Fig. 1.

FLIPR-based hBK assay. (A) Fluorescence emission from HEK293 cells stably overexpressing BK and loaded with BTC-AM followed over time on a fluorescence-imaging plate reader (FLIPR). In the first addition, cells are stimulated with either Cl⁻-free buffer (full line) or Cl⁻-free buffer containing a BK activator to be tested (dashed line). In the second addition, a Tl⁺-based stimulus buffer containing the Ca²⁺ ionophore A23187 and K₂SO₄ (for slight depolarization, because hBK channels are voltage- as well as Ca²⁺-sensitive) at final concentrations of 1.5 µM and 12.5 mM, respectively, is added (all traces). Note that, under control conditions, there is a minor increase in the fluorescence signal upon addition of stimulus buffer (full line), whereas in the presence of a hBK-positive modulator, there is a marked increase in the response after the second addition (dashed line). (B) A representative example of a screening plate showing the fluorescence traces over time. Wells A1-P22 were exposed to test compound at a concentration of 30 µM. One positive modulator hit (green square) was identified on the illustrated screening plate. Columns 23 and 24 contain control wells: buffer control (blue square, wells A23:H24) and a proprietary BK channel modulator used as positive control (red square, wells I23:P24). (C) Typical example of a concentration-response curve for NS19504 tested in 1/2-log dilutions ranging from 0.316 nM to 31.6 µM. Each compound concentration was tested in four individual wells, and responses were normalized toward the proprietary BK channel modulator. Symbols and error bars depict average responses and S.D. (D) Structure of NS19504. Ctrls, controls.