Binding of K ATP channel modulators in rat cardiac membranes

The binding of [ 3 H]‐P1075, a potent opener of adenosine‐5′‐triphosphate‐(ATP)‐sensitive K + channels, was studied in a crude heart membrane preparation of the rat, at 37°C. Binding required MgATP. In the presence of an ATP‐regenerating system, MgATP supported [ 3 H]‐P1075 binding with an EC 50 value of 100 μ m and a Hill coefficient of 1.4. In saturation experiments [ 3 H]‐P1075 binding was homogeneous with a K D value of 6±1 n m and a binding capacity (B max ) of 33±3 fmol mg −1 protein. Upon addition of an excess of unlabelled P1075, the [ 3 H]‐P1075‐receptor complex dissociated in a mono‐exponential manner with a dissociation rate constant of 0.13±0.01 min −1 . If a bi‐molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 n m −1 min −1 . From the kinetic experiments the K D value was calculated as 4.7±0.6 n m . Openers of the ATP‐sensitive K + channel belonging to different structural classes inhibited specific [ 3 H]‐P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta. Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea‐related carboxylate, AZ‐DF 265, inhibited [ 3 H]‐P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC 50 value of glibenclamide being ∼amp;90 n m ; affinities for the low affinity component were in the μ m concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC 50 value of 6 μ m , a maximum inhibition of 94% and a Hill coefficient of 1.5. It is concluded that binding studies with [ 3 H]‐P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP‐regenerating system. The pharmacological profile of the [ 3 H]‐P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR 2A ) and vascular smooth muscle cells (SUR 2B ), differs from that expected for SUR 2A and SUR 2B .British Journal of Pharmacology (1998) 123 , 1395–1402; doi: 10.1038/sj.bjp.0701756