Unanticipated Structural and Functional Properties of δ-Subunit-containing GABA_A Receptors^*

Abstract

GABA_A receptors mediate inhibitory neurotransmission in the mammalian brain via synaptic and extrasynaptic receptors. The delta (δ)-subunit-containing receptors are expressed exclusively extra-synaptically and mediate tonic inhibition. In the present study, we were interested in determining the architecture of receptors containing the δ-subunit. To investigate this, we predefined the subunit arrangement by concatenation. We prepared five dual and three triple concatenated subunit constructs. These concatenated dual and triple constructs were used to predefine nine different GABA_A receptor pentamers. These pentamers composed of α₁-, β₃-, and δ-subunits were expressed in Xenopus oocytes and maximal currents elicited in response to 1 mm GABA were determined in the presence and absence of THDOC (3α, 21-dihydroxy-5α-pregnane-20-one). β₃-α₁-δ/α₁-β₃ and β₃-α₁-δ/β₃-α₁ resulted in the expression of large currents in response to GABA. Interestingly, the presence of the neurosteroid THDOC uncovered α₁-β₃-α₁/β₃-δ receptors, additionally. The functional receptors were characterized in detail using the agonist GABA, THDOC, Zn²⁺, and ethanol and their properties were compared with those of non-concatenated α₁β₃ and α₁β₃δ receptors. Each concatenated receptor isoform displayed a specific set of properties, but none of them responded to 30 mm ethanol. We conclude from the investigated receptors that δ can assume multiple positions in the receptor pentamer. The GABA dose-response properties of α₁-β₃-α₁/β₃-δ and β₃-α₁-δ/α₁-β₃ match most closely the properties of non-concatenated α₁β₃δ receptors. Furthermore, we show that the δ-subunit can contribute to the formation of an agonist site in α₁-β₃-α₁/β₃-δ receptors.