External anions regulate stilbene-sensitive proton transport in placental brush border vesicles

The mechanism for HCO3-(-)independent proton permeability in microvillus membrane vesicles (MVV) isolated from human placenta was examined by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Proton fluxes (JH) across MVV were determined in response to induced pH and anion gradients from the time course of 6CF fluorescence, the MVV buffer capacity, and the 6CF vs. pH calibration. In the absence of anions, JH was 12 +/- 2 nequiv s-1 (mg of protein)-1 (pHin 7.4, pHout 6.0, MVV voltage-clamped with K+/valinomycin, 23 degrees C), corresponding to a proton permeability coefficient of 0.02 cm/s, with an activation energy of 9.1 +/- 0.3 kcal/mol. JH was inhibited 20% by dihydro-4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (H2DIDS) with KI = 8 microM [( Cl-]out = 0 mM). For a 0.5-unit pH gradient JH increased from 1.5 to 4.6 nequiv s-1 (mg of protein)-1 as the internal MVV pH was increased (5.5-7.5). External Cl-, Br-, and I- (but not SO4(2-) and PO4-) increased JH 1.3-2.5-fold for both inwardly and outwardly directed pH gradients with KD = 1.0 +/- 0.4 mM (Br-) and greater than 100 mM (Cl-). This increase was blocked by 100 microM H2DIDS but not by amiloride or furosemide. Internal Cl- did not alter JH induced by pH gradients nor were proton fluxes induced by anion gradients in the absence of a pH gradient. Experiments in which JH was driven by membrane potentials (induced by valinomycin and K+ gradients) indicated that proton transport was voltage-sensitive. These experiments demonstrate a stilbene-sensitive electrogenic proton transport mechanism in MVV that is regulated allosterically by anions at an external binding site.