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 ␤-...

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greater donor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB is essentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Ca is due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.

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