Phospholemman-Phosphorylation Mediates the β-Adrenergic Effects on Na/K Pump Function in Cardiac Myocytes

Abstract

Abstract-Cardiac sympathetic stimulation activates ␤-adrenergic (␤-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na] i ), which in turn affects [Ca] i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na] i decline and current (I Pump ) to assess ␤-AR effects on NKA function in isolated myocytes. In WT myocytes, 1 mol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (K m decreased from 18.8Ϯ1.4 to 13.6Ϯ1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na] i (from 12.5Ϯ1.8 to 10.5Ϯ1.4 mmol/L). In PLM-KO mice under control conditions K m (14.2Ϯ1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower K m (Na) in PLM-KO versus WT (12.9Ϯ0.9 versus 16.2Ϯ1.5). Thus, PLM inhibits NKA activity by decreasing its [Na] i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation). Key Words: Na pump Ⅲ phospholemman Ⅲ signal transduction Ⅲ ion channels A ctivation of the sympathetic nervous system and cardiac ␤-adrenergic (␤-AR) receptors causes cAMP formation and activation of protein kinase A (PKA). In cardiac myocytes, PKA phosphorylates several targets with key roles in the control of excitation-contraction coupling (ECC), including L-type Ca 2ϩ channels, phospholamban (PLB) and troponin-I, as well as other sarcolemmal proteins such as voltage-gated Na and K channels and phospholemman (PLM).During sympathetic activation, the larger Ca influx via more frequent and larger Ca current must be balanced by enhanced Ca extrusion via the Na/Ca exchange (NCX) that is driven by larger Ca transients. This increases Na influx at each beat, along with more frequent and larger Na current, which increases intracellular [Na] ([Na] i ). To limit the rise in [Na] i , the greater Na influx must be compensated for by an enhanced Na extrusion via the Na/K pump (NKA). Indeed, many early studies indicated stimulation of the Na-pump by ␤-AR activation. 1-3 However, there is controversy at present because some recent studies in single myocytes using NKA pump current (I Pump ) found either stimulation, 4 -6 inhibition, 7,8 or no change 9 in I Pump on ␤-...

Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchanger's proximal linker domain

“…Phosphorylation of Ser 68 of PLM by PKA or PKC relieves its inhibition on Na ϩ -K ϩ -ATPase (7,11,12,28,32). By contrast, PLM phosphorylated at Ser 68 is the active species that inhibits NCX1 (34,39).…”

“…PLM regulates the activities of Na ϩ -K ϩ -ATPase (4,6,7,11,32) and Na ϩ /Ca 2ϩ exchanger (NCX1) (1,40) in the heart. Phosphorylation of Ser 68 of PLM by PKA or PKC relieves its inhibition on Na ϩ -K ϩ -ATPase (7,11,12,28,32).…”

Regulation of cardiac contractility: high time for FXYD

“…Phosphorylation of PLM by either PKA or PKC relieves its inhibition of Na ϩ -K ϩ -ATPase (5,7,13). Theoretically, inhibition of Na ϩ -K ϩ -ATPase by PLM is expected to raise intracellular Na ϩ concentration ([Na ϩ ] i ), thereby altering the thermodynamic driving force for Na ϩ / Ca 2ϩ exchange and resulting in increased intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and enhanced cardiac contractility.…”

“…Theoretically, inhibition of Na ϩ -K ϩ -ATPase by PLM is expected to raise intracellular Na ϩ concentration ([Na ϩ ] i ), thereby altering the thermodynamic driving force for Na ϩ / Ca 2ϩ exchange and resulting in increased intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and enhanced cardiac contractility. Myocytes isolated from congenic PLM-null mice, however, had similar resting [Na ϩ ] i (5) and contraction amplitudes at physiological extracellular Ca 2ϩ concentration (16) compared with wild-type C57BL/6 myocytes. Another complexity is that PLM has been shown to regulate cardiac Na ϩ /Ca 2ϩ exchange activity, independent of its effects on Na ϩ -K ϩ -ATPase (3).…”

“…What is the change in the phosphorylated fraction of PLM between basal and ␤-adrenergic-stimulated states? Are there different pools of PLM (and, by association, Na ϩ -K ϩ -ATPase) in the heart that respond separately to PKA or PKC stimulation (5,7,13)? What is the role of the TM segment when the cytoplasmic tail of PLM is sufficient to associate with and inhibit Na ϩ -K ϩ -ATPase (11)?…”