LABELING OF THE AMPHIUMA ERYTHROCYTE K+ H+ EXCHANGER WITH H2DIDS

Subsequent to swelling, the Amphiuma red blood cells lose K+, Cl-, and water until normal cell volume is restored. Net solute loss is the re sult of K+/H+ and Cl-/HCO3-, exchangers functionally coupled through c hanges in pH and therefore HCO3-. Whereas the Cl-/HCO3- exchanger is c onstitutively active, K+/H+ actively is induced by cell swelling. The constitutive Cl-/HCO3- exchanger is inhibited by low concentrations (< 1 mu M) of 4,4'-diisothiocyanostilbene-2,2'-disulfo acid (DIDS) or H2D IDS, yet the concentration of H2DIDS > 25 mu M irreversibly modifies t he K+/H+ exchanger in swollen cells. We exploited the volume-dependent irreversible low-affinity reaction between H2DIDS and the K+/H+ to id entify the protein(s) associated with K+/H+ exchange activity. Labelin g of the membrane proteins of intact cells with (H2DIDS)-H-3 results i n high-affinity labeling of a broad 100-kDa band, thought to be the an ion exchanger. Additional swelling-dependent low-affinity labeling at 110 kDa suggests the possibility of a volume-induced population of ani on exchangers. Finally, the correlation between volume-sensitive K+/H modification and low-affinity labeling suggests that transport activi ty is associated with a protein of similar to 85 kDa. Although a 55-kD a protein is also labeled, it is a less likely candidate, since label incorporation and transport modification are less well correlated than that of the 85- and 110-kDa proteins.