Determining the molecular and physiological actions of subtype-selective nanobodies of GABA_A receptors

γ-Aminobutyric acid type-A (GABA_A) receptors are the principal mediators of inhibitory neurotransmission in the human central nervous system. The α₂- and α₃-containing subtypes have tightly controlled spatial expression profiles, which influence anxiety, nociception, epilepsy, and autism. α₂/α₃-Selective small molecules compromise on strength of effect (efficacy) to avoid off-subtype modulation. To break this pharmacological deadlock, we study here a panel of nanobodies (NBs) raised against α₂- and α₃-containing GABA_A receptors. We identify subtype selective silent binders, positive allosteric modulators (PAMs), and inhibitors. Cryo-electron microscopy structures explain the binding modes and molecular mechanisms of action of representative NBs. Modulators exhibit distinct synaptic and extrasynaptic functional profiles in brain slices and neuronal networks and can reduce anxiety in vivo. These selective and efficacious NBs (whether inhibitors or positive modulators) enable strong yet precise pharmacological control of α₂/α₃-containing subtypes to advance basic research and as potential therapeutic leads to treat neuropsychiatric disorders.