The function of sodium/calcium ion exchanger in human platelets and megakaryocytes

Abstract

Previously work from our lab has predicted that Ca2+ release from intracellular Ca2+ stores occurs into a Na+/Ca2+ exchanger (NCX)-associated cytosolic nanodomain enclosed within the membrane complex. In this study, we aimed to test the hypothesis that Ca2+ release initially accumulates into an NCX-associated cytosolic nanodomain. We further aimed to examine whether Ca2+ accumulation within this nanodomain could be responsible for triggering the first stage of platelet activation - the platelet shape change. Experiments were also performed to assess whether CD34+-cultured megakaryocytes might utilize the NCX in a similar manner to platelets to start to establish this as a model system in which the structures of the cytosolic nanodomain could be assessed in the future.
In this study, we investigated whether removal of Ca2+ by the NCX is affected by DM-BAPTA loading. Experiments demonstrated that DM-BAPTA slowed but did not prevent NCX-mediated Ca2+ removal from Ca2+ released from intracellular stores. This effect could also be observed at the single-cell level. The NCX-mediated removal from DM-BAPTA-loaded cells was disrupted by disorganization of the dense tubular system by nicergoline, suggesting its presence within the membrane complex. Examination of the subcellular distribution of the NCX3 protein in human platelets demonstrated the presence of the NCX3 in a location consistent with being at the membrane complex. These data therefore provided the first demonstration of an NCX-associated cytosolic nanodomain in line with our hypothesis.
Additional experiments demonstrated that NCX inhibition triggers a Ca2+-dependent shape change in DM-BAPTA-loaded platelets. Furthermore, this appeared to be dependent upon the function of the IP3 receptor. These data therefore provide functional evidence for the close association of these two Ca2+-transporting proteins in a nanodomain of platelets. In addition, these data suggested that Ca2+ accumulation within the cytosolic nanodomain might be responsible for the rapid activation of the platelet shape in human platelets.
Investigations into the applicability of CD34+-cultured megakaryocytes as a model system in which to study platelet Ca2+ signalling demonstrated that megakaryocytes utilise NCX to regulate their thrombin-evoked Ca2+ signals. This effect was altered by changes in the cytokine level concentrations as well as the addition of calciferol to the platelet. These results demonstrate that whilst megakaryocytes also use NCX to critically control their signalling, this effect is modulated by the developmental condition of the megakaryocytes.
These data therefore are consistent with our previous hypothesis that the NCX3 is principally localised at the membrane complex, where it forms a close association with the IP3 receptor. The experiments on shape change suggests that localising different Ca2+-effectors in the membrane complex or in other subregions of the cell may play a key role in controlling the latency of activation of these process in platelets. These data support the possibility that the membrane complex is a key regulator of Ca2+ signalling in platelets, and may provide a useful target for future anti-platelet agents.