Abstract
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.
Highlights
Amiloride analogswith hydrophobic substitutionosn of many types of eukaryotic cells [1,2]
= 6.3 nM, Bma=X 1.2pmol/mg of protein) is present in mediate saturable transmembrane cationexchange, the Na+H+ exchanger must possess internal and external substratemicrovillus membrane vesicles but not in basolateral bindingsites for transportablecations,or asingle cationmembrane vesicles isolated from rabbit renal cortex, in accord withthe known membranloecalization of the Na+-H+exchanger in this tissue
This indicates that microvillus membrane vesicles contain at least one specific site for [3H]MIAbinding thatsaturatesat MIA concentrations below 6 X W 7 M. Binding to this site accounts for approximately 40% of the total [3H]MIAbinding observed when microvillus membrane that basolateralmembrane vesicles lack appreciable amiloride-sensitive Na+-H+exchange activity [12, 15]
Summary
Amiloride analogswith hydrophobic substitutionosn of many types of eukaryotic cells [1,2]. Na+-H+exchanger hasa high affinity internal binding microvillus membrane vesicles. These results suggest that the renal Na+-H' exchanger has a high affinity internal binding site foramilorideanalogs that is distinct
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