Influence of substrate structure on turnover of the organic cation/H+ exchanger of the renal luminal membrane.
We examined the influence of organic cation (OC) structure on the rate of turnover of the OC/H+ exchanger in rabbit renal brush-border membrane vesicles (BBMV). The rate of efflux of [14C]tetraethylammonium ([14C]TEA) from BBMV, measured in the presence of an inwardly directed chemical gradient for test agent, provided an indirect measure of activity of the OC/H+(OC) exchanger. The trans-stimulation of [14C]TEA efflux from BBMV was a saturable function of increasing extravesicular concentration of both unlabeled TEA and tetramethylammonium (TMA), with an apparent Michaelis constant (Kt) for the interaction of these compounds with the OC/H+(OC) exchanger of 25 microM and 1 mM, respectively. The effect on [14C]TEA efflux of saturating extravesicular concentrations of a series of n-tetraalkylammonium compounds was examined. Whereas the short-chain compounds TMA and TEA markedly stimulated [14C]TEA efflux (by 830% and 690%, respectively), the long-chain compounds tetrapropylammonium and tetrabutylammonium were less effective, increasing efflux by only 40% and 120%, respectively. When the exchanger was saturated with tetrapentylammonium, mediated efflux of [14C]TEA was reduced. Increasing alkyl chain length was also correlated with an increase in the inhibitory effect (as measured by the apparent inhibition constant, Ki, or the IC50 value) that these compounds had against transport of [14C]TEA by the OC/H+(OC) exchanger; i.e., there was a correlation between decreasing IC50 and decreasing turnover of the OC/H+(OC) exchanger. This same correlation was observed for a broader set of test agents of diverse molecular structure, including a series of n-tetraalkylammonium and -phosphonium compounds and the OCs, choline, N1-methyl nicotinamide, 1-methyl-4-phenylpyridinium, and amiloride. Because high affinity of substrates for the OC/H+(OC) exchanger is correlated with increasing substrate hydrophobicity, we conclude that the interaction of hydrophobic OCs with the renal OC/H+(OC) exchanger results in the formation of a substrate-exchanger complex that has a comparatively low rate of turnover.
Wright
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Department of Physiology, College of Medicine, University of Arizona, Tucson, AZ 85724, USA.
Wunz
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D000818:Animals; D017920:Antiporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001693:Biological Transport, Active; D002414:Cations, Monovalent / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D017136:Ion Transport / Q000502:physiology*; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D011485:Protein Binding; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D013329:Structure-Activity Relationship
D017920:Antiporters; D002414:Cations, Monovalent; D000644:Quaternary Ammonium Compounds; C027917:tetramethylammonium