The cation dimorphism of high K+ (HK) and low K+ (LK) sheep red cells is genetically associated with differences in a) Na+K+ pumps, b) ouabain-sensitive Cl- dependent K+ transport which is activated through reaction of N-ethylmaleimide (NEM) with its sulfhydryl (SH) groups or by cell swelling in hyposmotic media, and c) the presence of the M and L membrane antigens. Both HK and LK red cells possess a ouabain-insensitive Na+/Cl exchange flux whose role is unexplained. The process of maturation of the reticulocy to the prospective LK red cell may involve all of the above transport systems operating consecutively or simultaneously to exchange cellular K+ for Na+ (HK-LK transition). For the study of regulation and development of membrane transport and its pathophysiology and its genetic basis in disease, the HK/LK sheep red cell system offers a unique, genetically defined model. To understand the precise mechanism of the HK-LK transition a further analysis is proposed of the individual transport systems in mature HK and LK red cells as well as an in vitro follow-up study of the maturational changes of all transport activities. In particular, it is proposed: 1. A further characterization of the SH-dependent, volume sensitive K/Cl pathway in LK red cells with respect to a) its biochemical and antigenic nature as ascertained by covalent binding of 3H-NEM or 3H-bumetanide, an inhibitory loop diuretic, and attachment of radiolabeled anti-L1 specifically reducing SH-dependent K/Cl flux; b) the hypothesis of regulation of SH-dependent K/Cl flux by cytoplasmic factors (ATP) or cellular metabolism; and c) the physiologic basis of Cl-requirement and inhibition by loop diuretics. These analyses will shed new light on the mechanism of other coupled ion transporters recently described in renal and intestinal physiology. 2. An analysis of ouabain-insensitive Na+ pathways of HK and LK red cells, in particular of the Na+/Na+ counter transport as a potential candidate for Na+/H+ exchange that together with outward K/Cl flux may be important for the development of the LK cell. 3. An investigation of the specific site of action of cellular K+ to inhibit the Na+K+ pump in LK cells, and an evaluation of the role of protons. 4. A further characterization of the M/L surface antigens with the aid of new electrophoretic techniques (electro blotting). 5. To test experimentally the hypothesis that all of the above transporters studied simultaneously or consecutively down-regulate cellular K+ levels from that in HK reticulocytes to those in mature LK red cells, and to understand the mode of inactivation of SH-dependent K/Cl transport in HK and of K+ pump fluxes in LK erythrocytes.
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