Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman

Morales-Mulia, Marcela; Pasantes‐Morales, Herminia; Morán, Julio

doi:10.1016/s0167-4889(00)00023-9

Cited by 35 publications

(35 citation statements)

References 31 publications

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“…In support of the channel concept, recent structural studies indicate that synthetic peptides representing the TM domain of PLM form tetramers in reconstituted proteoliposomes (37). The channel-like properties of PLM is thought to be a major mechanism by which PLM regulates cell volume in non-cardiac tissues (38)(39)(40).…”

Section: Discussionmentioning

confidence: 96%

Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na⁺/Ca²⁺Exchanger

Wang¹,

Zhang²,

Ahlers³

et al. 2006

J. Biol. Chem.

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Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na + /Ca 2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the 1 st five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using GST pulldown assay, we demonstrated that the intracellular loop, but not the N-or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na + /Ca 2+ exchangers consisting of N-or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. While expression of Nterminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N-and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Coimmunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.

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Section: Discussionmentioning

confidence: 96%

Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na⁺/Ca²⁺Exchanger

Wang¹,

Zhang²,

Ahlers³

et al. 2006

J. Biol. Chem.

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“…Moreover, both when expressed in oocytes or added to lipid bilayers, PLM selectively transports the zwitterionic amino acid taurine (22), an osmolyte of animal cells. Recently, it was also shown that PLM stably transfected in HEK cells increases taurine efflux and regulatory volume decrease in response to cell swelling (39) and that in astrocytes taurine efflux can be inhibited after a decrease in the expression of endogenous PLM by antisense oligonucleotide (40). Thus, based on its function as a channel or a channel regulator, it is believed that PLM plays a specific role in muscle contractility and cell volume regulation.…”

Section: Discussionmentioning

confidence: 99%

Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

Crambert

Füzesi

Garty

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

253

285

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A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart-and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different ␣-␤ isozymes of NKA after coexpression in Xenopus oocytes, and with ␣1-␤, and less efficiently with ␣2-␤ isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K ؉ affinity of ␣1-␤1 and ␣2-␤1 isozymes and a nearly 2-fold decrease in the internal Na ؉ affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.

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“…We therefore reasoned that [Ca 2ϩ ] i transient and contractility changes associated with PLM downregulation would be sufficiently characterized with measurements at 0.6 and 5.0 mM [Ca 2ϩ ] o . Another limitation is that we ignored the observations in noncardiac tissues showing that PLM forms channels that are permeable to the zwitterion taurine (10) and participates in regulatory volume decrease of cells (11,12). Whole cell capacitance, however, was not changed in myocytes in which PLM was overexpressed (24) or downregulated (RESULTS).…”

Section: Discussionmentioning

confidence: 99%

Effects of phospholemman downregulation on contractility and [Ca²⁺]_itransients in adult rat cardiac myocytes

Mirza¹,

Zhang²,

Ahlers³

et al. 2004

American Journal of Physiology-Heart and Circulatory Physiology

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adult rat myocyte culture; patch clamp; fura-2; edge detection; excitation-contraction coupling PHOSPHOLEMMAN (PLM), a 72-amino acid membrane phosphoprotein with a single transmembrane domain (13), belongs to the FXYD gene family of small ion transporter regulators (18). In the heart and skeletal muscle, PLM is a major sarcolemmal substrate for protein kinases A and C (9, 14). Recent studies suggest that PLM regulates both Na ϩ -K ϩ -ATPase (4, 25) and Na ϩ /Ca 2ϩ exchanger (NCX1) (24) activities in cardiac muscle. In rat hearts that have survived myocardial infarction (MI), expression of PLM mRNA was increased twofold as early as 3 days after MI and remained elevated for at least 2 wk after MI (15). Interestingly, overexpression of PLM in normal adult rat cardiac myocytes affected myocyte contractility and cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) transients (17) in a pattern similar to the changes observed in post-MI rat myocytes (2,22). It is noteworthy that in post-MI rat myocytes, Na ϩ -dependent Ca 2ϩ uptake in sarcolemmal (SL) vesicles (5), NCX1 currents (27), and Na (6) were depressed. On the basis of the above observations, an attractive hypothesis is that overexpression of PLM in post-MI rat myocytes, by inhibiting two major SL ion transporters, resulted in abnormal [Ca 2ϩ ] i homeostasis and altered contractility. Downregulating PLM would therefore offer a rational approach to ameliorating contractile abnormalities post-MI. There are no published studies on the effects of PLM downregulation in cardiac tissues. The present study was undertaken to test the hypothesis that in adult rat cardiac myocytes, PLM downregulation alters cardiac myocyte contraction, [Ca 2ϩ ] i transient dynamics, and Na ϩ /Ca 2ϩ exchanger function. METHODSMyocyte isolation and culture. The protocol for myocyte isolation was approved by the Institutional Animal Care and Use Committee. Cardiac myocytes were isolated from the septum and left ventricular (LV) free wall of male Sprague-Dawley rats (ϳ280 g) as previously described (3). Isolated myocytes were seeded on laminin-coated coverslips and cultured with serum-free medum 199 (Earle's salts without L-glutamine and NaHCO3) supplemented with creatine, carnitine, taurine, and NaHCO 3 (17, 26). After 2 h, media were changed to remove nonadherent myocytes. Six hours after isolation, cultured myocytes were electrically paced {1 Hz, extracellular Ca 2ϩ concentration ([Ca 2ϩ ]o) ϭ 1.8 mM} (17,26). Culture media were changed daily over the course of experiments. Under continuous pacing culture conditions, we have previously demonstrated that myocyte contractility did not decline for at least 72 h (17).Construction of recombinant replication-deficient adenovirus expressing antisense PLM. The basic protocol has been described by He et al. (7). Initially, the coding sequence of dog heart PLM together with 5Ј-untranslated and 3Ј-untranslated sequences (13, 17) was

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Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman

Cited by 35 publications

References 31 publications

Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na⁺/Ca²⁺Exchanger

Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na⁺/Ca²⁺Exchanger

Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

Effects of phospholemman downregulation on contractility and [Ca²⁺]_itransients in adult rat cardiac myocytes

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Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman

Cited by 35 publications

References 31 publications

Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na+/Ca2+Exchanger

Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na+/Ca2+Exchanger

Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

Effects of phospholemman downregulation on contractility and [Ca2+]itransients in adult rat cardiac myocytes

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Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na⁺/Ca²⁺Exchanger

Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na⁺/Ca²⁺Exchanger

Effects of phospholemman downregulation on contractility and [Ca²⁺]_itransients in adult rat cardiac myocytes