Regulation of Erythrocyte Volume Homeostasis

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PROJECT SUMMARY/ABSTRACT PIEZO1 has recently been identified as the long sought after protein involved in mammalian mechano- sensation and stretch-activated cation channel activation. We have discovered mutations in PIEZO1 lead to hereditary xerocytosis, a hemolytic anemia characterized by primary erythrocyte dehydration, indicating PIEZO1 plays a critical role in cellular volume homeostasis. PIEZO1 is a candidate for the unidentified stretch- induced calcium-activated cation pathways in the red blood cell that play critical roles in erythrocyte aging, malaria invasion, and circulatory sheer stress. Preliminary data also indicate that PIEZO1 is an excellent candidate for Psickle, an unidentified cation permeability pathway induced by deoxygenation in sickle erythrocytes, at the initiation of the dehydration cascade. Therefore, Psickle is of fundamental importance to sickle cell pathobiology. Despite its importance, we have no knowledge of the mechanisms controlling PIEZO1 expression, structure, or function in erythroid cells. The overall goal of this proposal is to begin to elucidate the molecular mechanisms involved in PIEZO1 regulation and function in erythroid cells. Our preliminary studies indicate that variants of the PIEZO1 gene occur in hereditary xerocytosis and sickle cell disease patients and that these variants are associated with alterations in erythrocyte hydration. The goal of aim one is identification of genetic variants influencing erythrocyte hydration in patients with sickle cell disease and increased numbers of dense cells and characterization of the effect of these mutations on PIEZO1 expression, structure and function. The goal of aim two is to create a murine model of hereditary xerocytosis and analyze the influence of HX-associated Piezo1 gain of function mutations in vivo on wild type and sickle cell backgrounds. The goal of aim 3 is the characterization of the influence of Piezo1 loss of function on erythrocyte function in wild type and SCD erythrocytes to better understand molecular mechanisms regulating erythrocyte volume homeostasis, to address the hypothesis that PIEZO1 mediates the cation currents of Psickle, the unidentified, incipient transport pathway critical for dehydration of sickle erythrocytes, and to assess the influence of Piezo1 deficiency on parameters of sickle cell disease. To address the influence of mutations on PIEZO1 structure and function, functional cell-based assays of PIEZO1 membrane protein expression, trafficking, and electrophysiology in a novel, in vivo stably-transfected, single-copy, inducible cell model of PIEZO1 expression will be performed. Physiologic studies of mature erythrocytes from genetically modified mice and affected patients will be performed under a variety of cellular conditions. PIEZO1 is expressed in many cell types, indicating PIEZO1 likely mediates important functions in a wide variety of cells. Thus studies in erythroid cells may yield mechanistic or biological principles generalizable to many critical cellular processes or human diseases.