The Na+/Ca2+ exchanger's family of membrane transporters is widely distributed in cells and tissues of the animal kingdom and constitutes one of the most important mechanisms for extruding Ca2+ from the cell. Two basic properties characterize them. 1) Their activity is not predicted by thermodynamic parameters of classical electrogenic countertransporters (dependence on ionic gradients and membrane potential), but is markedly regulated by transported (Na+ and Ca2+) and nontransported ionic species (protons and other monovalent cations). These modulations take place at specific sites in the exchanger protein located at extra-, intra-, and transmembrane protein domains. 2) Exchange activity is also regulated by the metabolic state of the cell. The mammalian and invertebrate preparations share MgATP in that role; the squid has an additional compound, phosphoarginine. This review emphasizes the interrelationships between ionic and metabolic modulations of Na+/Ca2+ exchange, focusing mainly in two preparations where most of the studies have been carried out: the mammalian heart and the squid giant axon. A surprising fact that emerges when comparing the MgATP-related pathways in these two systems is that although they are different (phosphatidylinositol bisphosphate in the cardiac and a soluble cytosolic regulatory protein in the squid), their final target effects are essentially similar: Na+-Ca2+-H+ interactions with the exchanger. A model integrating both ionic and metabolic interactions in the regulation of the exchanger is discussed in detail as well as its relevance in cellular Cai2+ homeostasis.
In cardiac sarcolemmal vesicles, MgATP stimulates Na+/Ca2+exchange with the following characteristics: 1) increases 10-fold the apparent affinity for cytosolic Ca2+; 2) a Michaelis constant for ATP of ∼500 μM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 μM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5′- O-(3-thiotriphosphate), without the need for vanadate, but not by β,γ-methyleneadenosine 5′-triphosphate; and 6) not affected by unspecific protein alkaline phosphatase but abolished by a phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC effect is counteracted by phosphatidylinositol. In addition, in the absence of ATP,l-α-phosphatidylinositol 4,5-bisphosphate (PIP2) was able to stimulate the exchanger activity in vesicles pretreated with PI-PLC. This MgATP stimulation is not related to phosphorylation of the carrier, whereas phosphorylation appeared in the phosphoinositides, mainly PIP2, that coimmunoprecipitate with the exchanger. Vesicles incubated with MgATP and no Ca2+ show a marked synthesis ofl-α-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 μM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+exchanger through a fast phosphorylation chain: a Ca2+-independent PIP formation followed by a Ca2+-dependent synthesis of PIP2.
Intracellular Na+ and H+ inhibit Na+‐Ca2+ exchange. ATP regulates exchange activity by altering kinetic parameters for Ca2+i, Na+i and Na+o. The role of the Ca2+i regulatory site on Na+i‐H+i‐ATP interactions was explored by measuring the Na+o‐dependent 45Ca2+ efflux (Na+o‐Ca2+i exchange) and Ca2+i‐dependent 22Na+ efflux (Na+o‐Na+i exchange) in intracellular‐dialysed squid axons. Our results show that: (1) without ATP, inhibition by Na+i is strongly dependent on H+i. Lowering the pHi by 0.4 units from its physiological value of 7.3 causes 80 % inhibition of Na+o‐Ca2+i exchange; (2) in the presence of MgATP, H+i and Na+i inhibition is markedly diminished; and (3) experiments on Na+o‐Na+i exchange indicate that the drastic changes in the Na+i‐H+i‐ATP interactions take place at the Ca2+i regulatory site. The increase in Ca2+i affinity induced by ATP at acid pH (6.9) can be mimicked by a rise in pHi from 6.9 to 7.3 in the absence of the nucleotide. We conclude that ATP modulation of the Na+‐Ca2+ exchange occurs by protection from intracellular proton and sodium inhibition. These findings are predicted by a model where: (i) the binding of Ca2+ to the regulatory site is essential for translocation but not for the binding of Na+i or Ca2+i to the transporting site; (ii) H+i competes with Ca2+i for the same form of the exchanger without an effect on the Ca2+i transporting site; (iii) protonation of the carrier increases the apparent affinity and changes the cooperativity for Na+i binding; and (iv) ATP prevents both H+i and Na+i effects. The relief of H+ and Na+ inhibition induced by ATP could be important in cardiac ischaemia, in which a combination of acidosis and rise in [Na+]i occurs.
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