The plasma membrane Na+-Ca2+ exchange is primarily responsible for Ca2+ extrusion in most cells, particularly during the rise in Ca2+ ([Ca2+]i) following activation of cell function. One of the key features of this countertransport system is that it is highly modulated by intracellular substrates including ATP, Ca2+, H+ and lipids (for references, see Hilgemann, Philipson & Vassort, 1996). In squid axons as well as in cardiac cells, the major up-regulation mechanism of the Na+-Ca2+ exchange involves intracellular MgATP. In both preparations, this nucleotide causes a strong stimulation of the exchange activity, including an increase in the affinity of the intracellular Ca2+ regulatory and Na+ transport sites (DiPolo, 1974; Blaustein, 1977; DiPolo & Beaugé, 1986; Berberian & Beaugé, 1996).
We have found recently in squid nerve fibres a novel form of up-regulation of the Na+-Ca2+ exchanger induced by a high-energy non-nucleotide phosphagen: phosphoarginine (Pa) (DiPolo & Beaugé, 1995), a compound that is normally present at millimolar concentrations in the cytosol of all invertebrates. The Pa stimulation: (i) occurs in the complete absence of ATP or ADP, (ii) is independent of and additive to the MgATP-stimulated exchange, (iii) is largely, but not absolutely dependent on Mg2+ ions and (iv) is fully and rapidly reversible with a Km of around 7.7 mM. The large magnitude of the stimulating effect of Pa, combined with its strong dependence on Cai2+ (DiPolo & Beaugé, 1995), makes this process suitable for extruding [Ca2+]i from regions in neurons where [Ca2+]i can reach very high levels (Llinas, Sugimory & Siver, 1994). The MgATP modulation of Na+-Ca2+ exchange has been characterized with respect to transport (Na+o, Na+i, Ca2+o, Cai2+), regulatory (Cai2+) and other ligand (Mgi2+, inorganic phosphate, vanadate) interactions (DiPolo & Beaugé, 1991). No such information is available, however, for the Pa effect. This is crucial information needed for the characterization of the two pathways involved in the metabolic regulation of this transport system.
In this study we have investigated the effect of Pa on the steady-state kinetic parameters of two partial reactions of the exchanger (Na+o-Cai2+ and Ca2+o-Cai2+ exchange) in relation to both transporting and regulatory species. We also determined whether the mechanism of Pa activation has similar characteristics to the phosphorylation-dephosphorylation process suggested for MgATP modulation of the Na+-Ca2+ exchanger (DiPolo & Beaugé, 1991).
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This work constitutes a detailed study of the ATP and Pa modulation of Na+-Ca2+ exchange in squid axons. The marked differences between the effects of these two compounds on the steady-state ion dependency and ligand interactions demonstrate that they are indeed two different metabolic pathways in the regulation of the exchanger. Two of the crucial differences in support of our conclusion are: (i) the CrATP experiments, which demonstrate that even with the nucleotide effect blocked, Pa can induce its usual activation, and (ii) the long dialysis experiments, which show a clear run-down of the MgATP effect while the Pa stimulation remains unimpaired. This is in agreedment with our recent report that a dialysable soluble 13 kDa cytoplasmic protein is required for the MgATP stimulation of the Na+-Ca2+ exchanger (Beaugéet al. 1996; DiPolo et al. 1997). Furthermore, the lack of run-down of the Pa effect after prolonged dialysis is in agreement with our recent finding that the phosphagen can stimulate a Na+ gradient-dependent 45Ca2+ uptake in membrane vesicles from squid optic nerve without the presence of any cytosolic component (G. Berberián, R. DiPolo & L. Beaugé, unpublished observations).
An interesting finding of this study is the marked differences in the way in which ATP and Pa affect the interaction of the exchanger with Na+ and Ca2+ ions. The three major differences are related to Na+o, Na+i and Cai2+. For Na+o, no effect of internal Pa on the affinity for external Na+o was found, in contrast to the well-known increase in the Na+o affinity induced by ATP (Baker & Glitsch, 1973; DiPolo, 1974; Blaustein, 1977). In relation to the Na+i interaction with the exchanger, the release of Na+i inhibition is much more pronounced with ATP than with Pa (Table 1). In fact, at low [Na+]i we detected a very small ATP stimulation of the Na+-Ca2+ exchange (30 %) compared with a dramatic activation by Pa (8.5-fold); this indicates that the ATP effect is associated with a release of Na+i inhibition on the Cai2+ transport site (Requena, 1978), while that of Pa is not. This is also in agreement with the fact that at high [Na+]i, the percentile activation of the Na+o-Cai2+ by Pa and ATP are about the same (Table 1). Another similarity between these two processes is the increase in the apparent affinity for Cai2+. Nevertheless, the Cai2+ dependency is significantly steeper for Pa than for ATP.
One unexpected finding of this study is that Pa affects the Na+o-Cai2+ exchange much more than the Ca2+o-Cai2+ exchange mode. In fact, in the absence of Pa, both partial reactions have similar magnitudes compared with the preferential increase in the Na+o-Cai2+ over the Ca2+o-Cai2+ exchange in the presence of Pa. This finding could be at least partially explained if, in the absence of Pa, the rate-limiting step resides in the efflux of Ca2+ while during Pa stimulation the influx of Ca2+ becomes rate limiting. In squid axons there is evidence that at alkaline pH (pH 8–9) there is a large increase in the Na+o-Cai2+ and Na+o-Na+i partial reactions with small activation of the Ca2+o-Cai2+ exchange mode (DiPolo & Beaugé, 1987). Similarly, in isolated sarcolemmal vesicles the rate of Na+-Ca2+ exchange increases with increasing pH (deprotonation), while Ca2+-Ca2+ exchange decreases with increasing pH (Khananshvilli & Weil-Maslansky, 1994). Although attractive, at present we do not know whether the increase in the Na+o-Cai2+ to Ca2+o-Cai2+ exchange ratio induced by Pa could be related to the mechanism of regulation of the Na+-Ca2+ exchanger by protons. Interestingly, both ATP and Pa activate Na+-Ca2+ exchange preferentially. This suggests that although these compounds seem to work through different metabolic pathways, the effect in terms of activation of partial reactions of the Na+-Ca2+ exchanger is similar for both.
In squid axons several experimental findings strongly support the view that the regulation of the Na+-Ca2+ exchange by MgATP involves a process of phosphorylation- dephosphorylation, either of the exchanger itself or of another regulatory structure (DiPolo & Beaugé, 1991). This includes the absolute requirement for Mg2+, activation by ATPγS, and activation by vanadate and inorganic phosphate. Another strong argument in favour of this hypothesis is that CrATP, a substrate of most kinases that acts as an end-product inhibitor, completely blocks the ATP effect, whereas Co (NH3)ATP, a poor substrate of most kinases (Dunaway-Mariano & Cleland, 1980), does not. In our study, all the commonly used protein kinase inhibitors and activators failed to induce any change either in the ATP or Pa stimulation, suggesting that whatever the mechanism of activation of the Na+-Ca2+ exchange by these two high energy compounds, it does involve the classical serine-threonine protein kinases. This is also the case for the commonly used phosphatase inhibitors of phosphatases (PP1 and PP2), which showed no effect on either pathway. Nevertheless, the MgATP effect is enhanced by two compounds that inhibit phosphatases: vanadate and p-NPP (see Table 1). An interesting finding is that an alkaline phosphatase, which has been reported to dephosphorylate mainly phosphotyrosine residues (Swarup, Cohen & Garbest, 1981) inhibits both the ATP and the Pa stimulation.
At present it is unknown whether the regulation of Na+-Ca2+ exchange by nucleotide and non-nucleotide high energy phosphate compounds is only present in invertebrates. In mammalian cardiac cells, the MgATP stimulation of Na+-Ca2+ exchange is related to the production of phosphatidylinositol 4, 5-bisphosphate (PIP2) through the inositol phosphate cascade (Hilgemann & Ball, 1996). In the invertebrate squid axon, the MgATP effect requires a soluble, low molecular weight cytoplasmic protein. It remains to be explored whether a similar phosphagen metabolic pathway is also involved in the regulation of the Na+-Ca2+ exchanger in vertebrates in which phosphocreatine would be the naturally occurring substrate.