Abstract
When red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca2+-signaling by confocal microscopy of Fluo-4 loaded RBCs. We were able to confirm our in vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca2+ when RBCs were passing through small capillaries in vivo. Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca2+-signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca2+ to enter the cell, leading to a transient activation of the Gardos-channel associated with K+, Cl−, and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constriction.
Highlights
The physiological function of the Gardos-channel, a Ca2+-activated K+-channel (Gardos, 1958) in the red blood cell (RBC), that was later identified as the hSK4 (KCNN4) channel (Hoffman et al, 2003), was obscure for decades
In an initial experiment we aimed to check if basic mechanical stimulation by osmotic swelling leads to an activity of mechanosensitive channels in RBCs
In contrast to hormonal-like stimulations (Wang et al, 2013), RBCs showed qualitatively a very homogeneous response, i.e., all cells responded immediately with an increase in intracellular Ca2+, just the extent of the increase varied between the cells. This Ca2+-increase could be completely blocked by preincubation of the RBCs with 2.5 μM GsMTx-4 (Figure 1Bb, green box), a widely used inhibitor of Piezo1 (Bae et al, 2011), whereas GsMTx-4 itself had no effect on cells in isosmotic solution (Figure 1Bb, red box)
Summary
The physiological function of the Gardos-channel, a Ca2+-activated K+-channel (Gardos, 1958) in the red blood cell (RBC), that was later identified as the hSK4 (KCNN4) channel (Hoffman et al, 2003), was obscure for decades It was regarded as a RBC suicidal mechanism (Andrews and Low, 1999; Kaestner and Bernhardt, 2002; Lang and Qadri, 2012) including the process of dehydration associated with Gardos-channel activity (Begenisich et al, 2004; Lew et al, 2005). In addition within the recent years hereditary anemic disorders have been associated with mutations in the Gardos-channel (Glogowska et al, 2015; Fermo et al, 2017).
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