Irreversible modification of the voltage-sensitive calcium channel by N-ethoxycarbonyl-2-ethyoxy-1,2-dihydroquinoline (EEDQ)

Abstract

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inhibited, in vitro, the specific binding of three structurally distinct L-type Ca 2+ channel ligands, (+)[ 3H]PN 200 110, [ 3H]desmethoxyverapamil and [ 3H] cis-diltiazem to guinea pig ileal longitudinal smooth muscle. Maximum tension responses to Ca 2+ in a K +-depolarized functional smooth muscle preparation were reduced in a concentration-dependent manner following pretreatment with EEDQ and washout. Microsomal membranes prepared from smooth muscle pretreated with EEDQ followed by extensive washout showed a significant reduction in the amount of (+)[ 3H]PN 200 110 bound without change of ligand affinity. Similar results were obtained in cardiac ventricle microsomes. Preincubation with verapamil (1 × 10 −5 M) largely prevented this reduction in [ 3H]PN 200 110 binding sites by EEDQ. 45Ca 2+ uptake in cortical synaptosomes during 1-sec depolarization following 68.5 mM K + was also inhibited by EEDQ. Specific binding of [ 125I]ω-conotoxin GVIA to rat cerebral cortex membranes was inhibited by EEDQ, also in an apparently irreversible manner as seen by the marked reduction in binding site density with no significant change in the K D value. These observations indicate that EEDQ blocks Ca 2+ channel function and reduces irreversibly both 1,4-dihydropyridine and ω-conotoxin GVIA binding sites.