Beverly J. Hamilton

Stable expression of cloned rat GABAA receptor subunits in a human kidney cell line

A predominant form of the GABAA/benzodiazepine receptor-Cl- channel complex is believed to consist of three different 48-55 kDa subunits (alpha, beta, gamma) with unknown stoichiometry. Plasmids containing the rat GABAA receptor cDNAs coding for alpha 1, beta 2, and gamma 2 were co-transfected, along with a plasmid encoding G418 resistance, into human embryonic kidney cells previously transformed with Adenovirus 5 (HEK-293) [J. Gen. Virol., 36 (1977) 59-72]. Four percent of the G418 resistant colonies were found to express mRNA for all three of the GABAA subunits constitutively. A single cell clone derived from one of the alpha 1 beta 2 gamma 2 expressors has demonstrated stable electrophysiological characteristics over 25 passages. The GABA-activated Cl- current in this cell line is blocked by picrotoxin and bicuculline, and is modulated by a variety of agonist and inverse agonist ligands including diazepam, Ro 154513, zolpidem, and beta-CCE. The cell line has been used successfully over a 12-month period as a screen for novel drugs modulating GABA-mediated polarization of neuronal cells.

Selective effects of dieldrin on the GABAA receptor-channel subunits expressed in human embryonic kidney cells.

We have recently demonstrated that the cyclodiene insecticide dieldrin modulates the kinetics of the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons in a complex manner, causing both stimulatory and inhibitory effects. We now report that the differential effects of dieldrin on the GABA-induced chloride current of human embryonic kidney cells expressing three different combinations of alpha, beta and gamma subunits. The EC50 values for GABA induction of current were estimated to be 9.8 microM for the alpha 1 beta 2 gamma 2s combination, 2.0 microM for the alpha 1 beta 2 combination and 3.0 microM for the alpha 6 beta 2 gamma 2s combination. When co-applied with GABA, dieldrin exerted a dual effect, enhancement and suppression, on the GABA-induced chloride currents in the alpha 1 beta 2 gamma 2s and alpha 6 beta 2 gamma 2s combinations. However, only suppression was observed in the alpha 1 beta 2 combination, indicating that the gamma subunit is necessary for dieldrins enhancing effect. Dieldrin was more efficacious in enhancing the current in the alpha 6 beta 2 gamma 2s combination than in the alpha 1 beta 2 gamma 2s combination, indicating some specific role of alpha subunits in the dieldrin enhancement of current. Dieldrin suppressed the GABA-induced current in a non-competitive manner, with an EC50 value of 2.1 microM for alpha 1 beta 2 gamma 2s, 2.8 microM for alpha 1 beta 2 and 1.0 microM for alpha 6 beta 2 gamma 2s combination. These results indicated that dieldrin suppression did not require specific subunit combinations among the three tested.

Selective potentiation of GABA-mediated Cl− current by lanthanum ion in subtypes of cloned GABAA receptors

The effect of lanthanum ion (La3+) on gamma-aminobutyric acid (GABA)-mediated Cl- currents was examined in the alpha 1 beta 2 or alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in a human kidney cell line (A293), using a whole-cell configuration of patch-clamp techniques. La3+ dose-dependently stimulated the Cl- currents in the alpha 1 beta 2 gamma 2 subtype with an EC50 of 21.3 +/- 3.5 microM with a maximal potentiation of 240 +/- 16% as normalized to the GABA response at 5 microM. In the alpha 1 beta 2 subtype, however, the ion marginally potentiated GABA response, a maximal stimulation being less than 70% with an EC50 for La3+ near 200 microM. The stimulation of GABA response by La3+ in the alpha 1 beta 2 gamma 2 subtype was due to a decrease in the half maximal concentration for GABA and was more pronounced at the negative membrane potentials. This selectivity of La3+ toward the subtypes of GABAA receptors contrasts to that of Zn2+ which inhibits the currents in the alpha 1 beta 2, but not in the alpha 1 beta 2 gamma 2 subtype (Neuron, 5: (1990) 781-788). It appears that these polyvalent cations are useful in understanding the molecular basis for the functional diversity and in characterizing the molecular organization of native GABAA receptors.

Alcohol modulation of cloned GABAA receptor-channel complex expressed in human kidney cell lines.

The effects of n-octanol on GABA-induced currents were examined on the alpha 1 beta 2 gamma 2s and alpha 1 beta 2 combinations of GABAA receptor subunits expressed in a human kidney cell line (HEK 293), using the whole-cell variation of the patch clamp technique. The EC50 of the GABA dose-response curve for the alpha 1 beta 2 combination was lower than that for the alpha 1 beta 2 gamma 2s combination. n-Octanol at 100 microM augmented the GABA-induced currents in a dose-dependent manner, decreasing the EC50 of the GABA dose-response curve without affecting the maximal response. The magnitude of n-octanol potentiation was nearly the same in both combinations. In contrast, a benzodiazepine agonist, chlordiazepoxide, augmented the currents of the alpha 1 beta 2 gamma 2s combination only. We conclude that the potentiation of GABAA receptor-mediated currents by a long carbon chain n-alcohol does not require the gamma 2 subunit.

Differential effects of hexachlorocyclohexane isomers on the GABA receptor subunits expressed in human embryonic kidney cell line

We have recently demonstrated by patch clamp experiments that the four isomers of hexachlorocyclohexane (HCH), alpha-, beta-, gamma- and delta-HCH insecticides, modulated the kinetics of the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons. The present paper reports the differential effects of HCH isomers of the GABA-induced chloride currents in three combinations of alpha, beta and gamma subunits of GABAA receptor expressed in human embryonic kidney cells. When co-applied with GABA, gamma-HCH strongly suppressed the peak amplitude of GABA-induced current, and delta-HCH strongly enhanced it in the alpha 1 beta 2 gamma 2s, alpha 1 beta 2, alpha 6 beta 2 gamma 2s combinations in a dose-dependent manner. There was little or no difference in the dose dependence of the effects between gamma- and delta-HCH in any of the three subunit combinations. However, alpha- and beta-HCH showed differential effects on GABA-induced chloride currents in the three subunit combinations tested. alpha-HCH showed enhancing effects on the peak current in alpha 1 beta 2 gamma 2s, small enhancing effects on alpha 1 beta 2, and biphasic effects on alpha 6 beta 2 gamma 2s subunit combinations. beta-HCH had little or no effect on the peak current in alpha 1 beta 2 gamma 2s and alpha 1 beta 2 combinations, but suppressed currents in the alpha 6 beta2 gamma 2s subunit combination in a dose-dependent manner. The differential actions of HCH isomers may produce variable effects on different regions of the nervous systems and in different species of animals.

Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the α1β2γ2 and α6β2γ2 subtypes of cloned GABAA receptors

1 In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the α1β2γ2 and the α6β2γ2 subtypes of cloned γ‐aminbutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines. 2 The prototype compounds, U‐85575 (12‐chloro‐5‐(5‐cyclopropyl‐1′,2′,4′‐oxadiazol‐3′‐yl)‐2,3‐dihydro‐diimidazo[1,5‐a;1,2‐c]quinazoline), and U‐92330 (5‐acetyl‐3‐(5′‐cyclopropyl‐1′,2′,4′‐oxadiazole‐3′‐yl)‐7‐chloro‐4,5‐dihydro[1,5‐a]quinoxaliné), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA‐mediated Cl− currents in both subtypes were sensitive to Ro 15–1788, a classical benzodiazepine antagonist. 3 Minor changes in the ring substituents of the drugs reduced their affinity to the α6β2γ2 subtype more pronouncedly than to the α1β2γ2 subtype. The diimidazoquinazoline containing a 2‐methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the α6β2γ2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5‐benzoyl group showed a lower affinity to the α6β2γ2 subtype than the 5‐acetyl counterpart. In particular, the 5‐benzoyl analogue containing a 6‐fluoro group showed no interaction with the α6β2γ2 subtype even at the concentration of 10 μm, probably due to stabilization of the benzoyl group in the out‐of‐plane region by the steric and electrostatic effects of the 6‐fluoro group. 4 We propose that the benzodiazepine site of the α6β2γ2 subtype shares overlapping regions with that of the α1β2γ2 subtype, but has a sterically restricted out‐of‐plane region, which may be also incompatible with the 5‐phenyl group of classical benzodiazepines.

Characterization of U‐97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes

1 U‐97775 (tert‐butyl 7‐chloro‐4,5‐dihydro‐5‐[(1‐(3,4,5‐trimethyl)piperazino)carbonyl]‐imidazo[1,5‐a])quinoxaline‐3‐carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2 The drug produced a bell‐shaped dose‐response profile in the α1β2γ2 receptor subtype as monitored with GABA‐induced C1− currents in the whole cell patch‐clamp technique. At low concentrations (<0.5 μM), U‐97775 enhanced the currents with a maximal increase of 120% as normalized to 5 μM GABA response (control). An agonist interaction of U‐97775 with the benzodiazepine site is suggested, because Ro 15–1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]‐flunitrazepam binding was inhibited by U‐97775 with a Ki of 1.2 nM. 3 The enhancement of GABA currents progressively disappeared as the U‐97775 concentration was raised above 1 μM, and the current amplitude was reduced to 40% below the control at 10 μM U‐97775. The current inhibition by U‐97775 (10 μM) was not affected by Ro 15–1788. It appears that U‐97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4 U‐97775 at low concentrations reduced and at high concentrations enhanced [35S]‐TBPS binding. Ro 15–1788 selectively blocked the effect of U‐97775 at low concentrations. Analysis of the binding data in the presence of Ro 15–1788 yielded a single low affinity site with an estimated Kd of 407 nM. 5 In other αβγ receptor subtypes, U‐97775 at low concentrations enhanced C1− currents in the α3β2γ2 but not in the α6β2γ2 subtype. On the other hand, U‐97775 at high concentrations reduced C1− currents in all the receptor subtypes we examined, including those of two subunits, α1β2, β2γ2 and α1gamma;2 subtypes. 6 Therapeutically, U‐97775 could be unique among benzodiazepine ligands because of its ability to limit its own agonistic activity such that, at high doses the appearance of agonistic activity would be delayed until occupancy of its second site wanes. This property should make the total agonistic activity of U‐97775 relatively constant over a wide range of drug doses, and may minimize its liability to abuse.

Differential potentiation of GABAA receptor function by two stereoisomers of diimidazoquinazoline analogues

1 U‐84935, diimidazo[1,5‐a;1′,2′‐C]quinazoline,5‐(5‐cyclopropyl‐1,2,4‐oxidiazol‐3yl)‐2,3‐dihydro, is a ligand of high affinity for the benzodiazepine site of the GABAA receptor composed of α1β2γ2 subunits. 2 The efficacy of its analogues was measured with their ability to potentiate GABA‐mediated Cl− currents in the whole cell configuration of the patch clamp techniques in human kidney cells (A293 cells) expressing the subtype of the GABAA receptor. 3 The analogues displayed various levels of efficacy including agonists, partial agonists and antagonists without marked changes in their affinity for the receptors. 4 The major determinant of their efficacy was the spacial configuration of a methyl substituent of the C2 atom of the rigid and planar diimidazoquinazoline ring: U‐90167, containing the methyl substituent projected below the plane of the ring, markedly enhanced the GABA current with a maximal potentiation of 220 ± 25%, while its stereoisomer, U‐90168, marginally increased the GABA response with a maximal potentiation of 45 ± 10%, to which its methyl group appeared to contribute very little. 5 U‐90167 potentiated the GABA response with an EC50 of 8.1 nm and a Hill coefficient of 1.1 and did not alter the reversal potential for the Cl− current. 6 From computational modelling, the sensitive methyl group of U‐90167 could be assigned to the general region for the 5‐phenyl group of diazepam. The diimidazoquinazoline, because of its rigid and plantar ring structure, may be useful to define further the out‐of‐plane region responsible for agonistic activity and to pinpoint other areas pivotal to the functionality of benzodiazepine ligands.