G protein-gated Inwardly-Rectifying K+ (GIRK) channels are critical mediators of cell excitability in the brain and heart. GIRK channels are homo- and heterotetrameric complexes formed by four subunits (GIRK1-4). While evidence from gene ablation studies in mice suggests that specific GIRK channel subtypes make discrete contributions to organ physiology and behavior, the lack of subtype-selective GIRK channel probes has prevented rigorous evaluation of the consequences, and therapeutic potential, associated with GIRK channel activation. Recently, we identified a promising GIRK channel activator scaffold using a high-throughput screening approach. This scaffold was used to develop ML297, the first subtype-selective, small-molecule GIRK channel activator. ML297 is potent and strongly-selective for GIRK1-containing relative to GIRK1-lacking heteromers. ML297 reduces anxiety-related behavior in a GIRK1-dependent manner in mice, and suppresses seizures in rat epilepsy models. However, ML297 suffers from poor aqueous solubility, as well as low brain penetration and rapid clearance following systemic administration. Moreover, ML297 exhibits only modest selectivity for the predominant neuronal GIRK channel (GIRK1/2) relative to other GIRK1-containing subtypes, including the cardiac GIRK channel (GIRK1/4). These features of ML297 limit its utility as an in vivo probe for elucidating the relevance and therapeutic potential associated with GIRK1/2 channel activation. Accordingly, the goal of this project is to use the ML297 scaffold to develop new GIRK1/2 activators with an improved channel subtype selectivity profile and pharmacokinetic properties required of a brain-penetrant, in vivo probe. The project combines state-of-the-art approaches in medicinal chemistry and compound characterization, and will be conducted by an investigative team with complementary expertise in GIRK channel biology and in vivo probe development. The work is framed around three Specific AIMs: 1) To develop potent and selective GIRK1/2 channel activators, 2) To develop GIRK1/2 channel activators with optimized pharmacokinetic properties, and 3) To characterize the pharmacodynamic properties of GIRK1/2 channel activators in vivo. Work in AIMs 1 and 2 will involve an iterative parallel medicinal chemistry synthesis and testing strategy, and will yield GIRK1/2 activators with pharmacodynamic and pharmacokinetic properties superior to those of ML297. In AIM 3, these compounds will be characterized for in vivo efficacy, selectivity, and potency in the stress-induced hyperthermia (SIH) test, a paradigm with strong predictive validity for anxiolytic compounds. A select group of compounds exhibiting GIRK1/2-dependent efficacy in this test will undergo further evaluation in another anxiety model, and tests that explore the effects of the GIRK1/2 probes on motor activity and reward-related behavior. Completion of this research project will yield selective and effective in vivo probes for GIRK1/2 channels. These probes will facilitate future research into the relevance and therapeutic potential of GIRK1/2 channels, including more rigorous evaluation of their utility for treatment of anxiety-related disorders.