NCX1 phosphorylation dilemma: a little closer to resolution. Focus on "Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A".

NA /CA2 EXCHANGER (NCX) proteins constitute a family of solute carriers that provide the major pathway for removal of calcium from the cytosol in exchange for three times as many sodium ions. The vertebrate NCX proteins are expressed as tissue-specific subtypes coded by multiple genes (NCX1, NCX2, and NCX3 in mammals) and further diversified by variable splicing (e.g., NCX1.1, NCX1.2, and NCX1.4). These subtypes show subtle differences in regulation caused by the transported cations and possibly by adrenergic transmitters. -Adrenergic signaling is mediated by the cAMP-dependent protein kinase A (PKA) or more significantly, CaM kinase II, which in heart muscle can phosphorylate the L-type calcium channels, phospholamban [regulating sarco(endo)plasmic reticulum Ca -ATPase (SERCA) activity], the ryanodine receptor, and myofibrils. Because NCX plays such an important role in calcium homeostasis in both the normal and failing heart, possible -adrenergic-mediated phosphorylation of NCX would be expected to have significant consequences on cardiac contractility. Unfortunately, the issue of adrenergic regulation of NCX remains controversial because of conflicting data in support of direct PKA-mediated phosphorylation of NCX. In this issue of AJP-Cell Physiol, using bioinformatics analysis and peptide arrays, Wanichawan et al. (30) present compelling data that mammalian cardiac NCX is not a direct target for PKA. Early studies using squid axons (5, 6) and sarcolemmal vesicles (2) did not differentiate between direct binding effects of ATP and ATP-dependent phosphorylation. Hilgemann et al. (13, 19, 20) addressed this problem but found no functional changes in NCX current (INCX) resulting from phosphorylation following application of PKA or PKC catalytic subunits to excised giant patches of cardiac myocytes or in dog cardiac NCX1-expressing Xenopus oocytes. While we and others (4) have confirmed the absence of -adrenergic and ATP effects on cloned mammalian cardiac NCX1 exchangers in various expression systems, some groups have found stimulatory effects in intact mammalian cardiomyocytes (10, 25). Studies using single cells (with welldeveloped sarcoplasmic reticulum) have been subject to criticism based on the possibility that PKA may exert its effect on NCX indirectly. This stresses the need for simultaneous measurements of INCX (including its reversal potential), intracellular Ca ([Ca ]i) [carried out at physiological temperatures (25)], and direct measurements of NCX1 phosphorylation. The dual challenge of demonstrating phosphorylation and conducting experiments at physiological temperatures was taken up by Ruknudin et al. (26, 27) who found enhancement of outward INCX of cardiac (NCX1.1) but not renal (NCX1.3) splice variants (27). Nevertheless, more critical studies by a number of labs showed no stimulation of cardiac NCX1 in rabbit (9) and rodents (18), consistent with our observations in the guinea pig, rat ventricular myocytes, and recombinant dog NCX expressed in human embryonic kidney (HEK)293 cell lines (11).