Related Experiment Video
Updated: Sep 30, 2026
Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability
Published on: June 2, 2023
Fluorescence-based functional assay for sarcolemmal ATP-sensitive potassium channel activation in cultured neonatal
K L Whiteaker1, R Davis-Taber, V E Scott
1Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. kristi.l.whiteaker@abbott.com
Introduction:
Activation of ATP-sensitive K+ channels (K(ATP)) has been shown to induce ischemic preconditioning that serves as a protective mechanism in the heart. A high throughput assay for identifying K(ATP) channel openers would therefore be desirable.
Methods:
We describe a cell-based 96-well format fluorescence assay using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) to evaluate membrane potential changes evoked by K(ATP) channel openers and blockers in cultured neonatal rat ventricular myocytes.
Results:
Pinacidil and its analog P1075 (N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine), ZD6169 (N-(4-benzoylphenyl)-3,3,3,-trifluoro-2-hydroxy-2-methyl propionamide), and the enantiomers of cromakalim evoked concentration-dependent decreases in DiBAC4(3) fluorescence responses. Pretreatment with the K(ATP) channel blocker, glyburide attenuated opener-evoked decreases in fluorescence responses in a concentration-dependent manner. The rank order potency of openers in cardiac myocytes correlated well, but showed 6-10-fold higher potency in activating vascular smooth muscle K(ATP) channels in A10 cells.
Discussion:
Our studies demonstrate that the pharmacological modulation of sarcolemmal K(ATP) channels can be readily assessed in a high throughput manner by measuring glyburide-sensitive fluorescence changes in cardiac ventricular myocytes.
