Enhanced Ca2+ Influx in Mechanically Distorted Erythrocytes: Measurements with 19F Nuclear Magnetic Resonance Spectroscopy

Abstract

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-5fluorophenoxy)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.

Surface model of the human red blood cell simulating changes in membrane curvature under strain

“…We emulated the distortion of RBCs suspended in stretched gelatin gel by specifying that a geometrical strain field was applied in one direction, chosen to be along the z-axis. This equates to what applies in real NMR experiments [1,2]. It is not possible, experimentally, to align all the RBCs in gelatin media prior to or after gelation, so the members of the population of RBCs assume all possible orientations of their axes of symmetry in the strain field.…”

“…Then, correlations could be made with experimental measurements like those already reported on stretched/compressed gels [1,2], and in electrophysiological measurements on whole RBCs.…”

“…Our aim is to convey a sense of scale of the distribution of proteins in the membrane and adjacent cytoskeleton, relative to the whole human red blood cell (RBC); and to graphically represent changes in membrane curvature on the ~1 nm to ~10 m scale, brought about by the systematic straining of these cells. This study was motivated by the quest for the geometrical and mechanistic basis of recent findings on mechanically distorted RBCs, made by using nuclear magnetic resonance (NMR) spectroscopy with stretched and compressed gels [1,2].…”

“…The NMR-based cation-flux estimates are in the same range as measurements made by electrophysiological means, performed on whole RBCs and membrane patches (patch clamping) (e.g., [6]). However, because the electrophysiological measurements typically occur over seconds (of a transient response to shape change), while the NMR experiments can last for hours a different property of channel opening and closure is in operation [1,2,8]; the latter is referred to a 'channel fatigue' e.g., [9]. The Piezo1 three-arm (triskelion) structure spans a relatively large ~23 nm diameter membrane patch; this has been measured from images obtained with cryo-electron microscopy [10][11][12].…”