We present the first direct nuclear magnetic resonance (NMR) evidence of enhanced entry of Ca2+ ions into human erythrocytes (red blood cells; RBCs), when these cells are mechanically distorted. For this we loaded the RBCs with the fluorinated Ca2+ chelator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA), and recorded 19F 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 13C NMR spectroscopy with [1,6-13C]d-glucose as substrate showed active glycolysis albeit at a reduced rate in cell suspensions and gels. The enhancement of Ca2+ 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 Ca2+ ionophore, A23187.

We present mathematical simulations of shapes of red blood cells (RBCs) and their cytoskeleton when they are subjected to linear strain. The cell surface is described by a previously reported quartic equation in three dimensional (3D) Cartesian space. Using recently available functions in Mathematica to triangularize the surfaces we computed four types of curvature of the membrane. We also mapped changes in mesh-triangle area and curvatures as the RBCs were distorted. The highly deformable red blood cell (erythrocyte; RBC) responds to mechanically imposed shape changes with enhanced glycolytic flux and cation transport. Such morphological changes are produced experimentally by suspending the cells in a gelatin gel, which is then elongated or compressed in a custom apparatus inside an NMR spectrometer. A key observation is the extent to which the maximum and minimum Principal Curvatures are localized symmetrically in patches at the poles or equators and distributed in rings around the main axis of the strained RBC. Changes on the nanometre to micro-meter scale of curvature, suggest activation of only a subset of the intrinsic mechanosensitive cation channels, Piezo1, during experiments carried out with controlled distortions, which persist for many hours. This finding is relevant to a proposal for non-uniform distribution of Piezo1 molecules around the RBC membrane. However, if the curvature that gates Piezo1 is at a very fine length scale, then membrane tension will determine local curvature; so, curvatures as computed here (in contrast to much finer surface irregularities) may not influence Piezo1 activity. Nevertheless, our analytical methods can be extended address these new mechanistic proposals. The geometrical reorganization of the simulated cytoskeleton informs ideas about the mechanism of concerted metabolic and cation-flux responses of the RBC to mechanically imposed shape changes.

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