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.The Na ϩ /Ca 2ϩ exchanger, NCX1, is an integral membrane protein that is expressed in many tissues and is involved in cellular Ca 2ϩ homeostasis (1, 2). The expression level of NCX1 is modulated during development (3, 4) and under pathological conditions (5-9). The Na ϩ /Ca 2ϩ exchanger activity has been shown to be affected by the ions that it transports (Na ϩ and Ca 2ϩ ) (10 -13), by protons (14,15), by phosphatidylinositol 4,5-bisphosphate in the membrane (16), and by exogenous agents including intracellular application of an inhibitor peptide (XIP) (17). However, regulation of NCX1 by PKA 1 has remained controversial (18 -23).Early studies suggested that ATP-dependent regulation of NCX1 occurred in squid axons (24,25) and in cardiac sarcolemmal vesicles (26), but these studies did not distinguish between direct ATP binding and ATP-dependent phosphorylation. Several studies were designed to resolve this problem. Hilgemann and colleagues (11,12,27,28) investigated the question by measuring NCX1 currents in giant excised patches. The work used two preparations, NCX1-expressing Xenopus oocytes (27) or cardiac myocytes expressing native NCX1 (11,12,28). These investigations found no functional change in cardiac NCX1 activity following application of PKA or protein kinase C (PKC) catalytic subunits to...