The ability of differing subunit combinations of ␥-aminobutyric acid type A (GABA A ) receptors produced from murine ␣1, ␤2, and ␥2L subunits to form functional cell surface receptors was analyzed in both A293 cells and Xenopus oocytes using a combination of molecular, electrophysiological, biochemical, and morphological approaches. The results revealed that GABA A receptor assembly occurred within the endoplasmic reticulum and involved the interaction with the chaperone molecules immunoglobulin heavy chain binding protein and calnexin. Despite all three subunits possessing the ability to oligomerize with each other, only ␣1␤2 and ␣1␤2␥2L subunit combinations could produce functional surface expression in a process that was not dependent on Nlinked glycosylation. Single subunits and the ␣1␥2L and ␤2␥2L combinations were retained within the endoplasmic reticulum. These results suggest that receptor assembly occurs by defined pathways, which may serve to limit the diversity of GABA A receptors that exist on the surface of neurons.

A cDNA encoding a novel member of the G-protein-coupled receptor (GCR) superfamily. an ATP receptor, has been isolated from an embryonic chtck whole brain cDNA hbrary by hybridizatton screenmg. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) m Xenopus oocytes a slowly-developing inward current was observed in response to application of ATP. The pharmacology of this expressed protem defines it as a PZv purinoceptor.

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1. Modulation of GABAA receptors by external H+ was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABAA receptors composed of combinations of al, /31, /62, y2S and a subunits. 2. Changing the external pH from 7 4 reduced GABA-activated currents in sympathetic neurones. pH titration of the GABA-induced current was fitted with a pH model which predicted that H+ interact with two sites (pKa values of 6 4 and 7 2).3. For al/?1 GABAA receptors, low external pH (< 7 4) enhanced responses to GABA. pH titration predicted the existence of two sites with pKa values of 6X6 and 7 5. The GABA concentration-response curve was shifted to the left by low pH and non-competitively inhibited at high pH (> 7*4).4. al/lly2S receptor constructs were not affected by external pH, whereas exchanging the /31 subunit for /32 conferred a sensitivity to pH, with predicted pKa values of 5-16 and 9-44.5. Low pH enhanced the responses to GABA on alflc1 subunits, whilst high pH caused an inhibition (pKa values of 6-6 and 9 9). The GABA concentration-response curves were enhanced (pH 5-4) or reduced (pH 9-4) with no changes in the GABA EC50. 6. Immunoprecipitation with subunit and epitope-specific antisera to al, /11 and a subunits demonstrated that these subunits could co-assemble in cell membranes.7. Expression of alf11y2S8 constructs resulted in a 'bell-shaped' pH titration relationship.Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABAA receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alfi1 constructs at high pH (> 7 4) caused an increased peak and reduced steadystate current, with a correspondingly increased rate of desensitization.9. Modulation of GABAA receptor function was apparently unaffected by the internal pH. Moreover, pH values between 5 and 9.5 did not significantly affect the charge distribution on the zwitterionic GABA molecules. 10. In conclusion, this study demonstrates that external pH can either enhance, have little effect, or reduce GABA-activated responses, and this is apparently dependent on the receptor subunit composition. The potential importance of H+ sensitivity of GABAA receptors is discussed.y-Aminobutyric acid (GABA) is the major inhibitory studies have established that GABAA receptors are heteroneurotransmitter present in the mammalian CNS, a role oligomeric proteins composed of assemblies of different that is reliant upon the almost ubiquitous distribution of subunit polypeptides. On the basis of amino acid sequence 'type A' GABA receptors. Previous molecular cloning identities, these subunits are characterized into four t To whom correspondence should be addressed.