Characterization of two Mg21 transporters in sealed plasma membrane vesicles from rat liver

Cefaratti, Christie, Andrea Romani, and Antonio Scarpa. Characterization of two Mg21 transporters in sealed plasma membrane vesicles from rat liver. Am. J. Physiol. 275 (Cell Physiol. 44): C995–C1008, 1998.—The plasma membrane of mammalian cells possesses rapid Mg21 transport mechanisms. The identity of Mg21 transporters is unknown, and so are their properties. In this study, Mg21 transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg21 with negligible leak; 2) the addition of Na1 or Ca21 induces a concentrationand temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg21; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg21 ·μm22 ·min21 after Na1 and Ca21 addition, respectively); 4) coaddition of maximal concentrations of Na1 and Ca21 induces an additive Mg21 efflux; 5) both Na1and Ca21-stimulated Mg21 effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca21 channel blockers; 6) extracellular Na1 or Ca21 can stimulate Mg21 efflux in the absence of Mg21 gradients; and 7) Mg21 uptake occurs in LPM loaded with Na1 but not with Ca21, thus indicating that Na1/Mg21 but not Ca21/Mg21 exchange is reversible. These data are consistent with the operation of two distinct Mg21 transport mechanisms and provide new information on rates of Mg21 transport, specificity of the cotransported ions, and reversibility of the transport.