Document Detail

Dual control of cardiac Na+ Ca2+ exchange by PIP(2): analysis of the surface membrane fraction by extracellular cysteine PEGylation.
MedLine Citation:
PMID:  17540704     Owner:  NLM     Status:  MEDLINE    
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.
Chengcheng Shen; Mei-Jung Lin; Alp Yaradanakul; Vincenzo Lariccia; Joseph A Hill; Donald W Hilgemann
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2007-05-31
Journal Detail:
Title:  The Journal of physiology     Volume:  582     ISSN:  0022-3751     ISO Abbreviation:  J. Physiol. (Lond.)     Publication Date:  2007 Aug 
Date Detail:
Created Date:  2007-08-03     Completed Date:  2007-09-26     Revised Date:  2013-06-06    
Medline Journal Info:
Nlm Unique ID:  0266262     Medline TA:  J Physiol     Country:  England    
Other Details:
Languages:  eng     Pagination:  1011-26     Citation Subset:  IM    
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9040, USA.
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MeSH Terms
Cardiomegaly / physiopathology*
Cell Line
Cell Membrane / physiology*
Cysteine / analogs & derivatives*,  metabolism
Endocytosis / physiology
Fibroblasts / physiology
Genes, Reporter
Heart / physiology*
Luminescent Proteins / genetics
Mice, Inbred C57BL
Phosphatidylinositol 4,5-Diphosphate / physiology*
Polyethylene Glycols
Sodium-Calcium Exchanger / physiology*
Grant Support
Reg. No./Substance:
0/Luminescent Proteins; 0/NCX1 protein, mouse; 0/Phosphatidylinositol 4,5-Diphosphate; 0/Polyethylene Glycols; 0/Sodium-Calcium Exchanger; 52-90-4/Cysteine

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