Figure 5.

Development of a thallium flux assay for Kir7.1 (M125R). (A) Mean ± SEM % inhibition of wild type (closed bars; n = 6–7) or M125R mutant (open bars; n = 4–6) Kir7.1 by the indicated concentration of VU573.Note that the wild type data are reproduced from Figure 3. (B) Thallium flux-dependent FluoZin-2 fluorescence recorded from polyclonalKir7.1 (M125R) T-REx-HEK-293 cells cultured overnight in absence (−Tet) or presence (+Tet) of Tetracycline. The fluorescence emission was recorded before and after the addition of extracellular thallium (shaded box). (C) Representative traces for changes of Tl⁺-induced FluoZin-2 fluorescence following 20 min pre-treatment of cells with the indicated concentrations of VU573. (D) CRC for VU573-dependent inhibition of Kir2.3 activity. Values are mean ± SEM (n = 3). A fit of the CRC with a single-site four-parameter logistic function yielded IC₅₀ of 4.9 μM.