Biochemical Pharmacology, Vol. 40, No. 2, pp. 327-332, 1990. 0006-2952/90 $3.00 + 0.00 Printed in Great Britain. ~) 1990. Pergamon Press pie IRREVERSIBLE MODIFICATION OF THE VOLTAGE-SENSITIVE CALCIUM CHANNEL BY N-ETHOXYCARBONYL-2-ETHOXY-1,2- DIHYDROQUINOLINE (EEDQ) MURALI GOPALAKRISHNANand DAVID J. TRIGGLE* Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo, Buffalo, NY 14260, U.S.A. (Received 8 October 1988; accepted 7 December 1989) Abstract--N-Ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ) inhibited, in vitro, the specific binding of three structurally distinct L-type Ca 2+ channel ligands, (+)[3H]PN 200110, [3H]des- methoxyverapamil and [3H]c/s-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 200110 bound without change of ligand affinity. Similar results were obtained in cardiac ventricle microsomes. Preincubation with verapamil (1 x 10- 5M) largely prevented this reduction in [3H]PN 200 110 binding sites by EEDQ. 45Ca2+ uptake in cortical synaptosomes during 1-sec depolarization following 68.5 mM K ÷ was also inhibited by EEDQ. Specific binding of [~25I]to-conotoxinGVIA 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 Ko value. These observations indicate that EEDQ blocks Ca 2+channel function and reduces irreversibly both 1,4-dihydropyridine and tg-conotoxin GVIA binding sites. Ca 2+ influx through voltage-dependent Ca 2+ chan- nels is of particular importance to various cellular functions including excitation-contraction and stimulus-secretion coupling [1-3]. The L-type Ca 2+ channels are sensitive to phenylalkylamines, benzo- thiazapines and 1,4-dihydropyridine activators and antagonists, whereas the N-type Ca 2+ channels are sensitive to to-conotoxin. Considerable evidence now exists to demonstrate that Ca 2+ channels, especially the L-type, are subject to both homologous and heterologous regulatory influences during disease states and following cell lesions or chronic drug or hormone administration [4, 5]. The mechanisms of "up"- or "down-regulation" of Ca 2+ channels are, however, less well understood. Alterations in chan- nel expression and function presumably involve changes in the turnover of channel proteins. A useful approach for studying the metabolism of certain classes of receptors has been the analysis of their reappearance after irreversible inactivation. N- Ethoxycarbonyl-2- ethoxy- 1,2-dihydroquinoline (EEDQ) is a carboxyl group activating agent used in peptide synthesis [6] which has proved to be a useful probe for the irreversible inactivation of sev- eral neurotransmitter receptors including those for norepinephrine (or- and fl-adrenoceptors), acetyl- choline (muscarinic), dopamine and serotonin [7- 16]. The irreversible process presumably involves carboxyl group activation and subsequent cross-link- ing with nucleophilic groups at or adjacent to the * Address correspondence and reprint requests to: D. J. Triggle, Ph.D., School of Pharmacy, 126 Cooke Hall, State University of New York at Buffalo, Buffalo, NY 14260. receptor [17]. In this study, we have investigated the interaction of EEDQ at the L-type 1,4-dihydro- pyridine site and the N-type o~-conotoxin GVIA site associated with the voltage-dependent Ca 2÷ channel in ileal, cardiac and neuronal preparations. MATERIALS AND METHODS Contractile responses. Male albino guinea pigs (Buckberg Farms, Tomkins Cove, NY), weighing 300-500 g, were killed by decapitation, and the ter- minal ileum was removed and placed in physiological saline solution (PSS) of the following composition (mM): NaC1, 118.0; KC1, 4.7; NaHCO3, 25.0; KH2PO4, 1.0; MgC12, 1.0; CaCI2, 1.8; and glucose, 10.0; aerated with Oz-CO2 (95:5). Longitudinal muscle strips (1-2 cm length), prepared as described by Rosenberger et al. [18], were suspended under a resting tension of 0.5 g and allowed to equilibrate for 60 min in aerated (95% 02:5% CO2) PSS at 37 ° with changes of PSS every 15 min. Changes in tension were recorded isometrically using force displacement transducers (Grass FT03) and a Grass Polygraph (model 7B). After the initial equilibration period, tissues were incubated in Ca2+-free PSS, prepared without the addition of CaC12, with several changes for 20 min. Tissues were then exposed to Ca2+-free 80 mM K ÷ (isotonic substitution of NaCI by KCI) depolarizing PSS for 10 min at the end of which time no response was apparent. Cumulative responses to calcium were then determined. Plateau responses were attained before addition of the next higher concentration of calcium. Following re-equilibration in PSS, tissues were treated with various con- centrations of EEDQ for 60 min. After repeated 327