High affinity binding of amiloride analogs at an internal site in renal microvillus membrane vesicles.
Amiloride analogs with hydrophobic substitutions on the 5-amino nitrogen atom are relatively high affinity inhibitors of the plasma membrane Na(+)-H+ exchanger. We demonstrated that a high affinity-binding site for [3H]5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) (Kd = 6.3 nM, Bmax = 1.2 pmol/mg of protein) is present in microvillus membrane vesicles but not in basolateral membrane vesicles isolated from rabbit renal cortex, in accord with the known membrane localization of the Na(+)-H+ exchanger in this tissue. The rank order potency for inhibition of microvillus membrane [3H]MIA binding by amiloride analogs was: MIA (I50 approximately 10 nM) greater than amiloride (I50 approximately 200 nM) greater than benzamil (I50 approximately 1200 nM). This correlated with a qualitatively similar rank order potency for inhibition of Na(+)-H+ exchange: MIA (I50 approximately 4 microM) greater than amiloride (I50 approximately 15 microM) greater than benzamil (I50 approximately 100 microM), but did not correlate with the rank order potency for inhibition of the organic cation-H+ exchanger in microvillus membrane vesicles: MIA approximately benzamil (I50 approximately 0.5 microM) greater than amiloride (I50 approximately 10 microM). However, tetraphenylammonium, an inhibitor of organic cation-H+ exchange, inhibited the rate of [3H]MIA binding without an effect on equilibrium [3H]MIA binding; the dissociation of bound [3H]MIA was inhibited by preloading the membrane vesicles with tetraphenylammonium. These findings indicated that high affinity [3H]MIA binding to renal microvillus membrane vesicles takes place at an internal site to which access is rate-limited by the tetraphenylammonium-sensitive organic cation transporter. Equilibrium [3H]MIA binding was inhibited by H+ but was unaffected by concentrations of Na+ or Li+ that saturate the external transport site of the Na(+)-H+ exchanger. Binding of MIA to its high affinity binding site had no effect on the rate of Na(+)-H+ exchange. This study suggests that the renal Na(+)-H+ exchanger has a high affinity internal binding site for amiloride analogs that is distinct from the external amiloride inhibitory site.
Desir
G V
GV
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Cragoe
E J
EJ
Aronson
P S
PS
D000584:Amiloride / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D007672:Kidney Cortex / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; C013407:benzamil; D000584:Amiloride; C056369:5-(N-methyl-N-isobutyl)amiloride; D012964:Sodium