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

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.