The squid preparation as a general model for ionic and metabolic Na+/Ca2+ exchange interactions: physiopathological implications.

We propose an integrated kinetic model for the squid nerve Na+/Ca2+ exchanger based on experimental evidences obtained in dialyzed axons. This model satisfactorily explains the interrelationship between ionic (Na+(i)-H+(i)-Ca2+(i)) and metabolic (ATP, phosphoarginine (PA)) regulation of the exchanger. Data in dialyzed axons show that the Ca(i)-regulatory site located in the large intracellular loop plays a central role in the modulation by ATP by antagonizing the inhibitory Na+(i)-H+(i) synergism. We have used the Na(o)/Na(i) exchange mode to unequivocally measure the affinity of the Ca(i)-regulatory site. This allowed us to separate Ca(i)-regulatory from Ca(i)-transport sites and to estimate their respective affinities. In this work we show for the first time that under conditions of saturation of the Ca(i)-regulatory site (10 microM Ca2+(i), pH(i) 8.0), ATP have no effect on the Ca(i)-transport site. In addition, we have expanded our equilibrium kinetic model of ionic and metabolic interactions to a complete exchange cycle (circular model). This model, in which the Ca(i)-regulatory site plays a central role, accounts for the decrease in Na(i) inactivation, at high pH(i), high Ca2+(i,) and MgATP. Furthermore, the model also predicts the net Ca2+ movements across the exchanger based on the exchanger complexes redistribution both during physiological and pathological conditions (ischemia).

Breakdown of Na+/K+-exchanging ATPase phosphoenzymes formed from ATP and from inorganic phosphate during Na+-ATPase activity.

The reactivity towards Na+ and K+ of Na+/K+-ATPase phosphoenzymes formed from ATP and Pi during Na+-ATPase turnover and that obtained from Pi in the absence of ATP, Na+ and K+ was studied. The phosphoenzyme formed from Pi in the absence of cycling and with no Na+ or K+ in the medium showed a biphasic time-dependent breakdown. The fast component, 96% of the total EP, had a decay rate of about 4 s(-1) in K+-free 130 mm Na+, and was 40% inhibited by 20 mm K+. The slow component, about 0.14 s(-1), was K+ insensitive. Values for the time-dependent breakdown of the phosphoenzymes obtained from ATP and from Pi during Na+-ATPase activity were indistinguishable from each other. In K+-free medium containing 130 mm Na+, the decays followed a single exponential with a rate constant of 0.45 s(-1). The addition of 20 mm K+ markedly increased the decays and made them biphasic. The fast components had a rate of approximately 220 s-1 and accounted for 92-93% of the total phosphoenzyme. The slow components decayed at a rate of about 47-53 s(-1). A second group of experiments examined the reactivity towards Na+ of the E2P forms obtained with ATP and Pi when the enzyme was cycling. In both cases, the rate of dephosphorylation was a biphasic function of [Na+]: inhibition at low [Na+], with a minimum at about 5 mm Na+, followed by recovery at higher [Na+]. Although qualitatively similar, the phosphoenzyme formed from Pi showed slightly less inhibition and more pronounced recovery. These results indicate that forward and backward phosphorylation during Na+-ATPase turnover share the same intermediates.

Phosphatidyl inositol-4,5-bisphosphate bound to bovine cardiac Na+/Ca2+ exchanger displays a MgATP regulation similar to that of the exchange fluxes.

This work shows the existence of a phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross-immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns-4,5-P2. In addition, PtdIns-4,5-P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100-130% when membrane vesicles are incubated (15-20 s at 37 degrees C) with 1 mM MgATP and 1 microM Ca2+ (b) in the presence of 100 microM Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns-4,5-P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns-4,5-P2 is greatly reduced compared with that in the presence of 1 microM Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns-4,5-P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP-stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+-dependent levels of PtdIns-4,5-P2 bound to the exchanger molecule.

In squid nerves intracellular Mg(2+) promotes deactivation of the ATP-upregulated Na(+)/Ca(2+) exchanger.

We investigated the role of intracellular Mg(2+) (Mg(i)(2+)) on the ATP regulation of Na(+)/Ca(2+) exchanger in squid axons and bovine heart. In squid axons and nerve vesicles, the ATP-upregulated exchanger remains activated after removal of cytoplasmic Mg(2+), even in the absence of ATP. Rapid and complete deactivation of the ATP-stimulated exchange occurs upon readmission of Mg(i)(2+). At constant ATP concentration, the effect of intracellular Mg(2+) concentration ([Mg(2+)](i)) on the ATP regulation of exchanger is biphasic: activation at low [Mg(2+)](i), followed by deactivation as [Mg(2+)](i) is increased. No correlation was found between the above results and the levels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] measured in nerve membrane vesicles. Incorporation of PtdIns(4,5)P(2) into membrane vesicles activates Na(+)/Ca(2+) exchange in mammalian heart but not in squid nerve. Moreover, an exogenous phosphatase prevents MgATP activation in squid nerves but not in mammalian heart. It is concluded that 1) Mg(i)(2+) is an essential cofactor for the deactivation part of ATP regulation of the exchanger and 2) the metabolic pathway of ATP upregulation of the Na(+)/Ca(2+) exchanger is different in mammalian heart and squid nerves.

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

1. 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 Na+o-Ca2+i exchange) and [Ca2+]o-dependent Ca2+ efflux (Ca2+o-Ca2+i exchange) were measured in internally dialysed squid axons at 16-17 C. 2. Measurements of changes in the apparent affinity of the Na+-Ca2+ exchanger for transporting (Na+o, Na+i, Ca2+o, Ca2+i) and regulatory (Ca2+i) ions induced by ATP and Pa show marked differences for the two substrates: (i) ATP strongly alters the affinity for Na+o and Na+i, while Pa does not, and (ii) in the absence of Na+i, ATP has no stimulatiory effect; on the other hand, Pa causes a dramatic increase in Na+o-Ca2+i exchange with little activation of Ca2+o-Ca2+i exchange. 3. 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. 4. 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. 5. 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. 6. 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.

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles.

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 approximately 500 microM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 microM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5'-O-(3-thiotriphosphate), without the need for vanadate, but not by beta,gamma-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-alpha-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 of L-alpha-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 microM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2 increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+ exchanger through a fast phosphorylation chain: a Ca(2+)-independent PIP formation followed by a Ca(2+)-dependent synthesis of PIP2.

ATP-ADP exchange reaction catalyzed by Na+,K+-ATPase: dephosphorylation by ADP of the E1P enzyme form.

We studied the effects of Mg2+ and of ADP and other nucleoside diphosphates on the dephosphorylation of the E1P form of the partially purified pig kidney Na+,K+-ATPase at 20-22 degrees C. We report for the first time the rate of the reversal of ATP phosphorylation. The experiments were done on enzyme subjected to controlled chymotrypsin digestion consisting of a homogenous population of a truncated catalytic subunit. Under this condition the whole cycle is E1 <-- (f1.ATP, b1) --> E1ATP <-- (f2, b2) --> E1P.ADP <-- (fd, bd.ADP) --> E1P-(f3) --> E1. The values of f1, b1, f2, and f3 were independently estimated in the absence of ADP; those of fd, bd, and b2 were obtained from the fit of ADP-dependent dephosphorylation data to the differential equation set. When f2 = 0 or b1 is very large, the model predicts that dephosphorylation by ADP gives a single exponential; in all other cases it predicts a biphasic dephosphorylation in a semilogarithmic plot. The fast phase is governed by b2.ADP and the slow one by b1. This was experimentally verified. Also, ADP stimulates E1P breakdown without release of Pi, thus leading to ATP synthesis. The data indicate that the true substrate for ATP synthesis is free ADP, while Mg2+ inhibits mainly by a reduction in the free [ADP]; in addition, E1P has a very low affinity for MgADP. The nucleotide structure is also very important; all ADP analogues tested were much less effective than ADP due to a reduced affinity for the E1P and a poor capacity to reverse phosphorylation.

Phosphoryl Group Exchange between ATP and ADP Catalyzed by H+-ATPase from Oat Roots.

ATP-ADP exchange was estimated in the presence of plasma membrane H+-ATPase of oat (Avena sativa) roots partially purified with Triton X-100 by measuring [14C]ATP formation from [14C]ADP. Most studies were done at 0[deg]C. At pH 6.0 the exchange showed: (a) Mg2+ requirement with a biphasic response giving maximal activity at 152 [mu]M and (b) insensitivity to ionic strength, [Na+], and [K+]. ATP and ADP dependence were analyzed with a model in which nucleotide-enzyme interactions are at rapid-random equilibrium, whereas E1ATP [left right arrow] E1P-ADP transitions occur in steady state. The results indicated competition between ADP and ATP for the catalytic site, whereas ATP interaction with the ADP site was extremely weak. At 0[deg]C the exchange showed a 3-fold pH increase, from pH 5.5 to 9.0. At an alkaline pH the reaction was not affected by sodium azide and carbonyl cyanide p-trifluometoxyphenyl-hydrazone, had a biphasic response to Mg2+ (maximal at 513 [mu]m), and was insensitive to ionic strength. At 20[deg]C ATP-ADP exchange was pH insensitive. At both temperatures ATP hydrolysis displayed a bell-shaped response, with a maximum around pH 6.0 to 6.5. Because no adenylate kinase activity was detected under any condition, these results demonstrate the existence of an ATP-ADP exchange reaction catalyzed by the plant H+-ATPase.

Complex ATP-activation kinetics of plant H+-transporting ATPase may or may not require two substrate sites.

The complex ATP-activation kinetics of plant H+-ATPase requires two ATP effects on the enzyme. They may result from the simultaneous existence of two ATP sites or a single site that consecutively changes its properties. We describe here three main models for ATP binding to the plant H+-ATPase. Considering the experimental data there are some restrictions in their application. A system with two simultaneous catalytic sites with cooperative binding is possible provided the substrate exerts regulatory properties, and when ES (or SE) leads to a slower velocity path than SES (v1 < v2). In other words, simple cooperativity does not work. A system with two substrate sites, one of which is catalytic and the other is always and only regulatory (i.e. it affects the overall reaction rate), offers two alternatives: one with potential cooperative binding (the system does not discriminate between binding to the catalytic and regulatory sites; and the other with intrinsically different affinities of catalytic and regulatory sites (i.e. the system discriminates between binding to the two binding sites). Here it is also obligatory that ES (or SE) leads to a slower-velocity path than SES (v1 < v2). Thirdly, a system is possible with single ATP domain that is consecutively catalytic and regulatory as the cycle proceeds. These three mechanisms give rise to equivalent rate equations. Therefore, there is no way to distinguish between them on the basis of kinetic studies. Another conclusion drawn from modeling these schemes is that the form of the plots might resemble but not correspond to certain cooperativity type. For instance, for two substrate sites, a true negative cooperativity for substrate binding can mimic positive cooperativity if the v1 velocity pathway is much slower than the v2 one.

A novel 13 kDa cytoplasmic soluble protein is required for the nucleotide (MgATP) modulation of the Na/Ca exchange in squid nerve fibers.

The Na/Ca exchange is a highly regulated transport mechanism in which MgATP, a powerful modulatory intracellular substrate, has important implications for its function. As occurs with some preparations, in squid axons, nucleotide regulation is lost after membrane vesicle isolation. This has been a significant obstacle in the biochemical characterization of the MgATP effect. An important clue in solving this long-standing puzzle is presented in this work by showing that prolonged intracellular dialysis of squid axons produces a complete run down of the MgATP effect. Here we report that a soluble cytoplasmic factor isolated from fresh squid axoplasm and brain reconstitutes the MgATP stimulation of the Na-gradient-dependent 45Ca uptake in squid optic nerve membrane vesicles. Partial purification of this factor uncovers the presence of a novel 13 kDa soluble cytoplasmic protein (SCPr) which, when microinjected in ATP de-regulated dialyzed squid axons, completely restores the MgATP stimulation of Na(o)-dependent Ca efflux. We propose that in the squid preparation this SCPr constitutes the link between the nucleotide and target effector: the Na/Ca exchanger itself, or other plasma membrane structures which may secondarily interact with the exchanger.

Phosphoarginine stimulation of Na(+)-Ca2+ exchange in squid axons--a new pathway for metabolic regulation?

1. [Na+]o-dependent Ca2+ efflux (forward Na(+)-Ca2+ exchange), [32P]ATP wash-out curves and [ATP] were measured in internally dialysed squid giant axons at 17-18 degrees C. 2. We found that dialysing squid axons without ATP and with [Ca2+]i around 1 microM the basal levels of the [Na+]o-dependent Ca2+ efflux were significantly higher in the presence of N omega-phosphoarginine (PA). Phosphocreatine, a related phosphagen, is without effect. 3. PA stimulation of the Na(+)-Ca2+ exchange occurs in the complete absence of ATP (< 1 microM), being independent of, and additive to, the ATP-stimulated [Na+]o-dependent Ca2+ efflux. PA stimulation of [Na+]o-dependent Ca2+ efflux is fully and rapidly reversible with a Km around 7.7 mM. Activation by saturating [PA] is equivalent in magnitude to that of ATP. 4. PA stimulation of Na(+)-Ca2+ exchange is markedly dependent on intracellular Ca2+ and Mg2+ ions. Below 0.5 microM Ca2+i PA effect is negligible, becoming noticeable between 0.8 and 2 microM. In addition, Ca2+i considerably increases the rate at which PA activates the Na(+)-Ca2+ exchange. Although there is no absolute requirement of the PA effect for Mg2+ ions, this divalent cation largely stimulates the PA effect. 5. This work demonstrates, for the first time, the presence in squid axons of a new form of metabolic regulation of the Na(+)-Ca2+ exchange directly and solely related to PA and different from that of MgATP. This novel mechanism is likely to play a physiological role in Ca2+ extrusion through the Na(+)-Ca2+ exchanger, particularly at micromolar [Ca2+]i.