Phospholemman and the cardiac sodium pump: protein kinase C, take a bow.

In excitable tissues, the activity of the plasmalemmal sodium/potassium ATPase (Na/K pump) is vital for the maintenance of normal electrical activity and ion gradients. In cardiac muscle, the transsarcolemmal sodium (Na) gradient established by the Na/K activity is essential not only for generating the rapid upstroke of the action potential but also for driving a number of ion exchange and transport processes that are crucial for normal cellular function, excitation contraction coupling, ion homeostasis and the control of cell volume. These Na-dependent membrane transporters include those responsible for the regulation of other ions (such as the sodium calcium exchanger (NCX), Na/H exchanger and Na-HCO3 cotransporter), as well as those involved in the movement of substrates and amino acids.1 By determining the set point for NCX, the Na/K pump controls the predominant mechanism of transmembrane calcium (Ca) flux, and hence indirectly controls intracellular Ca load and myocardial contractility. Interventions that influence either the activity of the Na/K pump, or indirectly the transmembrane sodium gradient, can therefore profoundly affect normal cardiac function. Phospholemman, a type 1 transmembrane protein, is the predominant quantitative site of phosphorylation by protein kinase A (PKA) and protein kinase C (PKC) in cardiac sarcolemma.2 PKA phosphorylates serine 68 and PKC phosphorylates serines 63 and 68 in phospholemman. For a long time following its cloning in 1991,3 the physiological role of phospholemman was unclear. Indeed, writing in this journal in 1998, researchers noted that “As a major target for hormone-stimulated phosphorylation in the heart, the physiological function of phospholemman is likely to be an important one”,4 however they were unable to suggest what this function may be. Various roles have now been proposed: 1) regulation of cell volume; 2) regulation of cardiac NCX; and 3) regulation of cardiac Na/K pump. Despite its size (only 72 amino acids), phospholemman is certainly a busy protein in cardiac myocytes. The description in 2000 of a new gene family of ion transport regulators termed “FXYD” led to considerable progress in defining the physiological function of phospholemman.5 In cardiac myocytes, phospholemman (FXYD1) associates with the Na/K pump.6 Evidence from several laboratories supports the notion that phospholemman provides the link between cardiac kinases and the Na/K pump.7–9 Phosphorylation of phospholemman by PKA at serine 68 is associated with Na/K pump activation,7–10 and it has been proposed that phospholemman regulates the Na/K pump in a manner analogous to regulation of the calcium ATPase SERCA 2a by phospholamban: phosphorylation leading to disinhibition through elevated substrate affinity. In phospholemman deficient mice, -adrenergic stimulation of the Na/K pump is absent.9 Sympathetic stimulation of cardiac myocytes involves activation of PKA, via -adrenergic receptors, and PKC, via -adrenergic receptors. Whereas a considerable amount of effort has been dedicated to investigating the effect of PKA agonists on phospholemman and the cardiac Na/K pump, the effect of PKC phosphorylation of phospholemman has remained undefined. In this issue of Circulation Research, Han et al11 have investigated the effect of PKC activation on Na/K pump function, and the role played in this by phospholemman. Every laboratory has their favorite method of measuring Na/K pump activity. The power of the research presented by Han et al11 lies in their use of 3 different techniques to investigate Na/K pump in freshly isolated mouse cardiomyocytes. Measurement of intracellular Na using the Na sensitive dye SBFI assesses the Na/K pump-dependent extrusion of Na after a period of pump inhibition. Simultaneous measurement of intracellular Na and Na/K pump current using 3 to 5 M patch electrodes defines Na/K pump currents over a range of intracellular sodium. Low resistance patch electrodes measure Na/K pump currents at a single intracellular Na. So, not only is the transporting function of the Na/K pump assessed as the generation of an outward current (the exchange of 3Na for 2K), it is also assessed as the extrusion of its intracellular substrate (Na) as a function of that substrate concentration. All 3 techniques agree that PKC activation with phorbol ester increases Na/K pump Vmax, but is without effect on the enzyme’s Na affinity. Crucially, PKC-dependent stimulation of Na/K pump is absent in phospholemman deficient mice, indicating that, as for PKA, phospholemman provides the functional link between kinase and Na/K pump. Sympathetic stimulation of the heart activates both PKA and PKC. Han et al11 have gone on to investigate the interaction of these 2 pathways in regulating the Na/K pump. Interestingly, there appears to be little overlap between the 2. PKA activation reduces K0.5 for Na and PKC activation increases Vmax regardless of the prior state of the myocyte. The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Institute for Cardiovascular Research (W.F.), Ninewells Hospital, University of Dundee, UK; and the Cardiovascular Division (M.J.S.), King’s College London, UK. Correspondence to Dr William Fuller, University of Dundee, Vascular Diseases Unit, Institute for Cardiovascular Research, Ninewells Hospital & Medical School, Dundee DD1 9SY, United Kingdom. E-mail [email protected] (Circ Res. 2006;99:1290–1292.) © 2006 American Heart Association, Inc.