Neuronal nicotinic receptors: from protein structure to function.

Neuronal nicotinic acetylcholine receptors are a prototype of ligand-gated channels that mediate transmission in the central and peripheral nervous system. Structure-function studies performed at the amino acid level are now unraveling the determinant residues either for the properties of the ligand-binding domain or the ionic pore. In this work we review, in the light of the latest finding, the structure-function relationship of these receptors and their implication in neurological diseases.

Development of stable cell lines expressing different subtypes of GABAA receptors.

The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABAA receptor alpha and beta subunits. G 418 resistant colonies isolated at this step were screened for [3H] muscimol binding to select for those that coexpressed alpha- and beta-subunits. The best alpha and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABAA receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary alpha beta gamma GABAA receptor combinations were distinguished using [3H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABAA receptor clones, alpha 1 beta 2 gamma 2s, alpha 3 beta 2 gamma 2s and alpha 5 beta 3 gamma 2s, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes.

Protracted treatment with diazepam reduces benzodiazepine1 receptor-mediated potentiation of gamma-aminobutyric acid-induced currents in dissociated rat hippocampal neurons.

Tolerance to benzodiazepines (BZs) is thought to involve alterations of the gamma-aminobutyric acid (GABA)A receptor as a result of the prolonged occupancy of its modulatory BZ recognition site. We used the whole-cell patch-clamp technique to compare the functional and pharmacological properties of GABAA receptors in acutely dissociated hippocampal neurons from the control or diazepam-tolerant rats. Administration of diazepam (15 mg/kg p.o.) twice a day for 10 days induced tolerance as demonstrated by the decreased potency of acute diazepam i.p. injections to protect against pentylenetetrazole-induced clonictonic convulsions (10.5% of tolerant rats protected by 0.1 mg/kg of diazepam against 55% of nontreated rats, 48 hr after the last dose of the chronic treatment). The specific current induced by 1 microM GABA in acutely dissociated hippocampal neurons 48 hr after withdrawal (10.5 +/- 1.3 microA/cm2) was similar to that observed in the control rats (8.7 +/- 0.8 microA/cm2). The EC50 value for GABA was unchanged by the chronic treatment [6.3 (5.4-7.1) and 7.5 (6.2-8.7) microM in neurons from the control and treated rats, respectively]. The potency of the nonselective allosteric modulator diazepam to stimulate Cl- currents was identical in cells from treated rats [EC50 values of 25 (20-30) and 34 (26-41) nM in the control and treated rats, respectively; P < .05], but the potency of the selective BZ1-site ligand zolpidem was decreased [EC50 values of 99 (88-111) and 267 (221-313) nM in the control and treated rats, respectively; P < .05]. The maximal potentiation of the GABA-induced current was significantly decreased with diazepam (maximal potentiation: 168.0 +/- 16.2 and 124.0 +/- 8.9% in the control and treated rats, respectively). These results suggest that tolerance to diazepam is accompanied in hippocampal neurons by a decrease in BZ1 binding sites and in the functional coupling of BZ/GABA recognition sites.

Zolpidem functionally discriminates subtypes of native GABAA receptors in acutely dissociated rat striatal and cerebellar neurons.

The whole-cell patch-clamp technique was used to compare the properties of native GABAA receptors in Purkinje and striatal neurons acutely dissociated from neonatal rat brains (7-11 days old). In symmetrical chloride concentrations and at a negative holding voltage, GABA induced inward currents in a concentration-dependent manner with EC50 values of 4 and 8 uM in Purkinje and striatal neurons, respectively. Diazepam potentiated the current induced by 1 uM GABA in Purkinje and striatal neurons with EC50 values of 28 and 42 nM and maximal potentiations of 128 and 182%, respectively. Zolpidem potentiated this GABA-induced current in Purkinje and striatal neurons with EC50 values of 33 and 195 nM and maximal potentiations of 189 and 236%, respectively. These results show that zolpidem, in contrast to diazepam, functionally discriminates subtypes of native GABAA receptors. Zolpidem has greater affinity for GABAA receptors containing omega 1 (Purkinje cells) than for those with omega 2 (striatum) sites and has higher intrinsic activity at these receptors than diazepam. These properties of zolpidem may contribute to its hypnoselective profile.