Influence of substrate structure on turnover of the organic cation/H⁺ exchanger of the renal luminal membrane

Abstract

 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 (K _t) for the interaction of these compounds with the OC/H⁺(OC) exchanger of 25 µM 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, K _i, 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, N ¹-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.