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

Abstract

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 GABA A 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 ␣2, ␣4, ␣5, ␤1, ␤2, ␤3, ␥1, ␥2, and ␥3 GABA A receptor subunits, whereas the ZI cells were neuroepithelial cells or newly postmitotic neurons, expressing predominantly ␣4, ␣5, ␤1, and ␥2 subunits. Fluctuation analyses of macroscopic Cl Ϫ currents evoked by GABA revealed three kinetic components of GABA A receptor/Cl Ϫ 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 Ϫ 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 GABA A receptor/Cl Ϫ channels containing ␣4␤1␥2 and ␣5␤1␥2 subunits, respectively.

GABA: A Pioneer Transmitter That Excites Immature Neurons and Generates Primitive Oscillations

“…Patch-clamp recordings made from embryonic and early postnatal cortical neurons show that they express functional GABA receptors that change properties significantly with development. GABA A receptors expressed in proliferating precursors have higher apparent affinity, long channel kinetics (491), and long channel open times in neuroepithelial cells (414). The different properties of GABA A receptors are most likely due to different subunit composition, although other factors play an important role including intracellular calcium concentration, degree of receptor phosphorylation, association with anchoring proteins, and degree of clustering of the receptors.…”

Regulation of GABAAReceptor Subunit Expression by Pharmacological Agents

“…Embryonic hippocampal neuronal cultures include a heterogeneous mixture of pyramidal and nonpyramidal neurons of multiple different morphologies (Rothman and Cowan, 1981). The cultured cells express GABA A R subunits ␣1-␣5, ␤1-␤3, ␥1-␥3 and ␦ (Brooks-Kayal et al, 1998;Maric et al, 1999).…”

Binding Sites, Singly Bound States, and Conformation Coupling Shape GABA-Evoked Currents

“…The next step was to include the singly bound open and desensitized states in the scheme postulating two identical and independent binding sites. An estimation of the rate constants (␣ 1 , ␤ 1 ) for partially liganded open states was based on the evidence from single channel analysis (Macdonald et al 1989;Twyman et al 1990), as well as on assessments made in similar models (e.g., Jones and Westbrook 1995;Maric et al 1999). In all these studies, the rate constant of entrance into the singly bound state (␤ 1 ) has been found to be much slower than that leading into the fully bound state (␤ 2 ), and the rate of return into the closed state (␣ 1 ) was fast.…”