Reconstitution of human red blood cell Na/H and Na/Na exchange transport

Na/H and Na/Na exchange transport was measured using human erythrocyte membrane proteins solubilized with octyl glucoside and reconstituted into voltage clamped soybean phospholipid membrane vesicles. The uptake of Na in exchange for either H or Na was: 1) 8 to 10 times higher in proteoliposomes that contained erythrocyte proteins than in proteoliposomes that contained heat denatured proteins or in liposomes that contained no proteins; 2) not affected by ouabain, bumetanide, or 4.4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS); 3) inhibited by amiloride, 5-(n-ethyl-n-isopropyl)amitoride (EIPA), and 5-(n-ethyl-1-n-isobutyl)amiloride (MIA) but not phenamil; and 4) inhibited by lithium (Li) in a concentration-dependent manner. Incubation of erythrocyte proteins with a low concentration of immobilized trypsin resulted in a significant increase (52%) in Na/Na transport, but no change was seen in Na/H transport. A higher concentration of trypsin increased Na/H transport by more than 2.5 times but did not increase Na/Na transport further. Examination of these studies indicates that, as assayed in reconstituted proteoliposomes that contained erythrocyte proteins, there is a differential response between Na/H and Na/Na transport to trypsin.