TY - JOUR
T1 - Dual control of cardiac Na+-Ca2+ exchange by PIP2
T2 - Analysis of the surface membrane fraction by extracellular cysteine PEGylation
AU - Shen, Chengcheng
AU - Lin, Mei Jung
AU - Yaradanakul, Alp
AU - Lariccia, Vincenzo
AU - Hill, Joseph A
AU - Hilgemann, Donald W
N1 - Publisher Copyright:
© 2007 The Journal of Physiology.
PY - 2007/8/1
Y1 - 2007/8/1
N2 - We describe a new assay to determine the fraction of cardiac Na+-Ca2+ exchangers (NCX1) in the surface membrane of cells (Fsurf). 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 Fsurf is quantified from Western blots as the fraction of NCX1 that migrates at a higher molecular weight. Fsurf 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, Fsurf decreased by 35%, and whole-cell exchange currents decreased by > 50%. In both HEK 293 and BHK cell lines, expression of human hPIP5Iβ kinase significantly decreases Fsurf. In BHK cells expressing M1 receptors, a muscarinic agonist (carbachol) causes a 40% decrease of Fsurf in normal media. This decrease is blocked by a high wortmannin concentration (3 μm), suggesting that type III phosphatidylinositol-4-kinase (PI4K) activity is required. As predicted from functional studies, carbachol increases Fsurf when cytoplasmic Ca2 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 Fsurf 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 (PIP2) levels increase during the light phase and Fsurf 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. Fsurf decreases significantly within 10 days, and cardiac PIP and PIP2 levels are significantly increased. In summary, multiple experimental approaches suggest that PIP2 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.
AB - We describe a new assay to determine the fraction of cardiac Na+-Ca2+ exchangers (NCX1) in the surface membrane of cells (Fsurf). 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 Fsurf is quantified from Western blots as the fraction of NCX1 that migrates at a higher molecular weight. Fsurf 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, Fsurf decreased by 35%, and whole-cell exchange currents decreased by > 50%. In both HEK 293 and BHK cell lines, expression of human hPIP5Iβ kinase significantly decreases Fsurf. In BHK cells expressing M1 receptors, a muscarinic agonist (carbachol) causes a 40% decrease of Fsurf in normal media. This decrease is blocked by a high wortmannin concentration (3 μm), suggesting that type III phosphatidylinositol-4-kinase (PI4K) activity is required. As predicted from functional studies, carbachol increases Fsurf when cytoplasmic Ca2 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 Fsurf 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 (PIP2) levels increase during the light phase and Fsurf 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. Fsurf decreases significantly within 10 days, and cardiac PIP and PIP2 levels are significantly increased. In summary, multiple experimental approaches suggest that PIP2 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.
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U2 - 10.1113/jphysiol.2007.132720
DO - 10.1113/jphysiol.2007.132720
M3 - Article
C2 - 17540704
AN - SCOPUS:34547101945
SN - 0022-3751
VL - 582
SP - 1011
EP - 1026
JO - Journal of Physiology
JF - Journal of Physiology
IS - 3
ER -