Neurosteroid Structure-Activity Relationships for Functional Activation of Extrasynaptic δGABAA Receptors

Synaptic GABA(A) receptors are primary mediators of rapid inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurologic disorders. The delta-subunit GABA(A) receptors are expressed extrasynaptically in the dentate gyrus and contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. However, the neurosteroid structure-function relationship at delta GABA(A) receptors within the native hippocampus neurons remains unclear. Here we report a structure-activity relationship for neurosteroid modulation of extrasynaptic GABA(A) receptor-mediated tonic inhibition in the murine dentate gyrus granule cells. We recorded neurosteroid allosteric potentiation of GABA as well as direct activation of tonic currents using a wide array of natural and synthetic neurosteroids. Our results shows that, for all neurosteroids, the C3 alpha-OH group remains obligatory for extrasynaptic receptor functional activity, as C3 beta-OH epimers were inactive in activating tonic currents. Allopregnanolone and related pregnane analogs exhibited the highest potency and maximal efficacy in promoting tonic currents. Alterations at the C17 or C20 region of the neurosteroid molecule drastically altered the transduction kinetics of tonic current activation. The androstane analogs had the weakest modulatory response among the analogs tested. Neurosteroid potentiation of tonic currents was completely (approximately 95%) diminished in granule cells from delta-knockout mice, suggesting that delta-subunit receptors are essential for neurosteroid activity. The neurosteroid sensitivity of delta GABA(A) receptors was confirmed at the systems level using a 6-Hz seizure test. A consensus neurosteroid pharmacophore model at extrasynaptic delta GABA(A) receptors is proposed based on a structure-activity relationship for activation of tonic current and seizure protection.