NS309 (6,7-dichloro-1H-indole-2,3-dione-3-oxime) is widely used as a pharmacological tool to increase the activity of small- and intermediate-conductance calcium-activated potassium channels. NS309 is assumed to function as a positive allosteric gating modulator. However, its binding site and the molecular details of its action remain unknown. Here, we show that NS309 has a profound effect on the calcium-dependent gating of the intermediate-conductance Ca