Abstract

Although ATP-sensitive K⁺ channels continue to be explored for their therapeutic potential, developments in high-affinity radioligands to investigate native and recombinant K_ATP channels have been less forthcoming. This study reports the identification and pharmacological characterization of a novel iodinated 1,4-dihydropyridine K_ATP channel opener, [¹²⁵I]A-312110 [(9R)-9-(4-fluoro-3-¹²⁵iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno[2,3-e]pyridin-8(7H)-one-1,1-dioxide]. Binding of [¹²⁵I]A-312110 to guinea pig cardiac (K_D = 5.8 nM) and urinary bladder (K_D = 4.9 nM) membranes were of high affinity, saturable, and to a single set of binding sites. Displacement of [¹²⁵I]A-312110 by structurally diverse potassium channel openers (KCOs) indicated a similar rank order of potency in both guinea pig cardiac and bladder membranes (K_i, heart): A-312110 (4.3 nM) > N-cyano-N′-(1,1-dimethylpropyl)-N″-3-pyridylguanidine (P1075) > (-)-N-(2-ethoxyphenyl)-N′-(1,2,3-trimethylpropyl)-2-nitroethene-1,1-diamine (Bay X 9228) > pinacidil > (-)-cromakalim > N-(4-benzoyl phenyl)-3,3,3-trifluro-2-hydroxy-2-methylpropionamine (ZD6169) > 9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione (ZM244085) ≫ diazoxide (16.7 μM). Displacement by K_ATP channel blockers, the sulfonylurea glyburide, and the cyanoguanidine N-[1-(3-chlorophenyl)cyclobutyl]-N′-cyano-N″-3-pyridinyl-guanidine (PNU-99963) were biphasic in the heart but monophasic in bladder with about a 100- to 500-fold difference in K_i values between high- and low-affinity sites. Good correlations were observed between cardiac or bladder-binding affinities of KCOs with functional activation as assessed by their respective potencies to either suppress action potential duration (APD) in Purkinje fibers or to relax electrical field-stimulated bladder contractions. Collectively, these results demonstrate that [¹²⁵I]A-312110 binds with high affinity and has an improved activity profile compared with other radiolabeled KCOs. [¹²⁵I]A-312110 is a useful tool for investigation of the molecular and functional properties of the K_ATP channel complex and for the identification, in a high throughput manner, of both novel channel blockers and openers that interact with cardiac/smooth muscle-type K_ATP channels.