptomology. Neither 1 nor 2 inhibits [3H]avermectin binding in house fly head membranes. Phenylpyrazoles 1 and 2 block [3H]EBOB binding in house fly head membranes by reducing the Bmax but not the Kd, indicating that they are irreversible or poorly reversible inhibitors or act at a distinct but coupled binding site. The insecticidal phenylpyrazoles appear to block the GABA-gated chloride channel with higher potency for a site in house fly brain than in mouse brain, thereby providing useful selective toxicity. These findings are consistent with an earlier pharmacological investigation on the action of a structurally related phenyltriazole as a potent antagonist of GABA-activated chloride currents in insects.

Current potentiation by diazepam but not GABA sensitivity is determined by a single histidine residue

The GABAA/benzodiazepine receptor is the principal inhibitory neurotransmitter receptor in the mammalian brain and is assembled from sequence-related subunits, such as alpha 1 beta 2 gamma 2. In contrast to alpha 1 beta 2 gamma 2 receptors, alpha 6 beta 2 gamma 2 receptors fail to exhibit high-affinity binding of allosteric positive modulators of GABA-activated chloride currents. The critical determinant responsible for this difference in ligand binding was previously traced to a position in the extracellular domain of the two alpha subunits (alpha 1 His100 and alpha 6 Arg 101). We now show by patch clamp analysis that this amino acid exchange also determines the diazepam potentiation. Thus, alpha 1(Arg101)beta 2 gamma 2 receptors do not, but alpha 6(His100)beta 2 gamma 2 receptors do exhibit diazepam potentiation. However, the same extracellular determinant is not responsible for the increased GABA sensitivity of alpha 6 beta 2 gamma 2 receptors relative to alpha 1 beta 2 gamma 2 receptors as revealed by electrophysiological analysis and by differential GABA sensitivity of [35S]TBPS binding.

Vasoactive intestinal polypeptide modulates GABAA receptor function in bipolar cells and ganglion cells of the rat retina.

1. The effect of vasoactive intestinal polypeptide (VIP) on bipolar cells and ganglion cells freshly dissociated from the rat retina was studied under voltage clamp with the use of patch-clamp recording in the whole-cell configuration. 2. Application of VIP (1-100 microM) by itself resulted in no detectable current response in either bipolar cells or ganglion cells. However, gamma-aminobutyric acid (GABA)-activated macroscopic current responses elicited in both neuronal populations were potentiated on superimposed exposure to the neuropeptide. 3. GABA-activated chloride currents and muscimol-induced current responses were similarly potentiated on exposure to VIP, suggesting a synergistic interaction between VIP and GABAA receptor mechanisms. 4. We postulate that VIP plays a neuromodulatory role by regulating the excitability of inner retinal neurons and in this way modulates the efficacy of synaptic transmission in the retina.

Mechanism of modulation of GABA-activated current by internal calcium in rat central neurons

GABA-activated currents in Purkinje cells isolated from rat cerebellum were investigated. Increase of intracellular Ca 2+ in the physiological range of concentrations caused a decrease in GABA-activated chloride currents. This effect resulted from a decrease of both the maximal values of GABA-activated currents and possibly from the affinity of GABA A receptors. Therefore, the mechanism of Ca 2+ effect on GABA A receptors in central rat neurons differs from that in bullfrog sensory neurons.

GABA activated chloride currents in cultured rat hippocampal and septal region neurons can be inhibited by curare and atropine

In order to study the influence of curare and atropine on the γ-aminobutyric acid (GABA)-evoked chloride current, we have investigated cultures of hippocampal and septal region neurons from embryonic rats (E18). The neurons were cultured under the trophic influence of spatially separated astrocytes in serum-free medium. By means of the patch clamp technique, the excitable cells displayed GABA induced chloride currents within 1–15 days in vitro. In both cultures picrotoxin or bicuculline as well as curare or atropine reversibly inhibited the chloride current in a dose dependent manner. We conclude that curare and atropine are not specifically anti-cholinergic for cultured central neurons. Our results provide evidence for common ligand binding properties shared by GABA A and acetylcholine (ACh) receptors supporting the recent concept of a receptor superfamily.

Effects of diuretics on GABA‐gated chloride current in frog isolated sensory neurones

1. Effects of three diuretics (furosemide, amiloride and alpha-human atrial natriuretic polypeptide (alpha-hANP] on GABA-activated chloride current (ICl) were investigated in frog isolated sensory neurones, following suppression of Na+, K+ and Ca2+ currents, by use of a 'concentration-clamp' technique. 2. Furosemide inhibited the GABA-activated ICl in a non-competitive manner and facilitated the inactivation phase, while amiloride inhibited the GABA response in a competitive manner, both inhibitions being concentration-dependent. Alpha-hANP had no effects on the GABA-induced ICl. 3. The reversal potential of GABA-activated ICl (EGABA) was not shifted in the presence of amiloride or furosemide. 4. The results suggest that amiloride may act at the GABA binding site while furosemide may act on the GABA-gated chloride channel.

Conductance properties of GABA-activated chloride currents recorded from cultured hippocampal neurons

The conductance characteristics of gamma-aminobutyric acid-activated single channel currents from cultured hippocampal neurons were examined using patch clamp techniques. GABA-activated currents had amplitudes which were linearly correlated to the membrane potentials over a range of -80 to +70 mV and an open time and burst time of 2.2 and 4.3 ms, respectively. The conductance of the gamma-aminobutyric acid-activated channels was 19 pS. These data demonstrate that cultured hippocampal neurons have channel conductances which have characteristics different from those of adult neurons.

O-77 - Diazepam action on GABA-activated chloride currents in internally perfused frog sensory neurons

O-77 - Diazepam action on GABA-activated chloride currents in internally perfused frog sensory neurons

Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographical features.

Penicillin-induced interictal discharges from the cat hippocampus. I. Characteristics and topographical features.