Luis Beaugé

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. On the other hand, prevention of Na(+)(i) inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P(2). Experiments on NCX1-PtdIns-4,5P(2) cross-coimmunoprecipitation show that the relevant PtdIns-4,5P(2) is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca(2+)(i) regulatory site is seen in the deprotonated and PtdIns-4,5P(2)-bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.

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. These observations have two important implications: On the one hand, they confirm the ping-pong translocation schemes for Na⁺/Ca²⁺ exchange. On the other, they call for caution in interpreting kinetic data in membrane transport systems possessing intracellular ionic and/or metabolic regulation.

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. Similar to squid nerve membrane vesicles, inside-out yeast plasma membrane vesicles (ISOV) showed a Ca(2+)(i) regulation of the forward mode that was modulated by previously phosphorylated regulatory cytosolic protein (ReP1-NCXSQ). On the other hand, a close association between NCXSQ1 and ReP1-NCXSQ, estimated by co-immunoprecipitation, was independent of ReP1-NCXSQ phosphorylation. An additional crucial observation was that in proteoliposomes containing only the ST-6HB-NCXSQ1-eGFP protein, Na(+)/Ca(2+) exchange was stimulated by phosphorylated ReP1-NCXSQ; i.e., this up-regulation needs no other requirement besides the lipid membrane and the exchanger protein. Finally, this work provides a potential approach to obtain enough purified NCXSQ1 for structural and biochemical studies which have been delayed due to the lack of sufficient material.

Optimal metabolic regulation of the mammalian heart Na+/Ca2+ 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 0.25mM MgATP and 0.2μM Ca(2+). In addition, DDT prevented coimmunoprecipitation of NCX1 and PtdIns(4)-5kinase. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be (i) functionally competent and (ii) set in the NCX1 microenvironment closely associated to the exchanger. This kind of supramolecular structure is needed to optimize binding of the newly synthesized PtdIns-4,5P2 to its target region in the exchanger protein.

In Bovine Heart Na+/Ca2+ Exchanger Maximal Ca2+i Affinity Requires Simultaneously High pHi and PtdIns-4,5-P2 Binding to the Carrier

Abstract:  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. MgATP reverts it only partially despite the fact that the PtdIns‐4,5‐P2 bound to NCX1 reaches the same levels as at pH 7.8. Extravesicular Na+‐stimulated and Ca2+‐dependent Ca2+efflux indicate the Ca2+ regulatory site is involved. Therefore, to display maximal affinity to Ca2+i, PtdIns‐4,5‐P2 binding and deprotonation of NCX1 are simultaneously need.