High throughput assay for discovering new inhibitors of the GIRK1/4 channel
In certain embodiments of the present disclosure, a method for determining an inhibitor of acetylcholine-activated potassium channel is described. The method includes incubating a cardiac cell in a solution comprising a test compound. The method further includes adding a muscarine (M2) receptor agonist to the cardiac cell in the solution and monitoring the cardiac cell for a change in membrane potential. A statistically insignificant change in the membrane potential following addition of the muscarine (M2) receptor to the solution signifies that the test compound is a K + channel blocker that inhibits opening of the acetylcholine-activated potassium channel.
1. A method for determining an inhibitor of acetylcholine-activated potassium channel comprising:
incubating a cardiac cell in a solution comprising a test compound and a compound to prevent calcium oscillations in the cardiac cell;
adding a muscarinic (M2) receptor agonist to the cardiac cell in the solution;
monitoring the cardiac cell for a change in membrane potential; wherein a statistically insignificant change in the membrane potential following addition of the muscarinic (M2) receptor to the solution signifies that the test compound is a K + channel blocker that inhibits opening of the acetylcholine-activated potassium channel.
2. The method according to claim 1 , wherein the membrane potential is monitored by use of a detection substance that is capable of generating a detectable signal upon binding to a cytoplasmic component of the cardiac cell.
3. The method according to claim 2 , wherein the detectable substance is a fluorescent membrane potential-sensitive dye.
4. The method according to claim 3 , wherein the fluorescent membrane potential-sensitive dye is an oxonol dye.
5. The method according to claim 3 , wherein the membrane potential is monitored by use of a fluorescent imaging plate reader.
6. The method according to claim 2 , wherein the change in the membrane potential is determined by the ratio of the detectable signal following addition of the muscarinic (M2) receptor agonist to the cardiac cell in the solution to the detectable signal prior to addition of the muscarinic (M2) receptor agonist to the cardiac cell in the solution.
7. The method according to claim 1 , wherein the muscarinic (M2) receptor agonist is acetylcholine.
8. The method according to claim 1 , wherein the muscarinic (M2) receptor agonist is carbachol.
9. The method according to claim 1 , wherein the cardiac cell is an atrial cell.
10. The method according to claim 9 , wherein the atrial cell is an HL-1 cell.
11. The method according to claim 1 , wherein the compound to prevent calcium oscillations in the cardiac cell is nisoldipine.
12. The method according to claim 1 , wherein the solution includes the test compound in a concentration between 10 nanomolar and 10 micromolar.
13. The method according to claim 1 , wherein the Z-factor of the method is between 0.5 and 1.