The intermediate-conductance Ca2+-activated K+ channel KCa3.1 is expressed in erythrocytes, vascular endothelial cells, and immune cells. KCa3.1 is involved in a wide variety of disease pathologies, suggesting that both positive and negative gating modulators would be of interest as pharmacological tools and potential drugs. NS309 is the most potent positive gating modulator for KCa3.1, with an EC50 of 75 nM, and interestingly, it is capable of increasing channel activity even at saturating calcium concentrations. However, its binding site is currently unknown. Previous X-ray crystallographic and solution-state NMR studies suggested that NS309 was binding between calmodulin's N-lobe and the C-terminal region of the calmodulin binding domain and interacting with residues M51 and R372. Unfortunately, the crystal dimer used in these studies was later found to be an artifact and disproved when the full-length crystal structure of KCa3.1 was published in 2018. This full-length structure suggested that the “real” binding site for positive gating modulators was located within the interface between calmodulin's N-lobe and the S4-S5 linker, a hypothesis we have now confirmed via site-directed mutagenesis and whole-cell patch clamp. In addition, we have also been investigating a second binding pocket between the distal end of the S4-S5 intracellular loop and the S6 transmembrane segment (a site highlighted by Schroedinger's SiteMap software). And while NS309 does not seem to be binding in this second pocket, several mutations within this pocket cause the channel to become hypersensitive to calcium, indicating that this pocket could serve as another functional binding site for positive gating channel modulators.