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. 2009 Sep 25;284(39):26749-57.
doi: 10.1074/jbc.M109.047357. Epub 2009 Jul 28.

Isoform specificity of the Na/K-ATPase association and regulation by phospholemman

Julie Bossuyt  1 , Sanda Despa, Fei Han, Zhanjia Hou, Seth L Robia, Jerry B Lingrel, Donald M Bers
Affiliations

Isoform specificity of the Na/K-ATPase association and regulation by phospholemman

Julie Bossuyt et al. J Biol Chem. .

Abstract

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-alpha1 or CFP-NKA-alpha2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 +/- 3.4% for NKA-alpha1 and 27.5 +/- 2.5% for NKA-alpha2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-alpha2 versus PLM-NKA-alpha1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving >or=3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-alpha1 is ouabain-sensitive and NKA-alpha2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-alpha1 and NKA-alpha2 function. Isoproterenol enhanced internal Na(+) affinity of both isoforms (K((1/2)) decreased from 18.1 +/- 2.0 to 11.5 +/- 1.9 mm for NKA-alpha1 and from 16.4 +/- 2.5 to 10.4 +/- 1.5 mm for NKA-alpha2) without altering maximum transport rate (V(max)). Protein kinase C activation also decreased K((1/2)) for both NKA-alpha1 and NKA-alpha2 (to 9.4 +/- 1.0 and 9.1 +/- 1.1 mm, respectively) but increased V(max) only for NKA-alpha2 (1.9 +/- 0.4 versus 1.2 +/- 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-alpha1 and NKA-alpha2 in a comparable but not identical manner.

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Figures

FIGURE 1.
FIGURE 1.
FRET between CFP-NKA-α1/CFP-NKA-α2 and PLM-YFP transfected in HEK-293 cells detected with a progressive acceptor photobleaching protocol. A, CFP-NKA-α2 and PLM-YFP epifluorescence images before starting the photobleaching (Pre-bleach) and at 400 s during photobleaching (Post-bleach). The intensity of YFP emission decreases exponentially, whereas the CFP fluorescence increases with the photobleaching of YFP, indicating the existence of FRET. B and C, mean data for the CFP- NKA and PLM-YFP fluorescence during the progressive acceptor photobleaching protocol for cells transfected with PLM and CFP-NKA-α1 (B, 24 cells) or CFP-NKA-α2 (C, 40 cells). D, change in the donor (PLM-YFP) versus acceptor (CFP-NKA) fluorescence during the progressive acceptor photobleach. The relationship is linear for both NKA-α1 and NKA-α2.
FIGURE 2.
FIGURE 2.
Detection of FRET between PLM-CFP and PLM-YFP. A, relative change in the PLM-CFP and PLM-YFP fluorescence during the progressive acceptor photobleaching protocol. B, relative change in the donor (PLM-YFP) versus acceptor (PLM-CFP) fluorescence during the progressive acceptor photobleach.
FIGURE 3.
FIGURE 3.
Effect of PLM phosphorylation and NKA inhibition on the FRET between CFP-NKA-α1/CFP-NKA-α2 and PLM-YFP. A, effect of high (Forsk, 10 μm; mean of 47 and 20 cells for NKA-α1 and NKA-α2, respectively) and low concentrations (10 nm, mean of 37 and 47 cells for NKA-α1 and NKA-α2, respectively) of forskolin, 8-bromo-cAMP (10 μm, mean of 35 and 49 cells for NKA-α1 and NKA-α2, respectively), and 100 nm PDBu (n = 52 and 58 for NKA-α1 and NKA-α2, respectively); Ctl, control. B, effect of various concentrations of ouabain on the FRET between PLM-NKA-α1 and PLM-NKA-α2. More than 14 cells were used for each experimental condition. C and D, effect of ouabain treatment (10 mm for 10 min) on PLM co-immunoprecipitation with NKA-α1 and NKA-α2. The NKA isoform signal (at 110 kDa) was quantified in the immunoprecipitate (IP:PLM column) and normalized to the corresponding PLM signal (at 10 kDa). Ouabain (Oua) did not affect the total NKA-α1 and NKA-α2 amount (Load column).
FIGURE 4.
FIGURE 4.
Measurement of NKA-α1- and NKA-α2-mediated Na+ efflux in intact mouse myocytes. The ouabain-sensitive component (top, NKA-α2 in WT mice; bottom, NKA-α1 in SWAP mice) was completely blocked with 20 μm ouabain (Oua), present throughout the experiment, and we measured the Na+ dependence of the rate of Na+ extrusion mediated by the ouabain-insensitive NKA isoform. The ouabain-insensitive NKA was first blocked (0K, 145 Na+ external solution) causing [Na+]i loading, then reactivated in 4K,0Na solution. The protocol was repeated with 1 μm ISO present. ISO was applied 10 min before activating the pump and then throughout measuring the NKA function.
FIGURE 5.
FIGURE 5.
Effect of ISO (1 μm) on Na+ efflux through NKA-α1 and NKA-α2. A, [Na+]i dependence of Na+ extrusion by NKA-α1 (the ouabain-sensitive Na+/K+ pump in WT mice, n = 9 cells, 6 hearts) and NKA-α2 (in SWAP mice, n = 8 cells, 6 hearts) with and without 1 μm ISO. B, maximum Na+ extrusion rate, Vmax, of NKA-α1 and NKA-α2, with and without ISO. C, [Na+]i for the half-maximal activation of the pump, K½, of NKA-α1 and NKA-α2, with and without ISO.
FIGURE 6.
FIGURE 6.
Effect of PDBu (300 nm) on Na+ efflux through NKA-α1 and NKA-α2. A, [Na+]i dependence of Na+ extrusion by NKA-α1 (n = 5 cells, 3 hearts) and NKA-α2 (n = 6 cells, 3 hearts) with and without 300 nm PDBu. B, maximum Na+ extrusion rate, Vmax of NKA-α1 and NKA-α2, with and without PDBu. C, [Na+]i for the half-maximal activation of the pump, K½, of NKA-α1 and NKA-α2, with and without PDBu.
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References

    1. Sweadner K. J., Rael E. (2000) Genomics 68, 41–56 - PubMed
    1. Presti C. F., Jones L. R., Lindemann J. P. (1985) J. Biol. Chem. 260, 3860–3867 - PubMed
    1. Palmer C. J., Scott B. T., Jones L. R. (1991) J. Biol. Chem. 266, 11126–11130 - PubMed
    1. Crambert G., Fuzesi M., Garty H., Karlish S., Geering K. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11476–11481 - PMC - PubMed
    1. Feschenko M. S., Donnet C., Wetzel R. K., Asinovski N. K., Jones L. R., Sweadner K. J. (2003) J. Neurosci. 23, 2161–2169 - PMC - PubMed

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