Some Biochemical Properties of the Upregulation of the Squid Nerve Na⁺/Ca²⁺ Exchanger by MgATP and Phosphoarginine

Abstract

In squid nerve MgATP upregulation of Na+/Ca2+ exchange requires a soluble cytosolic regulatory protein (SCRP) of about 13 kDa; phosphoarginine (PA) stimulation does not. MgATP-gamma-S mimics MgATP. When a 30-10-kDa cytosolic fraction is exposed to 0.5 mM [32P]ATP in the same solution used for transport assays, and in the presence of native membrane vesicles, a 13-kDa and a 25-kDa band become phosphorylated. Membrane vesicles alone do not show these phosphorylated bands and heat denaturation of the cytosolic fraction prevents phosphorylation. Moreover, staurosporine, a general inhibitor of kinases, does not affect MgATP + SCRP stimulation of the exchanger or the phosphorylation of the 13 kDa but prevents phosphorylation of the 25-kDa cytosolic band. The 30-10-kDa fraction phosphorylated in the presence of staurosporine stimulates Na+/Ca2+ exchange in vesicles in the absence of ATP but with Mg2+ in the medium. The 30-10-kDa fraction is not phosphorylated by PA. In membrane vesicles two protein bands, at about 60 kDa and 70 kDa identified as the low molecular weight neurofilament (NF), are phosphorylated by PA, but not by MgATP. This phosphorylation is specific for PA, insensitive to staurosporine (similar to the PA-stimulated fluxes), and labile. In addition, co-immunoprecipitation was observed between the NF and the exchanger protein. Under the conditions of these experiments no phosphorylation of the exchanger is detected, either with MgATP or PA.

In the squid axon Na+/Ca2+ exchanger the state of the Cai-regulatory site influences the affinities of the intra- and extracellular transport sites for Na+ and Ca2+

“…The [Ca 2+ ] i went from zero to 20 μM and the concentration of Mg 2+ was either 0.5 or 8 mM. Figure 2a describes a typical experiment in which the Na þ o -dependent 22 Na + efflux was measured as a function of [Ca 2+ ] i (0, 5, 10 and 20 μM) in an axon dialyzed with a solution containing 40 mM Na + , 0 mM ATP and at a physiological pH i of 7.3. In this experiment, the Ca 2þ idependency of the Na o /Na i exchange was carried out first at 0.5 mM Mg 2+ and then at 8 mM Mg 2þ i .…”

“…Intracellular Mg 2+ is an absolute requirement for ATP upregulation of the squid Na + /Ca 2+ exchanger, a mechanism that almost certainly involves kinase-phosphatase interplay/s [22,38]. It is interesting to note that at high enough concentrations (>2 mM), Mg 2þ i inhibits ATP stimulation of the exchanger both in dialyzed axons and inside-out squid nerve membrane vesicles; the characteristics of its inhibition suggest it is likely to occur through phosphatase activation [38].…”

“…On the other hand, Ca 2þ i and ATP release these inhibitory processes [16,17]. Although the metabolic pathways for the ATP upregulation of the Na + /Ca 2+ exchanger are different in the mammalian heart (PIP2 production and binding to the exchanger [18][19][20] and in the squid nerve (phosphorylation of a soluble cytosolic protein [21,22]), the final kinetic effects on the exchanger are strikingly similar.…”

“…The 45 Ca had significant amounts of non radioactive CaCl 2 that had to be taken into account when preparing the solutions. On the other hand, the specific activity of 22 Na, as NaCl salt, was high enough to be ignored, considering also that the lowest [Na + ] i was 40 mM. For both isotopes the dialysis solutions contained about 200,000 cpm/μl and their specific activity varied with the concentrations of the ions.…”

Ionic regulation of the cardiac sodium-calcium exchanger

“…56 Indeed, the pre-phosphorylated 13 kDa protein can stimulate activity in the absence of ATP. 57 The divergence of ATP-dependent regulatory mechanisms between the squid and mammalian exchangers is surprising. Perhaps the further characterization of the 13 kDa protein and its mechanism of action on NCX-SQ1 will reveal some areas of commonality between the two types of exchangers.…”