Functional GABAA receptor heterogeneity of acutely dissociated hippocampal CA1 pyramidal cells.
CA1 pyramidal cells were voltage clamped, and GABA was applied to individual cells with a modified U-tube, rapid drug application system. With Vh = -50 mV, inward currents elicited by 10 microM GABA were inhibited by GABAA receptor (GABAR) antagonists and were baclofen insensitive, suggesting that GABA actions on isolated CA1 pyramidal cells were GABAR mediated. GABA concentration-response curves averaged from all cells were fitted best with a two-site equation, indicating the presence of at least two GABA binding sites, a higher-affinity site (EC50-1 = 11.0 microM) and a lower-affinity site (EC50-2 = 334.2 microM), on two or more populations of cells. The effects of GABAR allosteric modulators on peak concentration-dependent GABAR currents were complex and included monophasic (loreclezole) or multiphasic (diazepam) enhancement, mixed enhancement/inhibition (DMCM, zolpidem) or multiphasic inhibition (zinc). Monophasic (70% of cells) or biphasic (30% of cells) enhancement of GABAR currents by diazepam suggested three different sites on GABARs (EC50-1 =1.8 nM; EC50-2 = 75.8 nM; EC50-3 = 275.9 nM) revealing GABAR heterogeneity. The imidazopyridine zolpidem enhanced GABAR currents in 70% of cells with an EC50 = 222.5 nM, suggesting a predominance of moderate affinity alpha2 (or alpha3-) subtype-containing BZ Type IIA receptors. A small fraction of cells (10%) had a high affinity for zolpidem, something that is suggestive of alpha1 subtype-containing BZ Type I receptors. The remaining 30% of cells were insensitive to or inhibited by zolpidem, suggesting the presence of alpha5 subtype-containing BZ Type IIB receptors. Whether BZ Type I and Type II receptors coexist could not be determined. The beta-carboline methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibited GABAR currents in all cells at midnanomolar concentrations, but in addition, potentiated GABAR currents in some cells at low nanomolar concentrations, characterizing two groups of cells, the latter likely due to functional assembly of alpha5betaxgamma2GABARs. In all cells, GABAR currents were moderately sensitive (EC50 = 9 microM) to loreclezole, consistent with a relatively greater beta3 subtype, than beta1 subtype, subunit mRNA expression. Two populations of cells were identified based on their sensitivities to zinc(IC50 = 28 and 182 microM), suggesting the presence of at least two GABAR isoforms including alpha5beta3gamma2 GABARs. Consistent with the heterogeneity of expression of GABAR subunit mRNA and protein in the hippocampus and based on their differential responses to GABA and to allosteric modulators, distinct populations of CA1 pyramidal cells likely express multiple, functional GABAR isoforms.
Tietz
E I
EI
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
Kapur
J
J
Macdonald
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RL
D000494:Allosteric Regulation; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D002243:Carbolines / Q000494:pharmacology; D003292:Convulsants / Q000494:pharmacology; D003975:Diazepam / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018757:GABA Modulators / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology*; D006993:Hypnotics and Sedatives / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D017966:Pyramidal Cells / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000737:chemistry / Q000502:physiology*; D014230:Triazoles / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology; D000077334:Zolpidem; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D000927:Anticonvulsants; D002243:Carbolines; D003292:Convulsants; D018757:GABA Modulators; D006993:Hypnotics and Sedatives; D011725:Pyridines; D011963:Receptors, GABA-A; D014230:Triazoles; C034818:methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate; D005680:gamma-Aminobutyric Acid; C066440:loreclezole; D000077334:Zolpidem; D015032:Zinc; D003975:Diazepam