Differential up‐regulation of Na+‐Ca2+ exchange by phosphoarginine and ATP in dialysed squid axons

The aim of this study was to characterize further the two main metabolic pathways of regulation of the Na+-Ca2+ exchanger in squid axons induced by its two naturally ocurring high-energy compounds: ATP and phosphoarginine (Pa). [Na+]o-dependent Ca2+ efflux (forward Nao+-Cai2+ exchange) and [Ca2+]o-dependent Ca2+ efflux (Cao2+-Cai2+ exchange) were measured in internally dialysed squid axons at 16–17 °C. Measurements of changes in the apparent affinity of the Na+-Ca2+ exchanger for transporting (Nao+, Nai+, Cao2+, Cai2+) and regulatory (Cai2+) ions induced by ATP and Pa show marked differences for the two substrates: (i) ATP strongly alters the affinity for Nao+ and Nai+, while Pa does not, and (ii) in the absence of Nai+, ATP has no stimulatiory effect; on the other hand, Pa causes a dramatic increase in Nao+-Cai2+ exchange with little activation of Cao2+-Cai22+ exchange. The MgATP analogue chromium-ATP (CrATP) completely inhibits MgATP stimulation of the Na+-Ca2+ exchanger. Nevertheless, even with the effects of the nucleotide blocked, Pa exhibits its usual activation of the [Na+]o-dependent Ca2+ efflux. None of the classical serine-threonine-tyrosine kinase inhibitors, nor the PP1 and PP2 phosphatase inhibitors, affects either the ATP or the Pa effect. However, intracellular microinjections of an exogenous phosphatase (alkaline phosphatase) completely reverses the stimulation of the Na+-Ca2+ exchange induced by ATP and Pa. Prolonged intracellular dialysis with highly permeable porous capillaries (18 kDa molecular weight cut-off), which normally induces a complete run-down of the MgATP effect, does not alter the Pa stimulation of the exchanger, even after 6 h of continuous dialysis. We conclude that the ATP and Pa modulation of Na+-Ca2+ exchange in an invertebrate nerve fibre are two genuinely different mechanisms, which affect the carrier properties in very different ways. An interesting similarity between ATP and Pa is that a phosphorylation-dephosphorylation process seems to be a common feature of these two regulation modes. 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 & Beauge, 1986; Berberian & Beauge, 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 & Beauge, 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 & Beauge, 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 (Nao+, Nai+, Cao2+, Cai2+), regulatory (Cai2+) and other ligand (Mgi2+,inorganic phosphate, vanadate) interactions (DiPolo & Beauge, 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 (Nao+-Cai2+ and Cao2+-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 & Beauge, 1991).