Enhanced Ca(2+) influx in mechanically distorted erythrocytes measured with (19)F nuclear magnetic resonance spectroscopy

We present the first direct nuclear magnetic resonance (NMR) evidence of enhanced entry of Ca(2+) ions into human erythrocytes (red blood cells; RBCs), when these cells are mechanically distorted. For this we loaded the RBCs with the fluorinated Ca(2+) chelator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA), and recorded (19)F NMR spectra. The RBCs were suspended in gelatin gel in a special stretching/compression apparatus. The 5FBAPTA was loaded into the cells as the tetraacetoxymethyl ester; and (13)C NMR spectroscopy with [1,6-(13)C]d-glucose as substrate showed active glycolysis albeit at a reduced rate in cell suspensions and gels. The enhancement of Ca(2+) influx is concluded to be via the mechanosensitive cation channel Piezo1. The increased rate of influx brought about by the activator of Piezo1, 2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine (Yoda1) supported this conclusion; while the specificity of the cation-sensing by 5FBAPTA was confirmed by using the Ca(2+) ionophore, A23187.