Abstract
delta subunit-containing gamma-aminobutyric acid, type A (GABA(A))receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with alpha(1) and/or alpha(6) subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA(A) receptor pentamers by concatenation. These receptors composed of alpha(1), alpha(6), beta(3), and delta subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3alpha,21-dihydroxy-5alpha-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that delta can assume multiple positions in a receptor pentamer made up of alpha(1), alpha(6), beta(3), and delta subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of delta subunits between two alpha subunits in alpha(1)alpha(6)beta(3)delta receptors. This property is shared by alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors, but there are differences in the additionally expressed isoforms.
Highlights
Summary—In conclusion, we have shown that GABAA receptors containing the ␣1, ␣6, 3, and ␦ subunits exhibit the ability to promiscuously assemble into different arrangements, at least after expression in Xenopus oocytes
Further we show that one of the ␦ subunit-containing receptors remains relatively silent in the absence of neurosteroid
It is intriguing to hypothesize that a neuron directs assembly of different receptor isoforms from the same set of subunits, depending on the actual functional needs
Summary
Construction of Concatenated ␦ Subunit-containing cDNAs— We thank Dr Luddens for the cDNA coding for the rat ␦ subunit. CRNA coding for each dual and triple subunit concatemer was injected either alone or in different combinations in oocytes, resulting in a total of nine different concatenated receptors. The injected oocytes were incubated in modified Barth’s solution (35) at 18 °C for about 72 h for the determination of Imax and for at least 24 h before the measurements for the detailed characterization of the functional receptors. Non-concatenated and concatenated receptors containing the ␦ subunit showed a pronounced decrease in response to GABA with time. This decrease amounted to about 50% and did not recover. It should be noted that there is an up to 4-fold variation between two batches of oocytes in the mean current amplitude expressed from ␦ subunit-containing receptors. The perfusion system was cleaned between two experiments by washing with 100% DMSO after application of THDOC experiments to avoid contamination
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