Abstract
The sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane
protein essential in calcium homeostasis for many cells including
those in mammalian heart and brain. The function of NCX1 depends
on subcellular ("local") factors, the phosphorylation state of NCX1,
and the subcellular location of NCX1 within the cell. Here we investigate
the molecular organization of NCX1 within the cardiac myocyte. We
show that NCX1 is dynamically phosphorylated by protein kinase A
(PKA)-dependent phosphorylation in vitro. We also provide evidence
that the regulation of this phosphorylation is attributed to the
existence of an NCX1 macromolecular complex. Specifically, we show
that the macromolecular complex includes both the catalytic and regulatory
subunits of PKA. However, only the RI regulatory subunit is found
in this macromolecular complex, not RII. Other critical regulatory
enzymes are also associated with NCX1, including protein kinase C
(PKC) and two serine/threonine protein phosphatases, PP1 and PP2A.
Importantly, the protein kinase A-anchoring protein, mAKAP, is found
and its presence in the macromolecular complex suggests that these
regulatory enzymes are coordinately positioned to regulate NCX1 as
has been found in diverse cells for a number of channel proteins.
Dual immunocytochemical staining showed the colocalization of NCX1
protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally,
leucine/isoleucine zipper motifs have been identified as possible
sites of interaction. Our finding of an NCX1 macromolecular complex
in heart suggests how NCX1 regulation is achieved in heart and other
cells. The existence of the NCX1 macromolecular complex may also
provide an explanation for recent controversial findings.
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