The Molecular Basis for Altered Cation Permeability in Hereditary Stomatocytic Human Red Blood Cells

Abstract

Normal human RBCs have a very low basal permeability (leak) to cations, which is continuously corrected by the Na,K-ATPase. The leak is temperature-dependent, and this temperature dependence has been evaluated in the presence of inhibitors to exclude the activity of the Na,K-ATPase and NaK2Cl transporter. The severity of the RBC cation leak is altered in various conditions, most notably the hereditary stomatocytosis group of conditions. Pedigrees within this group have been classified into distinct phenotypes according to various factors, including the severity and temperature-dependence of the cation leak. As recent breakthroughs have provided more information regarding the molecular basis of hereditary stomatocytosis, it has become clear that these phenotypes elegantly segregate with distinct genetic backgrounds. The cryohydrocytosis phenotype, including South-east Asian Ovalocytosis, results from mutations in SLC4A1, and the very rare condition, stomatin-deficient cryohydrocytosis, is caused by mutations in SLC2A1. Mutations in RHAG cause the very leaky condition over-hydrated stomatocytosis, and mutations in ABCB6 result in familial pseudohyperkalemia. All of the above are large multi-spanning membrane proteins and the mutations may either modify the structure of these proteins, resulting in formation of a cation pore, or otherwise disrupt the membrane to allow unregulated cation movement across the membrane. More recently mutations have been found in two RBC cation channels, PIEZO1 and KCNN4, which result in dehydrated stomatocytosis. These mutations alter the activation and deactivation kinetics of these channels, leading to increased opening and allowing greater cation fluxes than in wild type.

Keywords: ABCB6; KCNN4; PIEZO1; RhAG; SLC2A1; SLC4A1; familial pseudohyperkalemia; hereditary stomatocytosis.

Figure 1

Temperature dependence of “leak” potassium flux in different leaky membrane variants among the hereditary stomatocytosis conditions. Open symbols denote normal red cells. Closed symbols denote patients, detailed below and in the Table. Potassium influx was measured using ⁸⁶Rb as a tracer. The medium contained (mM): Na⁺, 145: K⁺, 5; Cl⁻, 150, MOPS, 15 (pH 7.4 at 20°C); glucose, 5; ouabain, 0.1; bumetanide, 0.1. Reproduced with permission from Stewart (2004).

Figure 2

Diagram of the membrane domain of band 3 showing the membrane spans and HSt mutations. The length of the membrane spans mimics that determined from the crystal structure by Arakawa et al. (2015). In the final structure the half transmembrane span 3 and half transmembrane span 10 form a full transmembrane domain (TM) jointly (Arakawa et al., 2015). The nine amino acid residues deleted in SAO band 3 are shown in blue. The amino acid residues associated with HSt are shown in orange. The Asp705Tyr (TM9), Arg730Cys (TM10), Ser731Pro (TM10), His734Arg (TM10) Glu758Lys (TM11), Arg760Gln (TM11), and Ser762Arg (TM11) mutations all occur close to the anion binding site of band 3. The Gly796Arg mutation is in TM12 and may affect helix-helix packing. The Leu687Pro mutation is near the C-terminus of TM8 which is at the interface of the core and gate domains. Movement between these two domains is required for anion transport and substitution of Leu687 with a proline residue may restrict this movement (Arakawa et al., 2015).