ATP‐dependent vesiculation in red cell membranes from different hereditary stomatocytosis variants

Abstract

Summary. The hereditary stomatocytoses are a group of dominant haemolytic anaemias that show two main features: invaginated, 'stomatocytic' morphology; and a membrane leak to the univalent cations Na and K. A patient with the most severe variant of these conditions was reported to show a defect in an in vitro process of ATPdependent endocytic vesiculation (ADEV), which is found in normal red cells. We have examined this endocytosis process in 11 leaky red cell pedigrees available to us in the UK. ADEV in broken membranes was absent only in the two most severely affected, 'overhydrated' pedigrees studied, both of which showed a deficiency in the membrane raft protein, stomatin. The process was present, although typically diminished by about 10-20% compared with normal red cells, in all others. The cross-linker dimethyl adipimate (DMA), which could correct the cation leak in some of these patients, also corrected the ADEV defect in the same patients. In those patients in whom DMA had no effect on the ion leak, ADEV was not absent. In normal cells, this process of vesiculation was inhibited by inhibitors of membrane 'raft' function, by an antistomatin antibody and by vanadate and N-ethyl maleimide, but not by inhibitors of a number of kinases. These data highlight the heterogeneity of these conditions. A mechanism is discussed by which a defect in raft-based endocytosis could lead to the exaggerated surface exposure of an ion channel, which could then function constitutively, i.e. 'leak'.

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

“…The F65S mutation has been reported so far in 6 families and 4 patients while the I61R variant is found in one single individual [27], [28]. The two mutations were associated to distinct sub-types of OHSt on the basis of phenotypic difference, including defective in vitro process of ATP-dependent endocytic vesiculation, which is found in normal red cells [35]. Our data also suggest a difference in both variants, in their impact on K + conductance in yeast.…”

The monovalent cation leak in overhydrated stomatocytic red blood cells results from amino acid substitutions in the Rh-associated glycoprotein

“…Since in this heterozygous condition both the wild-type and Ser65-RhAG proteins should be present, the altered epitope was probably formed by changes in the oligomeric interactions of RhAG, and suggests that WT-RhAG forms hetero-oligomers with Ser65-RhAG. Another observation indicating long-range structural alterations was the inhibitory effect of DMA on these Phe65Ser RhAG cells, 4 suggesting that the position or orientation of extracellular lysine residue(s) are altered in the Phe65Ser RhAG, creating a DMA binding site that is not found in wild-type or I61R RhAG. OHSt cells are deficient in the raft protein stomatin, and stomatin expression is also reduced in a subtype of cryohydrocytosis.…”

“…The physiologic function of RhAG is disputed. Its homology to the Amt proteins suggests that it may act as a cation channel transporting NH 4 ϩ , or as a gas channel moving NH 3 or CO 2 . However, early studies of the transport properties of human RhAG gave conflicting results, variously describing RhAG as a NH 3 /NH 4 ϩ exchanger or ammonium transporter, [28][29][30][31] or as an ammonia channel, [32][33][34] or suggesting that RhAG transports CO 2 , 35 or both CO 2 and NH 3 .…”

“…OHSt (type 1) ghost membranes show a defect in a process of ATP-dependent endocytic vesiculation that can be corrected by the cross-linker dimethyl adipimidate (DMA), which also corrects the cation leak in these cells. 4 OHSt (type 2) ghost membranes do not have the defect in ATPdependent endocytic vesiculation, and the cation leak is not corrected by DMA. 4 We were prompted to study the Rh-associated glycoprotein (RhAG) because we found that a related, less leaky condition known as cryohydrocytosis was caused by mutations in SLC4A1, which codes for the band 3 anion exchanger.…”

“…4 OHSt (type 2) ghost membranes do not have the defect in ATPdependent endocytic vesiculation, and the cation leak is not corrected by DMA. 4 We were prompted to study the Rh-associated glycoprotein (RhAG) because we found that a related, less leaky condition known as cryohydrocytosis was caused by mutations in SLC4A1, which codes for the band 3 anion exchanger. 5 Band 3 is the focus of a macrocomplex of proteins that includes RhAG, and RhAG can be coimmunoprecipitated with anti-band 3 antibodies and thus associates directly with band 3.…”