The Exchanger Inhibitory Peptide Region-Dependent Inhibition of Na /Ca Exchange by SN-6 [2-[4-(4- Nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic Acid Ethyl Ester], a Novel Benzyloxyphenyl Derivative

We investigated the properties and interaction domains of SN-6 [2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester], a newly synthesized and selective Na /Ca exchange (NCX) inhibitor. SN-6 (0.3–30 M) inhibited preferentially intracellular Na -dependent Ca uptake (i.e., the reverse mode) compared with extracellular Na -dependent Ca efflux (i.e., the forward mode) in NCX1-transfected fibroblasts. SN-6 was 3to 5-fold more inhibitory to Ca uptake in NCX1 (IC50 2.9 M) than to that in NCX2 or NCX3 but not to that in NCKX2. We searched for regions that may form the SN-6 receptor by NCX1/ NCX3-chimeric analyses and determined that amino acid regions 73 to 108 and 193 to 230 in NCX1 are mostly responsible for the differential drug response between NCX1 and NCX3. Further sitedirected mutagenesis revealed that double substitutions of Val227 and Tyr228 in NCX1, which exist within the exchanger inhibitory peptide (XIP) region, mimicked the different drug response. In addition, F213R, G833C, and N839A mutations in NCX1 resulted in loss of drug sensitivity. Exchangers with mutated XIP regions, which display either undetectable or accelerated Na -dependent inactivation, had markedly reduced sensitivity or hypersensitivity to SN-6, respectively. Cell ATP depletion enhanced the inhibitory potency of SN-6. Therefore, SN-6 at lower doses (IC50 0.63 M) potently protected against hypoxia/reoxygenation-induced cell damage in renal tubular cells overexpressing NCX1, suggesting that this drug predominantly works under hypoxic/ischemic conditions. These properties of SN-6, which may be derived from its interaction with the XIP region, are advantageous to developing it as a new anti-ischemic drug. The Na /Ca exchanger (NCX) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na and Ca (Blaustein and Lederer, 1999; Philipson and Nicoll, 2000; Shigekawa and Iwamoto, 2001). In cardiac muscle, the exchanger plays the primary role in the extrusion of intracellular Ca (Ca i) during each excitation-contraction coupling (Bers, 2002). Under pathological conditions such as cardiac ischemia/reperfusion injury, the exchanger is believed to cause Ca overload attributable to elevated intracellular Na (Na i) concentration (Tani, 1990), leading to mechanical and electrical dysfunction of cardiomyocytes. Mammalian NCX forms a multigene family comprising NCX1, NCX2, and NCX3. NCX1 is highly expressed in the heart, kidney, and brain and at much lower levels in other tissues, whereas the expression of NCX2 and NCX3 is limited mainly to the brain and skeletal muscle (Quednau et al., 1997). These three isoforms presumably have similar molecular topologies consisting of nine transmembrane segments and a large central cytoplasmic loop (Iwamoto et al., 1999a; Nicoll et al., 1999). The former part, particularly the -repeat regions, may participate in ion transport (Nicoll et al., 1996; Doering et al., 1998; Iwamoto et al., 2000); the latter part, possessing the exchanger inhibitory This work was supported by a Grant-in-Aid 14570097 for scientific research (to T.I.) from the Ministry of Education, Science and Culture of Japan and grant No.02 from the Salt Science Research Foundation (to T.I.). ABBREVIATIONS: NCX, Na /Ca exchanger; SN-6, 2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester; KB-R7943, 2-[2[4-(4-nitrobenzyloxyl)phenyl]ethyl]isothiourea methanesulfonate; SEA0400, 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; XIP, exchanger inhibitory peptide; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; BSS, balanced salt solution; QNB, L-quinuclidinyl benzilate; LDH, lactate dehydrogenase; MOPS, 3-(N-morpholino)propanesulfonic acid. 0026-895X/04/6601-45–55$20.00 MOLECULAR PHARMACOLOGY Vol. 66, No. 1 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3225/1156958 Mol Pharmacol 66:45–55, 2004 Printed in U.S.A. 45 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from peptide (XIP) region (Li et al., 1991; Matsuoka et al., 1997) and regulatory Ca binding sites (Levitsky et al., 1994; Matsuoka et al., 1995), is primarily involved in various regulatory properties. NCX1 has been shown to be secondarily regulated by the transport substrates Na and Ca (Hilgemann et al., 1992a,b). Ca i at the submicromolar level activates NCX activity by promoting the recovery of the exchanger from the “I2 inactivation state”, whereas high Na i restrains the exchange by facilitating the entry of the exchanger into the “I1 inactivation state”. A selective NCX inhibitor will be very useful in the study of NCX’s physiological and pathophysiological roles and to clarify the reaction mechanism of this transporter. Moreover, such an inhibitor may have therapeutic potential as a new remedy for several ischemic diseases, arrhythmias, heart failure, and essential hypertension. KB-R7943, a benzyloxyphenyl derivative, was first introduced in 1996 as a selective NCX inhibitor (Iwamoto et al., 1996; Watano et al., 1996). In 2001, Matsuda et al. reported on SEA0400, a newly developed, potent, and selective inhibitor of NCX. The inhibitory potency of SEA0400 is 80 to 100 times more powerful than that of KB-R7943. SEA0400 has an excellent specificity; KBR7943 possesses nonspecific actions against ion channels, neuronal nicotinic acetylcholine receptors, the N-methyl-Daspartate receptor, and the norepinephrine transporter (Watano et al., 1996; Sobolevsky and Khodorov, 1999; Pintado et al., 2000; Matsuda et al., 2001). However, Reuter et al. (2002) suggested recently that SEA0400, too, has unknown nonspecific effects. These NCX inhibitors have several interesting features: KB-R7943 and SEA0400 inhibit the reverse mode (i.e., Ca Fig. 1. Chemical structures of benzyloxyphenyl derivatives SN-6 and KB-R7943.