d Subunit Inhibits Neurosteroid Modulation of GABAA Receptors

Neurosteroid modulation of GABAA receptors has been observed with all subunit combinations investigated; however, hetero-oligomeric GABAA receptors containing d subunits were not studied previously. We describe the effect of d subunit expression on 3a,21-dihydroxy-5a-pregnan-20-1 (THDOC)induced potentiation of GABA-gated currents in transfected HEK 293 cells and in cerebellar granule cells in vitro. THDOC (100 nM) significantly potentiated GABA-gated currents in cells transfected with combinations of a1, a6, b3, and g2 subunit cDNAs, whereas cotransfection of d subunit cDNA inhibited this potentiation. In contrast, the direct Cl channel activation by THDOC at higher concentrations (1–10 mM) was not significantly dependent on d subunit cotransfection. These results suggest that the presence of the d subunit inhibits GABAA receptor modulation but not the direct activation by neurosteroids. Cotransfection with d subunit also affected the negative allosteric modulation by pregnenolone sulfate. THDOC potentiation of GABA-gated currents was greater in cerebellar granule cell cultures at 4 d in vitro (DIV) compared with those at 14 DIV. Single-cell reverse transcription-PCR analysis of the mRNAs expressed in cultured cerebellar granule cells shows that an increased number of granule cells at 14 DIV express d subunit mRNAs as compared with 4 DIV granule cells. The presence of d subunit mRNAs detected in individual cells correlated well with the lack of sensitivity to THDOC. These results suggest that developmental expression of GABAA receptor d subunits may play an important role in determining the region-specific neurosteroid-induced modification of fast inhibitory synaptic function.