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

Summary

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⁻>Cl⁻>HCO ⁻₃ =F⁻≫I⁻=NO ⁻₃ =CNS⁻, and hence consistent with a reported Br⁻>Cl⁻>NO ⁻₃ sequence of the volume-dependent K⁺Cl⁻ transport. Alloimmune anti-L_l antibodies known to decrease passive K⁺ fluxes in low K⁺ cells reduced by 51% both volume-and NEM-stimulated, furosemidesensitive 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 (C _s) and a volume (V _s)-stimulated domain, both interfacing with the L₁ surface antigen. Attachment of alloanti-L₁ from the outside reduces K⁺Cl⁻ transport stimulated throughC _s by NEM orV _s by cell swelling.