Effects of Phospholemman Expression on Swelling-Activated Ion Currents and Volume Regulation in Embryonic Kidney Cells

Davis, Cristina E.; Patel, Manoj K.; Miller, James R.; John, J. Edward; Jones, Larry R.; Tucker, Amy L.; Mounsey, J. Paul; Moorman, J. Randall

doi:10.1023/b:nere.0000010447.24128.ac

Cited by 25 publications

(30 citation statements)

References 57 publications

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“…1D). In agreement with previous studies, neither NCX1 (8) nor PLM (19) was detected in HEK293 cells transfected with only the empty pAdTrack expression plasmid (image not shown; Western blots shown in Fig. 2).…”

Section: Resultssupporting

confidence: 93%

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Identification of an Endogenous Inhibitor of the Cardiac Na+/Ca2+ Exchanger, Phospholemman

Ahlers

Zhang

Moorman

et al. 2005

Journal of Biological Chemistry

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Rapid and precise control of Na؉ /Ca 2؉ exchanger (NCX1) activity is essential in the maintenance of beatto-beat Ca 2؉ homeostasis in cardiac myocytes. Here, we show that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, is a novel endogenous protein inhibitor of cardiac NCX1. Using a heterologous expression system that is devoid of both endogenous PLM and NCX1, we first demonstrated by confocal immunofluorescence studies that both exogenous PLM and NCX1 co-localized at the plasma membrane. Reciprocal co-immunoprecipitation studies revealed specific protein-protein interaction between PLM and NCX1. The functional consequences of direct association of PLM with NCX1 was the inhibition of NCX1 activity, as demonstrated by whole-cell patch clamp studies to measure NCX1 current density and radiotracer flux assays to assess Na ؉ -dependent 45 Ca 2؉ uptake. Inhibition of NCX1 by PLM was specific, because a single mutation of serine 68 to alanine in PLM resulted in a complete loss of inhibition of NCX1 current, although association of the PLM mutant with NCX1 was unaltered. In native adult cardiac myocytes, PLM co-immunoprecipitated with NCX1. We conclude that PLM, a member of the FXYD family of small ion transport regulators known to modulate Na ؉ -K ؉ -ATPase, also regulates Na ؉ /Ca 2؉ exchange in the heart.

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Section: Resultssupporting

confidence: 93%

“…pcDNA3-expressing PLM also demonstrated the minor bands (19). The identities of these minor bands are at present unknown.…”

Section: Inhibition Of Na ϩ /Ca 2ϩ Exchanger By Phospholemmanmentioning

confidence: 93%

Identification of an Endogenous Inhibitor of the Cardiac Na+/Ca2+ Exchanger, Phospholemman

Ahlers

Zhang

Moorman

et al. 2005

Journal of Biological Chemistry

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“…There are strong interactions between various residues occluding the central pore of the tetramer; thus, we believe that any conformational change to an open pore would require major structural rearrangements and disruption of interresidue interactions, which are incompatible with the typical delicate balance between open-close conformations in voltage-or ligand-gated transmembrane channels. However, it has been shown that hyperpolarization activates anion currents through PLM (40), and based on further experiments with embryonic kidney cells, it has been hypothesized that PLM may facilitate osmolyte influx in renal tissue (18). Indeed, the clustering of four negative charges at the extracellular entrance would provide a binding site for the zwitterionic osmolyte taurine.…”

Section: Discussionmentioning

confidence: 99%

“…Electrical measurements of PLM in artificial lipid bilayers and oocytes showed that PLM facilitates the membrane flux of ions (16) and taurine transport (17), which leads to the possibility that PLM has a function in the regulation of cell volume either as a modulator of a swelling-activated signal transduction pathway or directly facilitating osmolyte influx in noncardiac tissues (18).…”

Section: Human Phospholemman (Plm)mentioning

confidence: 99%

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Phospholemman Transmembrane Structure Reveals Potential Interactions with Na+/K+-ATPase

Beevers

Kukol

2007

Journal of Biological Chemistry

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Phospholemman (PLM) is a 72-residue bitopic cardiac transmembrane protein, which acts as a modulator of the Na ؉ /K ؉ -ATPase and the Na ؉ /Ca 2؉ exchanger and possibly forms taurine channels in nonheart tissue. This work presents a high resolution structural model obtained from a combination of site-specific infrared spectroscopy and experimentally constrained high throughput molecular dynamics (MD) simulations. Altogether, 37 experimental constraints, including nine long range orientational constraints, have been used during MD simulations in an explicit lipid bilayer/water system. The resulting tetrameric ␣-helical bundle has an average helix tilt of 7.3°a nd a crossing angle close to 0°. It does not reveal a hydrophilic pore, but instead strong interactions between various residues occlude any pore. The helix-helix packing is unusual, with Gly Human phospholemman (PLM)2 is a member of a family of single-span transmembrane proteins characterized by the invariant extracellular motif FXYD (1) and is also known as FXYD1. It is found in liver, skeletal muscle, and most abundantly in the cardiac sarcolemma (2). Over recent years, conclusive experimental evidence has been presented that PLM acts as a tissue-specific modulator of the Na ϩ /K ϩ -ATPase similar to the other members of the FXYD family, each of which is prevalent in a different tissue, summarized in several recent reviews (3, 4). The Na ϩ /K ϩ -ATPase consists of a catalytic ␣-subunit with 10 transmembrane segments and a ␤-subunit involved in membrane insertion and as a modulator of transport properties (5). In particular, cross-linking and modeling studies revealed the direct interaction between the Na ϩ /K ϩ -ATPase ␣-subunit and members of the FXYD family (␥ and CHIF), which involves the binding of a single transmembrane domain into a groove formed by M2, M6, and M9 transmembrane helices of the Na ϩ /K ϩ -ATPase (6, 7). PLM appears to be a major control point in the function of heart cells, although its specific physiological role is unclear, complicated by the possibility that its regulatory effect on the Na ϩ /K ϩ -ATPase may depend on the phosphorylation state of PLM (8) as well as other less well established functional roles (e.g. interaction with the Na/Ca 2ϩ exchanger (9, 10) or independent channel formation). PLM knock-out mice expressed a complex response, including increased cardiac mass, larger cardiomyocytes, and ejection fractions in the absence of hypertension, whereas the overall Na ϩ /K ϩ -ATPase activity was reduced by 50% (11). There is growing evidence that PLM regulates the activity of Na/Ca 2ϩ exchanger 1 (NCX1) from heterologous expression studies of PLM and NCX1 in HEK239 cells (10) and overexpression of PLM in rat cardiomyocytes (12, 13) using different approaches to measure NCX1 activity. It appears that phosphorylation of PLM abolishes its inhibitory effect on the Na ϩ /K ϩ -ATPase (14, 15), whereas the phosphorylated form of PLM inhibits NCX1 (9, 12).Electrical measurements of PLM in artificial lipid bilayers and oocytes sh...

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Coordinated Regulation of Cardiac Na+/Ca2+ Exchanger and Na+-K+-ATPase by Phospholemman (FXYD1)

Cheung

Zhang

Song

et al. 2012

Advances in Experimental Medicine and Biology

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Phospholemman (PLM) is the founding member of the FXYD family of regulators of ion transport. PLM is a 72-amino acid protein consisting of the signature PFXYD motif in the extracellular N terminus, a single transmembrane (TM) domain, and a C-terminal cytoplasmic tail containing three phosphorylation sites. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. The TM domain of PLM interacts with TM9 of the α-subunit of Na(+)-K(+)-ATPase, while its cytoplasmic tail interacts with two small regions (spanning residues 248-252 and 300-304) of the proximal intracellular loop of Na(+)/Ca(2+) exchanger. Under stress, catecholamine stimulation phosphorylates PLM at serine(68), resulting in relief of inhibition of Na(+)-K(+)-ATPase by decreasing K(m) for Na(+) and increasing V(max), and simultaneous inhibition of Na(+)/Ca(2+) exchanger. Enhanced Na(+)-K(+)-ATPase activity lowers intracellular Na(+), thereby minimizing Ca(2+) overload and risks of arrhythmias. Inhibition of Na(+)/Ca(2+) exchanger reduces Ca(2+) efflux, thereby preserving contractility. Thus, the coordinated actions of PLM during stress serve to minimize arrhythmogenesis and maintain inotropy. In acute cardiac ischemia and chronic heart failure, either expression or phosphorylation of PLM or both are altered. PLM regulates important ion transporters in the heart and offers a tempting target for development of drugs to treat heart failure.

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Effects of Phospholemman Expression on Swelling-Activated Ion Currents and Volume Regulation in Embryonic Kidney Cells

Cited by 25 publications

References 57 publications

Identification of an Endogenous Inhibitor of the Cardiac Na+/Ca2+ Exchanger, Phospholemman

Identification of an Endogenous Inhibitor of the Cardiac Na+/Ca2+ Exchanger, Phospholemman

Phospholemman Transmembrane Structure Reveals Potential Interactions with Na+/K+-ATPase

Coordinated Regulation of Cardiac Na+/Ca2+ Exchanger and Na+-K+-ATPase by Phospholemman (FXYD1)

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