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A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive K+ channel; K(ATP)) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference K(ATP) channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.

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A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive K+ channel; K(ATP)) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference K(ATP) channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.

1 The pharmacological properties of K ATP channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K + channel Kir6.2 were characterized in HEK-293 cells. 2 [ 3 H]-Glyburide (glibenclamide) bound to transfected cells with a B max value of 18.5 pmol mg 71 protein and with a K D value of 0.7 nM. Speci®c binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3 Functional activity of K ATP channels was assessed by whole cell patch clamp, cation e ux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 mM diazoxide. The currents showed weak inward recti®cation and were sensitive to inhibition by glyburide (IC 50 =0.92 nM). 4 Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered 86 Rb + e ux from transfected cells that was sensitive to inhibition by glyburide (IC 50 =3.6 nM). 5 Diazoxide, but not levcromakalim, evoked concentration-dependent decreases in DiBAC 4 (3) uorescence responses with an EC 50 value of 14.1 mM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding a nities. 6 In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K ATP channels described in pancreatic and neuronal tissues.