Solubilization and reconstitution of an amiloride-inhibited sodium transporter from rabbit kidney medulla

An octyl glucoside extract has been formed from rabbit kidney medulla microsomes from which reconstituted proteoliposomes can be formed by lipid addition and dialysis to remove detergent. These proteoliposomes are capable of amiloride-inhibited 22Na+ transport. The amiloride-inhibited Na+ transport process is complete within 10 min and directly proportional to the vesicle protein concentration. Sodium accumulation by the proteoliposomes has been proven to represent transport by the demonstration that all Na+ taken up by the vesicles can be removed by the ionophore nigericin. The process has been shown to be specific for amiloride by the demonstration that the effect of amiloride on Na+ transport could not be reproduced by the similar compound sulfaguanidine nor by pyrazine, 2-pyrazinecarboxamide, 2-pyrazinecarboxylate, or 3-amino-2-pyrazinecarboxylate. The relationship between Na+ uptake into proteoliposomes and Na+ concentration was similar to the relationship between Na+ uptake and concentration observed with medulla microsomes. The concentration of amiloride required for half-maximal inhibition of Na+ uptake into either proteoliposomes or medulla microsomes was also the same. The evidence seems clear that the protein responsible for amiloride-inhibited Na+ transport into rabbit kidney medulla microsomes has been extracted from the membranes and incorporated into purified lipid vesicles.