GABA(A) receptor subunit composition and functional properties of Cl- channels with differential sensitivity to zolpidem in embryonic rat hippocampalcells

Using flow cytometry in conjunction with a voltage-sensitive fluorescent in dicator dye (oxonol), we have identified and separated embryonic hippocampa l cells according to the sensitivity of their functionally expressed GABA, receptors to zolpidem. Immunocytochemical and RT-PCR analysis of sorted zol pidem-sensitive (ZS) and zolpidem-insensitive (ZI) subpopulations identifie d ZS cells as postmitotic, differentiating neurons expressing alpha 2, alph a 4, alpha 5, beta 1, beta 2, beta 3, gamma 1, gamma 2, and gamma 3 GABA(A) receptor subunits, whereas the ZI cells were neuroepithelial cells or newl y postmitotic neurons, expressing predominantly alpha 4, alpha 5, beta 1, a nd gamma 2 subunits. Fluctuation analyses of macroscopic Cl- currents evoke d by GABA revealed three kinetic components of GABA(A) receptor/Cl- channel activity in both subpopulations. We focused our study on ZI cells, which e xhibited 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 aff inity for GABA, and low sensitivity to diazepam, and the other with long-la sting openings, low affinity for GABA, and high sensitivity to diazepam. Bo th types of channel activity were found in the same cell. Channel kinetics were well modeled by fitting dwell time distributions to biliganded activat ion and included two open and five closed states. We propose that short- an d long-lasting openings correspond to GABA(A) receptor/Cl- channels contain ing alpha 4 beta 1 gamma 2 and alpha 5 beta 1 gamma 2 subunits, respectivel y.