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Departments of
¹ Physiology
² Internal Medicine-Cardiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA

We describe a new assay to determine the fraction of cardiac Na⁺–Ca²⁺ 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 hPIP5I 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 µM), suggesting that type III phosphatidylinositol-4-kinase (PI4K) activity is required. As predicted from functional studies, carbachol increases F_surf 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 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 (PIP₂) 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 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.

(Received 18 March 2007; accepted after revision 24 May 2007; first published online 31 May 2007)
Corresponding author D. W. Hilgemann: Department of Physiology, UTSouthwestern Medical Center, Dallas, TX 75390-9040, USA. Email: donald.hilgemann{at}utsouthwestern.edu

This article has been cited by other articles:

				B. Robertson Regulation of ion channels and transporters by phosphatidylinositol 4,5-bisphosphate J. Physiol., August 1, 2007; 582(3): 901 - 902. [Full Text] [PDF]

				D. W. Hilgemann On the physiological roles of PIP2 at cardiac Na+ Ca2+ exchangers and KATP channels: a long journey from membrane biophysics into cell biology J. Physiol., August 1, 2007; 582(3): 903 - 909. [Abstract] [Full Text] [PDF]