Binding of K_ATP channel modulators in rat cardiac membranes

Abstract

1 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 378C.

Reinterpreting the Action of ATP Analogs on KATP Channels

“…However, an early study showed that AMP-PNP alone did not affect GBC binding, but could partially rescue or reverse ATP-induced switching (58). Extending these observations showed that MgAMP-PNP alone, at concentrations far in excess of the IC 50 for MgATP, does not support conformational change, but will reverse ATP-induced switching. These results are consistent with a structural study of an asymmetric bacterial ABC protein, TM287-TM288 from T. maritima, which shows that one molecule of AMP-PNP binds to the noncanonical NBD, where it impairs subsequent NBD dimerization and thus switching to outward-facing conformations (36) and with data on cystic fibrosis transmembrane conductance regulator showing preferential binding at NBD1 (48,49).…”

Two Neonatal Diabetes Mutations on Transmembrane Helix 15 of SUR1 Increase Affinity for ATP and ADP at Nucleotide Binding Domain 2

“…Experiments with MgATP included a creatine phosphokinasebased ATP-regenerating system to maintain a constant concentration of ATP over the 30-min incubation (34). The stabil-ity of ATP levels was verified using luciferase assays (Sigma; see supplemental material).…”

[125I]A-312110, a Novel High-Affinity 1,4-Dihydropyridine ATP-sensitive K+Channel Opener: Characterization and Pharmacology of Binding

“…Membranes (300 g/tube) were incubated in a total assay volume of 0.25 ml in assay buffer containing an ATP regeneration system (20 mM creatine phosphate, 50 U of creatine phosphokinase, and 1 mM Na 2 ATP) at 37°C for 90 min. These conditions have been previously employed wherein the levels of ATP are consistently maintained because free ADP is converted back to ATP by creatine phosphate and creatine is regenerated to creatine phosphate by the enzyme creatine phosphokinase (Gopalakrishnan et al, 1991;Loffler-Walz and Quast, 1998). To determine the optimal ATP dependence for maximal specific binding, the assay mixture was incubated with varying concentrations of Na 2 ATP (0.01-3 mM).…”