The endogenous inhibitory factor. (NCXIF) of the cardiac Na/Ca exchanger (NCXI) is a low molecular weight substance, which has a strong capacity to modulate the ventricle muscle contractility. Previously, we have shown that NCXIF can completely inhibit either the forward (Na-i-dependent Ca-uptake) or reverse (Na-o-dependent Ca-release) mode of Na/Ca exchange as well as its partial reaction, the Ca/Ca exchange. Although the preliminary studies have shown that NCXIF can rapidly (within few milliseconds) interact with a putative inhibitory site of the Na/Ca exchanger protein (or within its vicinity), it was not clear whether the NCXIF can directly interact with the ion transport sites of the exchanger protein or the interaction site of NCXIF is distinct from the ion-binding/transport site of NCXI. In order to segregate between these possibilities the NCXIF was tested for its capacity to compete with Ca at the cytosolic side by using the preparation of sarcolemma vesicles having predominantly the inside-out orientation. For this goal, the initial rates of Nai-dependent 45 Ca-uptake were measured in the presence of extravesicular (cytosolic) NCXIF under conditions in which the concentration of extravesicular Ca was varied (2-200 muM) and intravesicular Na was kept fixed at saturating concentration (160 mM). Under these conditions the NCXIF results in several fold decrease in V-max values, while having no significant effect on the K-m. Taking into account the molecular weight of 350-550 Da (derived from the gel-filtration and mass-spectra data), the experimentally measured inhibitory potency of NCXIF can be estimated as the IC50 = 0.3-0.6 muM. Therefore, it is concluded that the NCXIF is reasonably potent blocker, which interacts with cytosolic domain thereby preventing the ion-translocation (and not ion-binding) events. (C) 2004 Elsevier Inc. All rights reserved.