Subunit selective modulation of GABAA receptors using pharmacogenetically tethered neurosteroids

Neurosteroids are powerful endogenous modulators of inhibition and emerging therapeutics for anxiety, epilepsy, and mood disorders, yet their actions at defined receptor subtypes and within specific neuronal populations remain poorly resolved. Here, we engineer a neurosteroid DART (Drug Acutely Restricted by Tethering) platform to deliver neuroactive steroid (NAS) activity with cellular precision and receptor-subunit selectivity. From a screen of seventeen NAS analogs, we identified seven scaffolds suitable for further engineering, and we discovered that linker attachment at the steroid C11 position uniquely preserves NAS positive allosteric modulation of GABAA receptors, whereas C2 and C17 attachment abolished activity. C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The lead DART compound, YX85.1DART.2, enhanced GABA-evoked currents in neurons expressing engineered 4/{delta}-containing GABAA receptors but spared {gamma}2-containing receptors. A complementary benzodiazepine DART BZP.1DART.2 showed the opposite selectivity. Together, these tools enable cell-restricted, subunit-resolved interrogation of neurosteroid action on inhibitory microcircuits and provide a strategy to dissect how distinct GABAA receptor subclasses contribute to circuit function and therapeutic outcomes.