Electrogenic proton-regulated oxalate/chloride exchange by lobster hepatopancreatic brush-border membrane vesicles

Abstract

SUMMARYThe transport of [14C]oxalate (Ox2–) by epithelial brush-border membrane vesicles (BBMV) of lobster (Homarus americanus) hepatopancreas, formed by a magnesium precipitation technique, was stimulated by an outward Cl– gradient (in >out). By contrast, Ox2– uptake was not enhanced by an inward Na+ or K+ transmembrane gradient. Generation of an inside-positive membrane potential by K+ in the presence of valinomycin stimulated Ox2–/Cl– exchange,while an inside-negative membrane potential generated by K+ efflux in the presence of valinomycin inhibited this process. Neither Ox2–/Ox2– nor Ox2–/SO42– transport exchange were affected by alterations of transmembrane potential. An inwardly directed proton gradient, or the presence of low bilateral pH, enhanced Ox2–/Cl– exchange, yet the H+gradient alone could not stimulate Ox2– uptake in Cl–-equilibrated BBMV or in vesicles lacking internal Cl–. The stilbenes 4-acetamido-4′-isothiocyanotostilbene-2,2′-disulfonic acid (SITS)and 4,4′-diisothiocyano-2,2′-disulfonic stilbene (DIDS) strongly inhibited Ox2–/Cl– exchange. Oxalate influx occurred by a combination of carrier-mediated transfer, exhibiting Michaelis–Menten kinetics, and nonsaturable `apparent diffusion'. Apparent kinetic constants for Ox2–/Cl–exchange were Kt=0.20 mmol l–1 and Jmax=1.03 nmol l–1 mg–1protein 7 s–1. 36Cl– influx into oxalate-loaded BBMV was stimulated by an inside-negative transmembrane potential compared with short-circuited vesicles. These results suggest that Ox2–/Cl– exchange in crustacean hepatopancreatic BBMV occurred by an electrogenic carrier mechanism exhibiting a 1:1 flux ratio that was modulated by an external proton-sensitive regulatory site.