Electrogenic proton-regulated oxalate/chloride exchange by lobster hepatopancreatic brush-border membrane vesicles.
The 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-/SO4(2-) 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(-1) protein 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.
Gerencser
George A
GA
Department of Physiology and Functional Genomics, College of Medicine, University of Florida, Gainesville, FL 32610, USA. gag@phys.med.ufl.edu
Robbins
Frank
F
Zhang
Jianliang
J
Ahearn
Gregory A
GA
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002250:Carbon Radioisotopes; D002712:Chlorides / Q000378:metabolism*; D043143:Hepatopancreas / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008121:Nephropidae; D010070:Oxalates / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism*
D002250:Carbon Radioisotopes; D002712:Chlorides; D010070:Oxalates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium