Rat α6β2δ GABAA receptors exhibit two distinct and separable agonist affinities

The onset of motor learning in rats coincides with exclusive expression of GABA(A) receptors containing alpha(6) and delta subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through alpha(6)-containing GABA(A) receptors, known as tonic inhibition. Here we report that the coexpression of alpha(6), beta(2), and delta 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 beta(2) to alpha(6) and delta subunits increased the prevalence of the high-affinity state. Comparative studies of alpha(6)beta(2)delta, alpha(1)beta(2)delta, alpha(6)beta(2)gamma(2), alpha(1)beta(2)gamma(2) and alpha(4)beta(2)delta receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to alpha(6)beta(2)delta receptors. The pharmacology of spontaneous channel activity arising from alpha(6)beta(2)delta receptor expression corresponded to that of tonic inhibition. For example, GABA(A) receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5 alpha-THDOC and classical glycine receptor agonists beta-alanine and taurine directly activated alpha(6)beta(2)delta receptors with high potency. Specific mutation within the GABA-dependent activation domain (beta(Y157F)) impaired both low- and high-affinity components of GABA agonist activity in alpha(6)beta(Y157F)delta receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the delta subunit (delta(R287M)) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the alpha(6)beta(2)delta receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.