Abstract

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⁺/Ca²⁺ exchange (NCX) inhibitor. SN-6 (0.3-30 μM) inhibited preferentially intracellular Na⁺-dependent ⁴⁵Ca²⁺ uptake (i.e., the reverse mode) compared with extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux (i.e., the forward mode) in NCX1-transfected fibroblasts. SN-6 was 3- to 5-fold more inhibitory to ⁴⁵Ca²⁺ uptake in NCX1 (IC₅₀ = 2.9 μM) 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 site-directed 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 μM) 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.