HX due to heterozygous PIEZO1 mutation. Human PIEZO1 is a 2521 amino acid (287 kDa) protein with ∼38 transmembrane helices. Cryoelectron microscopy studies of the highly homologous mouse Piezo1 reveal that PIEZO1 trimers form an elegant 3-bladed propeller structure with a curved transmembrane region creating an inverted membrane dome and a central ion pore formed by the C-terminal domains of the subunits, in which most of the disease-causing mutations are located.^103-106  This unique structure of PIEZO1 senses changes in membrane tension to alter gating of the ion channel.^106,107  (A) Top view of the homotrimeric PIEZO1 channel showing its 3-bladed, propeller-like architecture (from the extracellular space looking down through the central pore). The 3 subunits of the trimer are color coded. (B) Side view of the homotrimeric PIEZO1 channel showing the curved transmembrane region, which creates a membrane dome.^106,107  The position of the membrane is roughly indicated with white dotted lines. Two of the mutation “hot spot” areas described by Picard et al⁵⁶  are highlighted in chain A (dark blue); the sequence p.R2456-P2510 in the pore domain (coded within exon 51) is colored orange, and one of the most common HX mutations in this region (p.R2456) is labeled in red; the sequence p.S1994-V2201 (coded within exons 42-45) is colored yellow, and one of the mutations in this area (p.L2023V) is indicated in magenta. A third hot-spot region for mutations is located N-terminally in exons 14 to 18; the structure of this area has not yet been modeled. Patients demonstrating a more severe phenotype are more likely to have mutations in the PIEZO1 pore domain.⁵⁶  Images created using UCSF Chimera,¹⁰⁸  with the Protein Data Bank structure model 3JAC. (C) Sketch of interaction between PIEZO1 and KCNN4 as RBCs travel through the vasculature. In narrow capillaries and sinusoids, mechanical stress (represented by the red arrowheads) results in activation of PIEZO1 and Ca²⁺ entry. Increased intracellular Ca²⁺ leads to activation of KCNN4 (a calcium ion binds to each of the 4 calmodulin molecules tightly bound to the cytoplasmic domains of the 4 KCNN4 subunits),¹⁰⁹  and K⁺ efflux ensues. Subsequent water loss results in a temporary decrease in cell volume and facilitates passage.^57-59  (D) Blood smear from a patient heterozygous for p.R2456H with macrocytosis (MCV 96 fL) showing occasional stomatocytes (arrows), target cells (arrowheads), and dense cells (thin arrows). (E) Osmotic gradient ektacytometry showing the typical HX curve with left shift due to decreased O_min and O_hyp, indicating RBC dehydration. Ekatacytometry profiles are shown for 2 patients with HX due to PIEZO1 p.R2456H and p.L2023V.