Metabolic regulation of the squid nerve Na(+)/Ca (2+) exchanger: recent developments.

In squid nerves, MgATP modulation of the Na(+)/Ca(2+) exchanger requires the presence of a cytosolic protein which becomes phosphorylated during the process. This factor has been recently identified. Mass spectroscopy and Western blot analysis established that it is a member of the lipocalin superfamily of lipid-binding proteins (LBP or FABP) of 132 amino acids.

Structural and functional studies of ReP1-NCXSQ, a protein regulating the squid nerve Na+/Ca2+ exchanger.

The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na(+)/Ca(2+) exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily.

Metabolic regulation of the squid nerve Na⁺/Ca²⁺ exchanger: recent kinetic, biochemical and structural developments.

The Na⁺/Ca²⁺ exchangers are structural membrane proteins, essential for the extrusion of Ca²⁺ from most animal cells. Apart from the transport sites, they have several interacting ionic and metabolic sites located at the intracellular loop of the exchanger protein. One of these, the intracellular Ca²⁺ regulatory sites, are essential and must be occupied by Ca²⁺ to allow any type of ion (Na⁺ or Ca²⁺) translocation.

In dialyzed squid axons oxidative stress inhibits the Na+/Ca2+ exchanger by impairing the Cai2+-regulatory site.

The Na(+)/Ca(2+) exchanger, a major mechanism by which cells extrude calcium, is involved in several physiological and physiopathological interactions. In this work we have used the dialyzed squid giant axon to study the effects of two oxidants, SIN-1-buffered peroxynitrite and hydrogen peroxide (H(2)O(2)), on the Na(+)/Ca(2+) exchanger in the absence and presence of MgATP upregulation. The results show that oxidative stress induced by peroxynitrite and hydrogen peroxide inhibits the Na(+)/Ca(2+) exchanger by impairing the intracellular Ca(2+) (Ca(i)(2+))-regulatory sites, leaving unharmed the intracellular Na(+)- and Ca(2+)-transporting sites.

Optimal metabolic regulation of the mammalian heart Na(+)/Ca(2+) exchanger requires a spacial arrangements with a PtdIns(4)-5kinase.

In inside-out bovine heart sarcolemmal vesicles, p-chloromercuribenzenesulfonate (PCMBS) and n-ethylmaleimide (NEM) fully inhibited MgATP up-regulation of the Na(+)/Ca(2+) exchanger (NCX1) and abolished the MgATP-dependent PtdIns-4,5P2 increase in the NCX1-PtdIns-4,5P2 complex; in addition, these compounds markedly reduced the activity of the PtdIns(4)-5kinase. After PCMBS or NEM treatment, addition of dithiothreitol (DTT) restored a large fraction of the MgATP stimulation of the exchange fluxes and almost fully restored PtdIns(4)-5kinase activity; however, in contrast to PCMBS, the effects of NEM did not seem related to the alkylation of protein SH groups. By itself DTT had no effect on the synthesis of PtdIns-4,5P2 but affected MgATP stimulation of NCX1: moderate inhibition at 1mM MgATP and 1μM Ca(2+) and full inhibition at 0.

Key role of a PtdIns-4,5P2 micro domain in ionic regulation of the mammalian heart Na+/Ca2+ exchanger.

Phosphatidylinositol biphosphate (PtdIns-4,5P(2)) plays a key role in the regulation of the mammalian heart Na(+)/Ca(2+) exchanger (NCX1) by protecting the intracellular Ca(2+) regulatory site against H(+)(i) and (H(+)(i)+Na(+)(i)) synergic inhibition. MgATP and MgATP-gamma-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P(2) incubated in the absence of MgATP and at normal [Na(+)](i), alkalinization increases the affinity for Ca(2+)(i) to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca(2+)(i) any further.

Squid nerve Na+/Ca2+ exchanger expressed in Saccharomyces cerevisiae: up-regulation by a phosphorylated cytosolic protein (ReP1-NCXSQ) is identical to that of native exchanger in situ.

This work shows, for the first time, a properly metabolically regulated squid nerve Na(+)/Ca(2+) exchanger (NCXSQ1) heterologous expressed in Saccharomyces cerevisiae. The exchanger was fused to the enhanced green fluorescence protein (eGFP) on its C-terminus and had two tags, a Strep-tag II and 6 histidines, added to the N-terminal region (ST-6HB-NCXSQ1-eGFP). The eGFP fluorescence signal co-localized with that of the plasma membrane marker FM1-43 in whole cells that displayed an uptake of Ca(2+) with the expected characteristics of the reverse Na(+)/Ca(2+) exchange mode.

The squid axon Na/Ca2+ exchanger shows ping pong kinetics only when the Ca2+(i)-regulatory site is saturated.

In a previous work we demonstrated that, in dialyzed squid axons, an impairment of the Ca2+(i)-regulatory site affected the apparent affinities for external Na+ and Ca2+ in a way opposite to that predicted by the exiting (ping-pong) models for the exchangers. In the present work, we used model simulations and actual experiments where the Ca2+(i)-regulatory remained always saturated while [Ca2+](i) was either limiting or near saturating for the internal Ca2+ transport sites. Under these conditions, both the theoretical and experimental transport activation curves for external Na+ and Ca2+ were those expected from the current kinetic schemes.

A novel lipid binding protein is a factor required for MgATP stimulation of the squid nerve Na+/Ca2+ exchanger.

Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na(+)/Ca(2+) exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ).

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins.

Giant protoplasts of Saccharomyces cerevisiae of 10-35 microm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and "patchability" of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation.

Dual effect of Nai+ on Ca2+ influx through the Na+/Ca2+ exchanger in dialyzed squid axons. Experimental data confirming the validity of the squid axon kinetic model.

We propose a steady-state kinetic model for the squid Na(+)/Ca(2+) exchanger that differs from other current models of regulation in that it takes into account, within a single kinetic scheme, all ionic [intracellular Ca(2+) (Ca(i)(2+))-intracellular Na(+) (Na(i)(+))-intracellular H(i)(+)] and metabolic (ATP) regulations of the exchanger in which the Ca(i)(2+)-regulatory pathway plays the central role in regulation. Although the integrated ionic-metabolic model predicts all squid steady-state experimental data on exchange regulation, a critical test for the validity of it is the predicted dual effect of Na(i)(+) on steady-state Ca(2+) influx through the exchanger. To test this prediction, an improved technique for the estimation of isotope fluxes in squid axons was developed, which allows sequential measurements of ion influx and effluxes.

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

In squid axons, intracellular Mg2+ reduces the activity of the Na+/Ca2+ exchanger by competing with Ca2+ i for its regulatory site. The state of the Ca i-regulatory site (active-inactive) also alters the apparent affinity of intra- and extracellular transport sites. Conditions that hinder the binding of Ca2+ i (low pH i, low [Ca2+]i, high [Mg2+]i) diminish the apparent affinity of intracellular transport sites, in particular for Na i due to its synergism with H+ inhibition, but less noticeably for Ca2+ i because of its antagonism towards (Ha i + Na+ i) and Mg2+ i inhibitions.

In bovine heart Na+/Ca2+ exchanger maximal Ca2+i affinity requires simultaneously high pHi and PtdIns-4,5-P2 binding to the carrier.

Na+ i-dependent Ca2+ uptake, Na+-dependent Ca2+ release, and PtdIns-4,5-P2 binding to Na+/Ca2+ exchanger (NCX1) as a function of extravesicular (intracellular) [Ca2+] were measured. Alkalinization increases Ca2+ i affinity and PtdIns-4,5-P2 bound to NCX1; these effects are abolished by pretreatment with PtdIns-PLC and are insensitive to MgATP. Acidification reduces Ca2+ i affinity.

Some biochemical properties of the upregulation of the squid nerve Na+/Ca2+ exchanger by MgATP and phosphoarginine.

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.

Maximal Ca2+i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger.

Using bovine heart sarcolemma vesicles we studied the effects of protons and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) on the affinity of the mammalian Na(+)/Ca(2+) exchanger (NCX1) for intracellular Ca(2+). By following the effects of extravesicular ligands in inside-out vesicles, their interactions with sites of NCX1 facing the intracellular medium were investigated. Two Na(+)-gradient-dependent fluxes were studied: Ca(2+) uptake and Ca(2+) release.

Sodium/calcium exchanger: influence of metabolic regulation on ion carrier interactions.

The Na(+)/Ca(2+) 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 Ca(2+) 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 Ca(2+)) and nontransported ionic species (protons and other monovalent cations).

SEA-0400, a potent inhibitor of the Na+/Ca2+ exchanger, as a tool to study exchanger ionic and metabolic regulation.

The effects of a new, potent, and selective inhibitor of the Na(+)/Ca(2+) exchange, SEA-0400 (SEA), on steady-state outward (forward exchange), inward (reverse exchange), and Ca(2+)/Ca(2+) transport exchange modes were studied in internally dialyzed squid giant axons from both the extra- and intracellular sides. Inhibition by SEA takes place preferentially from the intracellular side of the membrane. Its inhibition has the following characteristics: it increases synergic intracellular Na(+) (Na(i)(+)) + intracellular H(+) (H(i)(+)) inactivation, is antagonized by ATP and intracellular alkalinization, and is enhanced by intracellular acidification even in the absence of Na(+).

Phosphoarginine regulation of the squid nerve Na+/Ca2+ exchanger: metabolic pathway and exchanger-ligand interactions different from those seen with ATP.

In squid nerves the Na(+)-Ca(2+) exchanger is up-regulated by ATP and phosphoarginine (PA). ATP regulation involves drastic alterations in the Na(+)(i), H(+)(i) and Ca(2+)(i) interactions with the large intracellular cytoplasmic loop of the exchanger protein. In this work we explored the mechanisms associated with PA regulation in intracellular dialysed squid axons and squid optic nerve membrane vesicles.

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase.

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular.

Regulation of phosphatidylinositol-4,5-biphosphate bound to the bovine cardiac Na+/Ca2+ exchanger.

Western blot and cross immunoprecipitation analysis with specific antibodies demonstrate that in bovine heart sarcolemmal vesicles phosphatidylinositol-4,5-biphosphate (PtdIns-4,5-P(2)) binds strongly to the Na(+)/Ca(2+) exchanger (NCX1). This binding is modulated by ATP, Ca(2+), vanadate, exchanger inhibitory peptide (XIP), and PLC-PtdIns specific in a way resembling the ATP regulation of the exchange fluxes. With 1 microM Ca(2+), 3 mM Mg(2+), and 0.

Intracellular ionic and metabolic regulation of squid nerve Na+/Ca2+ exchanger.

Intracellular Na(+) and H(+) synergistically inhibit the squid Na(+)/Ca(2+) exchanger by reducing the affinity for Ca(2+) of its regulatory site. MgATP antagonizes H(+)(i) and Na(+)(i) inhibition; this effect must occur through a phosphorylation-dephosphorylation process, because exogenous protein phosphatases prevent MgATP activation of the exchanger. Protection by ATP against H(+)(i) and Na(+)(i) inhibition happens by decreasing the apparent affinity for the synergistic binding of these cations to the carrier.

Ionic ligand interactions with the intracellular loop of the sodium-calcium exchanger. Modulation by ATP.

In the last decade, there has been a large increase in the study of the Na(+)/Ca(2+) exchanger due to its implications in physiological and pathophysiological processes at the cell and organ levels. Key areas of these studies have been molecular biology, regulation and physiology-pathophysiology of the exchanger. There are three main types of regulation that take place at the large intracellular loop of the Na(+)/Ca(2+) exchanger: (i) ionic (sodium inactivation, calcium regulation and proton inhibition), (ii) metabolic (ATP as phosphoryl group donor), and (iii) genetic (alternative splicing).

MgATP and phosphoinositides activate Na(+)/Ca(2+) exchange in bovine brain vesicles. Comparison with other Na(+)/Ca(2+) exchangers.

We investigated the metabolic modulation of the Na(+)/Ca(2+) exchanger in membrane vesicles obtained from bovine brain. The Na(+)/Ca(2+) exchanger was activated by MgATP with a K(0.5) of 336 micro M.

MgATP counteracts intracellular proton inhibition of the sodium-calcium exchanger in dialysed squid axons.

Intracellular Na(+) and H(+) inhibit Na(+)-Ca(2+) exchange. ATP regulates exchange activity by altering kinetic parameters for Ca(2+)(i), Na(+)(i) and Na(+)(o). The role of the Ca(2+)(i)regulatory site on Na(+)(i)-H(+)(i)-ATP interactions was explored by measuring the Na(+)(o)-dependent (45)Ca(2+) efflux (Na(+)(o)-Ca(2+)(i) exchange) and Ca(2+)(i)-dependent (22)Na(+) efflux (Na(+)(o)-Na(+)(i) exchange) in intracellular-dialysed squid axons.