Characteristics of concatemeric GABAA receptors containing a4/d subunits expressed in Xenopus oocytes

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, a4 and d subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant d subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure d and a4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (a1 + b2 + g2L), and with free-subunit a4/d receptors, expressed in Xenopus oocytes. KEY RESULTS delta-beta 2 - alpha 4 + beta 2-alpha 4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of alpha 4/delta-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[ 2-(2-thienyl) imidazo[ 1,2-a] pyridine-3-yl benzamide; a delta-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, delta-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for alpha 4/d-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as alpha 1/beta 2/gamma 2-containing receptors.