Evidence for Inhibition Mediated by Coassembly of GABAAand GABACReceptor Subunits in Native Central Neurons

Abstract

Fast inhibition in the nervous system is commonly mediated by GABA_Areceptors comprised of 2α/2β/1γ subunits. In contrast, GABA_Creceptors containing onlyρ subunits (ρ1-ρ3) have been predominantly detected in the retina. However, here using reverse transcription-PCR andin situhybridization we show that mRNA encoding the ρ1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the ρ1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA_Creceptor agonistcis-4-aminocrotonic acid (100-800 μM) requires the ρ1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA_A(bicuculline, 10 μM) and GABA_C[(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 μM] receptors. Responses to GABA_Cagonists were also enhanced by the GABA_Areceptor modulator pentobarbitone (300 μM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA_Aand GABA_Csubunits formed a heteromeric receptor. Immunohistochemistry indicated that ρ1 and α1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA_Creceptor agonists were enhanced by the GABA_Areceptor modulator zolpidem (500 nm), which acts on the α1 subunit when the γ2 subunit is also present. Finally, coimmunoprecipitation indicated that the ρ1 subunit formed complexes that also containedα1 and γ2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA_Aand GABA_Creceptor subunits.