Dual control of cardiac Na+ Ca2+ exchange by PIP(2): analysis of the surface membrane fraction by extracellular cysteine PEGylation.
We describe a new assay to determine the fraction of cardiac Na(+)-Ca(2+) exchangers (NCX1) in the surface membrane of cells (F(surf)). An extracellular NCX1 disulphide bond is rapidly reduced by tris(2-carboxyethyl)phosphine hydrochloride (TCEP), cysteines are 'PEGylated' by alkylation with an impermeable conjugate of maleimide and a 5000 MW polyethylene glycol (MPEG), and F(surf) is quantified from Western blots as the fraction of NCX1 that migrates at a higher molecular weight. F(surf) remains less than 0.1 when NCX1 is expressed via transient transfections. Values of 0.15-0.4 are obtained for cell lines with stable NCX1 expression, 0.3 for neonatal myocytes and 0.6-0.8 for adult hearts. To validate the assay, we analysed an intervention that promotes clathrin-independent endocytosis in fibroblasts. Using BHK cells, removal of extracellular potassium (K(+)) caused yellow fluorescent protein (YFP)-tagged NCX1 to redistribute diffusely into the cytoplasm within 30 min, F(surf) decreased by 35%, and whole-cell exchange currents decreased by > 50%. In both HEK 293 and BHK cell lines, expression of human hPIP5Ibeta kinase significantly decreases F(surf). In BHK cells expressing M1 receptors, a muscarinic agonist (carbachol) causes a 40% decrease of F(surf) in normal media. This decrease is blocked by a high wortmannin concentration (3 mum), suggesting that type III phosphatidylinositol-4-kinase (PI4K) activity is required. As predicted from functional studies, carbachol increases F(surf) when cytoplasmic Ca(2) is increased by removing extracellular Na(+). Phorbol esters are without effect in BHK cells. In intact hearts, interventions that change contractility have no effect within 15 min, but we have identified two long-term changes. First, we analysed the diurnal dependence of F(surf) because messages for cardiac phosphatidylinositol-4-phosphate (PIP) 5-kinases increase during the light phase in entrained mice (i.e. during sleep). Cardiac phosphatidylinositol-(4,5)-bis-phosphate (PIP(2)) levels increase during the light phase and F(surf) decreases in parallel. Second, we analysed effects of aortic banding because NCX1 currents do not mirror the increases of NCX1 message and protein that occur in this model. F(surf) decreases significantly within 10 days, and cardiac PIP and PIP(2) levels are significantly increased. In summary, multiple experimental approaches suggest that PIP(2) synthesis favours NCX1 internalization, that NCX1 internalization is probably clathrin-independent, and that significant changes of NCX1 surface expression occur physiologically and pathologically in intact myocardium.
Shen
Chengcheng
C
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9040, USA.
Lin
Mei-Jung
MJ
Yaradanakul
Alp
A
Lariccia
Vincenzo
V
Hill
Joseph A
JA
Hilgemann
Donald W
DW
D000818:Animals; D006332:Cardiomegaly / Q000503:physiopathology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D006224:Cricetinae; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D005347:Fibroblasts / Q000502:physiology; D017930:Genes, Reporter; D006321:Heart / Q000502:physiology*; D006801:Humans; D007668:Kidney; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D011092:Polyethylene Glycols; D019831:Sodium-Calcium Exchanger / Q000502:physiology*; D014162:Transfection
D008164:Luminescent Proteins; C499849:NCX1 protein, mouse; D019269:Phosphatidylinositol 4,5-Diphosphate; D019831:Sodium-Calcium Exchanger; D011092:Polyethylene Glycols; D003545:Cysteine