The low molecular weight inhibitor of NCX1 interacts with a cytosolic domain that differs from the ion-transport site of the Na/Ca exchanger

The endogenous inhibitory factor (NCX_IF) of the cardiac Na/Ca exchanger (NCX1) is a low molecular weight substance, which has a strong capacity to modulate the ventricle muscle contractility. Previously, we have shown that NCX_IF 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 NCX_IF 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 NCX_IF can directly interact with the ion transport sites of the exchanger protein or the interaction site of NCX_IF is distinct from the ion-binding/transport site of NCX1. In order to segregate between these possibilities the NCX_IF 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 Na_i-dependent ⁴⁵Ca-uptake were measured in the presence of extravesicular (cytosolic) NCX_IF under conditions in which the concentration of extravesicular Ca was varied (2-200 μM) and intravesicular Na was kept fixed at saturating concentration (160 mM). Under these conditions the NCX_IF 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 NCX _IF can be estimated as the IC₅₀ = 0.3-0.6 μM. Therefore, it is concluded that the NCX_IF is reasonably potent blocker, which interacts with cytosolic domain thereby preventing the ion-translocation (and not ion-binding) events.