Rat α6β2δ GABAA receptors exhibit two distinct and separable agonist affinities

摘要

The onset of motor learning in rats coincides with exclusive expression of GABAA receptors containing α6 and δ subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through α6-containing GABAA receptors, known as tonic inhibition. Here we report that the coexpression of α6, β2, and δ subunits produced receptor–channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high-affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of β2 to α6 and δ subunits increased the prevalence of the high-affinity state. Comparative studies of α6β2δ, α1β2δ, α6β2γ2, α1β₂γ₂ and α₄β₂δ receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to α₆β₂δ receptors. The pharmacology of spontaneous channel activity arising from α₆β₂δ receptor expression corresponded to that of tonic inhibition. For example, GABA_A receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5α-THDOC and classical glycine receptor agonists β-alanine and taurine directly activated α₆β₂δ receptors with high potency. Specific mutation within the GABA-dependent activation domain (β^Y157F) impaired both low- and high-affinity components of GABA agonist activity in α₆β^Y157Fδ receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the δ subunit (δ^R287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the α₆β₂δ receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.