Polycystins (PKD2, PKD2L1) are voltage-gated and Ca2+-modulated members of the transient receptor potential (TRP) family of ion channels. Loss of PKD2L1 expression results in seizure-susceptibility and autism-like features in mice, whereas variants in PKD2 cause autosomal dominant polycystic kidney disease. Despite decades of evidence clearly linking their dysfunction to human disease and demonstrating their physiological importance in the brain and kidneys, the polycystin pharmacophore remains undefined. Contributing to this knowledge gap is their resistance to drug screening campaigns, which are hindered by these channels unique subcellular trafficking to organelles such as the primary cilium. PKD2L1 is the only member of the polycystin family to form constitutively active ion channels on the plasma membrane when overexpressed. We leverage this feature to carry out high-throughput electrophysiology and identify potent antagonists with divergent chemical core structures. In-silico docking analysis and mutagenesis define their receptor sites within the pore vestibule of functioning channels. Results highlight striking similarities between the molecular pharmacology of PKD2L1 and voltage-gated sodium channels, including an open-state accessible lateral fenestration receptor within the pore, and a mechanism of inhibition that stabilizes the inactivated state. Outcomes establish the suitability of our approach to expand our chemical knowledge of polycystins and delineates novel receptor moieties for the development of channel-specific antagonists in TRP channel research.