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

Abstract

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 ∼500 μM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 μM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5′- O-(3-thiotriphosphate), without the need for vanadate, but not by β,γ-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-α-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 ofl-α-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 μM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+exchanger through a fast phosphorylation chain: a Ca2+-independent PIP formation followed by a Ca2+-dependent synthesis of PIP2.

Sodium/Calcium Exchanger (NCX1) Macromolecular Complex

“…Regulation by Phosphorylation-Early experiments suggested that ATP played an important regulatory role in NCX1 function (26,64,65). Possible roles for ATP were "direct" regulation or through phosphorylation of NCX1.…”

“…The intracellular loop also contains a regulatory Ca 2ϩ binding site, Na ϩ association site, sites for H ϩ regulation, and a "XIP" binding site. Although definite view on the spatial organization of the intracellular loop will await a structural investigation of this part of the protein, the preliminary data to date suggest that these regions of association in the intracellular loop interact.Regulation by Phosphorylation-Early experiments suggested that ATP played an important regulatory role in NCX1 function (26,64,65). Possible roles for ATP were "direct" regulation or through phosphorylation of NCX1.…”

Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions

“…The dominant pathway of PIP 2 synthesis, as in other cells, is probably the sequential phosphorylation in the sarcolemma of phosphatidylinositol (PI) at the 4-and then the 5-positions of inositol (66). As in other cells, PIP 2 is hydrolyzed by PLCs to generate IP 3 and DAG, or it can be dephosphorylated to PIP and PI. DAG can be phosphorylated to generate phosphatidic acid (PA) by DAG kinases, which may be regulated by translocation to the surface membrane (32,45), and dephosphorylation of PA can probably be an important source of DAG in heart (for example, see Ref.…”

“…Third, there are no reliable pharmacological tools to inhibit individual mechanisms involved in PIP 2 metabolism. Fourth, quantitative measurements of PIP 2 are more problematic than measurements of IP 3 . One recent innovation, which overcomes some of these problems, is to monitor the membrane association of green fluorescent proteincoupled PIP 2 -binding domains (57,62).…”

“…PIP 2 and IP 3 levels both plummet in response to cell isolation, and they remain depressed in cultured adult myocytes (71,72). An abundance of studies, in which neonatal and adult myocyte cultures were used, demonstrate increases of inositol phosphates, including IP 3 , with activation of G␣ q -coupled receptor pathways (72). However, basal IP 3 levels are so high in intact heart that it is difficult to demonstrate a rise of IP 3 with activation of G␣ qcoupled receptors.…”