Abstract
We examined the effects of quinidine, amiloride and Li+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. Quinidine reversibly inhibited the initial rate of Na+-H+ exchange (I50 200 microM). The plot of 1/V versus [quinidine] was curvilinear, with Hill coefficient greater than 1.0, indicating that the drug interacts at two or more inhibitory sites or at a single site on at least two different conformations of the transporter. Quinidine decreased the Vmax for Na+-H+ exchange and increased the Km for Na+, indicating a mixed-type mechanism of inhibition. In contrast, plots of 1/V versus [amiloride] and 1/V versus [Li+] were linear, indicating single inhibitory sites; amiloride and Li+ each increased the Km for Na+ with no effect on Vmax, indicating a competitive mechanism of inhibition. Addition of Li+ increased the intercept with no change in slope of the 1/V versus [amiloride] plot, indicating that Li+ and amiloride are mutually exclusive inhibitors of Na+-H+ exchange. Addition of quinidine increased the slopes of the plots of 1/V versus [amiloride] and 1/V versus [Li+], indicating that the binding of quinidine is not mutually exclusive with the binding of amiloride and Li+. Results from this and previous studies are consistent with the concept that the inhibitor amiloride and the transportable substrates Na+, H+, Li+, and NH+4 all mutually compete for binding to a single site, the external transport site of the renal Na+-H+ exchanger. However, our findings indicate that quinidine interacts with the Na+-H+ exchanger on at least one additional site that is not shared by Na+, Li+, or amiloride.
Highlights
Time Course of Nu+ I$lux-To determine a time point at which the initial rate of Na+ influx could be estimated, we measured the early time course of Na+ uptake, as illustrated in Fig. 1.In thisexperiment,the membrane vesicles werepreequilibrated in pH 6.8 buffer, andthen Na+ uptake was measured at external pH 7.4
Thedose-dependentinhibition by quinidine of Na+ influx ( l e f t panel ofFig. 2) indicates that consider the possibility that thequinidine inhibition of Na+H+ exchange observed in Fig. 2 was artifactual and resulted from quinidine entering the intravesicular space by nonionic diffusion, thereby collapsing the outward H+ gradient that had been imposed
In the present work we find that Li+ and amiloride each inhibit Na+influx by interacting at a single saturable site, we demonstrate that binding of Na+ is mutually exclusive with the binding of Li+ and amiloride, and necessarily require the presence of more than one binding site for an inhibitor
Summary
The lack of effect of preincubating and presumably preloading the vesicles with quinidine suggests that the drug probably inhibits Na+-H+ exchange by binding to the external face of the transporter. To evaluate the kinetic mechanism underlying quinidine inhibition of Na+-H+exchange, we measured the initial rate of Na+ influx as a function of Na+concentration in the presence and absence of 200 PM quinidine.
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