ATP-dependent vesiculation in red cell membranes from different hereditary stomatocytosis variants E. Jane H. Turner, 1 Helen G. Jarvis, 1 Margaret C. Chetty, 1 Giorgio Landon, 2 Peter S. Rowley, 2 M. M. Ho 1 * and G. W. Stewart 1 Departments of 1 Medicine and 2 Histopathology, University College London, Rayne Institute, London, UK Received 26 June 2002; accepted for publication 4 October 2002 Summary. The hereditary stomatocytoses are a group of dominant haemolytic anaemias that show two main fea- tures: 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 ATP- dependent endocytic vesiculation (ADEV), which is found in normal red cells. We have examined this endocytosis pro- cess 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 typ- ically 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 mem- brane ‘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 exagger- ated surface exposure of an ion channel, which could then function constitutively, i.e. ‘leak’. Keywords: stomatocytosis, erythrocytes, haemolytic anae- mia, endocytic vesiculation, cation leak. The ‘hereditary stomatocytoses and allied disorders’ (HSt) comprise a diverse group of dominantly inherited haemo- lytic anaemias, all of which show some kind of membrane leak to the cations Na and K (Stewart & Turner, 1999). These conditions vary markedly in severity, from the original, very frankly haemolytic form (first described by Lock et al, 1961) to mild asymptomatic conditions that present with ‘pseudohyperkalaemia’, artefactual hyperkal- aemia caused by a temperature-dependent loss of K from red cells on cooling to room temperature. The common link between all these conditions is an abnormality within the so-called ‘passive leak’ component of Na and K transport across the red cell membrane, operationally defined as that component of K flux that is resistant to the two inhibitors ouabain and bumetanide. The pathological mechanism that underlies these leaky conditions is not understood. In the most severe form of these conditions, now known as ‘overhydrated hereditary stomatocytosis’ (OHSt), a 32-kDa membrane protein, des- ignated ‘stomatin’ after these diseases, is missing from the membrane (Lande et al, 1982; Eber et al, 1989; Stewart et al, 1992). The gene for stomatin is not mutated in these conditions (Wang et al, 1992) but, nevertheless, the very striking molecular abnormality suggests that the protein is somehow implicated in the pathophysiology. This protein has emerged as the prototype of a family distributed across nature, some of which are implicated in systems that regulate protein turnover (Tavernarakis et al, 1999). It has been shown that, in cultured amniotic cells, stomatin is associated with sphingomyelin + cholesterol-rich ‘rafts’ of the cell membrane (Snyers et al, 1999). Mentzer et al (1978) observed many years ago that a process of ATP-dependent vesiculation (ADEV) was abnor- mal in a case of HSt. This observation assumes new importance, given the recognition of stomatin as a raft protein, the importance of rafts in cytoskeletal organization and endocytosis (Kurzchalia & Parton, 1999; Ikonen, 2001) Correspondence: G. W. Stewart, Department of Medicine, University College London, Rayne Institute, University Street, London WC1E 6JJ, UK. E-mail: g.stewart@ucl.ac.uk *Present address: Bacteriology Division, National Institute for Bio- logical Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK. British Journal of Haematology, 2003, 120, 894–902 894 Ó 2003 Blackwell Publishing Ltd