Molecular size of the Na+-H+ antiport in renal brush border membranes, as estimated by radiation inactivation

Abstract

The radiation inactivation method was applied to brush border membrane vesicles from rat kidney, in order to estimate the molecular size of the Na+-H+ antiporter. Sodium influx (1mM) driven by an acid intravesicular pH was unaffected by the high osmolarity of the cryoprotective solution. Initial rate of influx was estimated by linear regression performed on the first 10 seconds of transport: 0.512 pmol/micrograms protein/s. There was no binding component involved. Incubation performed in the presence of 1 mM amiloride, an inhibitor of the Na+-H+ antiport gave an initial rate of only 0.071 pmol/microgram/s, an 82% inhibition. Membrane vesicles were irradiated at -78 degrees C in a Gammacel Model 220. Sodium influx was reduced, as the dose of radiation increased, but the influx remained linear for the period of time (10s) during which the initial rate was estimated, indicating no alteration of the proton driving force during this time period. Amiloride-insensitive flux remained totally unaffected by the radiation dose, indicating that the passive permeability of the membrane towards sodium was unaffected. The amiloride-sensitive pathway presented a monoexponential profile of inactivation, allowing the molecular size to be estimated at 321 kDa. Based on DCCD-binding studies suggesting the molecular size of the monomer to be around 65 kDa for rat kidney, our results suggest that the functional transporter in the membrane to be a multimer.