Thiol-dependent passive K+Cl− transport in sheep red blood cells: VI. Functional heterogeneity and immunologic identity with volume-stimulated K+(Rb+) fluxes

Abstract

Ouabain-resistant (OR), volume- or N-ethylmaleimide (NEM)-stimulated K+(Rb+)Cl- fluxes were measured in low-K+ sheep red cells and found to be functionally separate but immunologically similar. In anisosmotic solutions both K+ effluxes and Rb+ influxes of NEM-treated and control cells were additive. In contrast to the NEM-stimulated K+Cl- flux, metabolic depletion did not reduce K+Cl- flux of normal or swollen cells. The anion preference of OR K+ efflux in NEM-treated cells was Br- greater than Cl- greater than HCO3- = F- much greater than I- = NO3- = CNS-, and hence consistent with a reported Br- greater than Cl- greater than NO3- sequence of the volume-dependent K+Cl- transport. Alloimmune anti-L1 antibodies known to decrease passive K+ fluxes in low K+ cells reduced by 51% both volume- and NEM-stimulated, furosemide-sensitive Rb+Cl- fluxes suggesting their immunologic identity, a conclusion also supported by anti-L1 absorption studies. Since pretreatment with anti-L1 prevented the activation of Rb+ influx by NEM, and the impermeant glutathionmaleimide-I did not stimulate Rb+Cl- influx, the NEM reactive SH groups must be located apart from the L1 antigen either within the membrane or on its cytoplasmic face. A model is proposed consisting of a K+Cl- transport path(s) regulated by a protein with two functional subunits or domains: a chemically (Cs) and a volume (Vs)-stimulated domain, both interfacing with the L1 surface antigen. Attachment of alloanti-L1 from the outside reduces K+Cl- transport stimulated through Cs by NEM or Vs by cell swelling.

Volume-sensitive K-Cl cotransport in inside-out vesicles made from erythrocyte membranes from sheep of low-K phenotype.

“…In LK sheep cells under isosmotic conditions, K influx is relatively independent of the metabolic state of the cells (7,41,42). However, in swollen or N-ethylmaleimide-treated cells, K influx is inhibited by metabolic depletion (7,41,43). Depletion ofATP was presumed, but not demonstrated, to be responsible.…”

“…Clindependent K fluxes in LK sheep cells and resealed human ghosts show saturation kinetics (47) and volume sensitivity (16,48); volume sensitivity of Cl-independent fluxes was (16). However, Cl-dependent K transport in untreated LK sheep erythrocytes is not only not inhibited by metabolic depletion, it may be stimulated (40,42,43). The explanation for these varying results is not clear and will be difficult to resolve in intact cells.…”

Volume-sensitive K transport in human erythrocytes.

“…In particular, swelling has been reported to activate Cldependent K transport in avian (Kregenow, 1974 ;Kregenow and Caryk, 1979 ;Haas and McManus, 1985), sheep Dunham and Ellory, 1981 ;Lauf, 1984), dog (Parker, 1983), and toadfish erythrocytes (Lauf, 1985), K/H exchange in Amphiuma erythrocytes (Gala, 1983), K conductance in human lymphocytes (Sarkadi et al ., 1985) and Ehrlich ascites tumor cells (Hoffmann et al ., 1984), and K loss through an unidentified mechanism in flounder erythrocytes (Gala, 1977).…”

Enhanced Volume-Sensitive K Flux in Patients on Chronic Hemodialysis

“…K influx was measured using 8bRb as a tracer, whereas K efflux was calculated by measuring Keffluent in the supernatant of the cell suspension. We did not use ^R b for measuring K efflux because the cells have to be loaded with 8bRb for more than 3 h, and the amount of SbRb taken up by erythrocytes may be too low for identification of small changes [18]. K efflux, measured by the methods we used in this study, showed little variation, and thus the data are thought to be reliable, as seen in figure 2.…”