Session: 101. Red Cells and Erythropoiesis, Excluding Iron: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research
Phosphatidylserine (PS) exposure on red blood cell (RBC) surface not only provides a procoagulant surface for coagulation complex assembly, but also serves as an “eat-me” signal for their clearance. The molecular mechanism underlying RBC PS exposure has been elusive. Through a recent study of hereditary xerocytosis (HX), a congenital disorder caused by gain-of-function mutations of the mechanosensitive PIEZO1 Ca2+ permeable channel, we established that the functional coupling between PIEZO1 and TMEM16F, a Ca2+-activated phospholipid scramblase, mediates RBC PS exposure. We also found that enhanced PIEZO1-TMEM16F coupling is responsible for increased tendency of PS exposure in HX RBCs. Our preliminary study demonstrated that enhanced PIEZO1-TMEM16F coupling contributes to excessive PS exposure in sickle RBCs, which increases the risk of thrombotic complications in sickle cell disease (SCD). Therefore, targeting PIEZO1-TMEM16F coupling to prevent PS exposure is an attractive strategy to mitigate SCD and HX complications. However, lack of effective and specific PIEZO1 and TMEM16F inhibitors hinders clinical translation of this strategy. Alternatively, we propose that Gardos Ca2+-activated K+ channel (also known as IK, KCa3.1 or SK4) is an amplifier of PIEZO1-TMEM16F coupling. Gardos has higher Ca2+ sensitivity than TMEM16F, and the Gardos-mediated K+ efflux following PIEZO1 activation induces membrane hyperpolarization, increasing driving force for Ca2+ influx through PIEZO1. We anticipate that Senicapoc, a Gardos inhibitor that has been used in clinical trials, can attenuate PIEZO1-TMEM16F coupling and reduce excessive PS exposure in RBC disorders.
Results:
We first quantified the effects of overexpressing PIEZO1 or PIEZO1 and Gardos on activating endogenous TMEM16F in HEK293T cells. We found that coexpression of Gardos significantly increases PIEZO1-mediated Ca2+ entry (2.6 fold; t(8) = 5.417, P = 0.0006) and TMEM16F-mediated PS exposure (8.6 fold; t(4) = 6.046, P = 0.0013) compared to overexpressing PIEZO1 alone upon stimulation with Yoda1. Our data thus demonstrated that heterologously expressed Gardos amplifies PIEZO1-TMEM16F coupling in HEK293T cells.
Next, we tested the role endogenous Gardos in regulating PIEZO1-TMEM16F coupling in HEL 92.1.7 cells, an erythroblast-like leukemia cell line. We found that PIEZO1 activation induces membrane hyperpolarization, which is reversed by Gardos inhibitor Senicapoc. Senicapoc also significantly reduces Yoda1-induced PS exposure (1.9 fold; t(4) = 8.923, P = 0.0009). The results indicate that Gardos amplifies endogenous PIEZO1-TMEM16F coupling in HEL 92.1.7 cells.
Finally, we tested Gardos’ role in regulating PIEZO1-TMEM16F coupling in RBCs. We found that SKA31, a Gardos agonist, promotes PS exposure in RBCs, supporting the amplification role of Gardos on PIEZO1-TMEM16F coupling. Paradoxically, Senicapoc promotes Yoda1-induced PS exposure instead of preventing PS exposure in RBCs (4.3 fold; t(4) = 9.913, P = 0.0006). Similar PS exposure potentiation effect also occurred in SCD RBCs. We did not find evidence of Senicapoc modulating the activity of PIEZO1 or TMEM16F. We postulate that Senicapoc may target an unknown protein that mediates PS exposure through a TMEM16F-independent mechanism.
Conclusion:
Gardos amplifies PIEZO1-TMEM16F coupling and subsequent PS exposure through mediating membrane hyperpolarization and enhancing Ca2+ entry. Although Gardos inhibition is a promising strategy to decouple PIEZO1-TMEM16F interaction and prevent PS exposure, Senicapoc is not a suitable Gardos inhibitor for this purpose due to its unknown off-target effect. More specific Gardos inhibitors are needed to prevent PS exposure in red cell disorders.
Disclosures: Arepally: Biokit: Research Funding; AstraZeneca: Research Funding; Veralox therapeutics: Research Funding; Annexon: Research Funding; Alexion Pharmaceuticals, Inc. a subsidiary of AstraZeneca: Research Funding; ABCAM PLC: Research Funding; Janssen Research $ Development: Research Funding; Sanofi: Research Funding.
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