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Using flow cytometry in conjunction with a voltage-sensitive fluorescent indicator dye (oxonol), we have identified and separated embryonic hippocampal cells according to the sensitivity of their functionally expressed GABAA receptors to zolpidem. Immunocytochemical and RT-PCR analysis of sorted zolpidem-sensitive (ZS) and zolpidem-insensitive (ZI) subpopulations identified ZS cells as postmitotic, differentiating neurons expressing α2, α4, α5, β1, β2, β3, γ1, γ2, and γ3 GABAA receptor subunits, whereas the ZI cells were neuroepithelial cells or newly postmitotic neurons, expressing predominantly α4, α5, β1, and γ2 subunits. Fluctuation analyses of macroscopic Cl− currents evoked by GABA revealed three kinetic components of GABAA receptor/Cl−channel activity in both subpopulations. We focused our study on ZI cells, which exhibited a limited number of subunits and functional channels, to directly correlate subunit composition with channel properties. Biophysical analyses of GABA-activated Cl− currents in ZI cells revealed two types of receptor-coupled channel properties: one comprising short-lasting openings, high affinity for GABA, and low sensitivity to diazepam, and the other with long-lasting openings, low affinity for GABA, and high sensitivity to diazepam. Both types of channel activity were found in the same cell. Channel kinetics were well modeled by fitting dwell time distributions to biliganded activation and included two open and five closed states. We propose that short- and long-lasting openings correspond to GABAA receptor/Cl−channels containing α4β1γ2 and α5β1γ2 subunits, respectively.

the journal of neuroscience/the journal of neuroscience

GABA<sub>A</sub>Receptor Subunit Composition and Functional Properties of Cl<sup>−</sup>Channels with Differential Sensitivity to Zolpidem in Embryonic Rat Hippocampal Cells

GABAAReceptor Subunit Composition and Functional Properties of Cl−Channels with Differential Sensitivity to Zolpidem in Embryonic Rat Hippocampal Cells

GABA_AReceptor Subunit Composition and Functional Properties of Cl⁻Channels with Differential Sensitivity to Zolpidem in Embryonic Rat Hippocampal Cells