Characterization of two Mg²⁺transporters in sealed plasma membrane vesicles from rat liver

Abstract

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ 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 Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

Differential Localization and Operation of Distinct Mg2+ Transporters in Apical and Basolateral Sides of Rat Liver Plasma Membrane

“…Plasma Membrane Isolation-Total LPM were isolated as described previously (21,22). Briefly, male Harlan Sprague-Dawley rats (250 -350 g) were anesthetized by intraperitoneal injection of 50 mg of pentobarbital/kg of body weight.…”

“…The pellets were recovered and resuspended at 6% (final concentration) in the isolation medium by four passes with the tight fitting pestle. Percoll (Amersham Pharmacia Biotech) was added to the resuspension in the proportion of 1.4 ml of Percoll to every 10.4 ml of resuspension (21), and the LPM were isolated by centrifugation at 34,500 ϫ g for 30 min. All operations following liver removal were carried out at 4°C.…”

“…Those studies also provided direct evidence that extracellular Na ϩ and Ca 2ϩ elicited a larger Mg 2ϩ efflux than either cation alone and suggested the presence of distinct Na ϩ /Mg 2ϩ and Ca 2ϩ /Mg 2ϩ transporters (21). Unfortunately, both putative transporters seemed to be inhibited by the same inhibitor.…”

“…Recent evidence in the literature demonstrates that the perfused liver or isolated hepatocytes can accumulate or release very large amounts of Mg 2ϩ upon specific metabolic stimulation (7,8). Recently, this laboratory was able to successfully obtain sealed liver plasma membranes (LPM) 1 to study Mg 2ϩ transport across the hepatocyte cell membrane (21). This model provides well defined and controllable experimental conditions in which membrane transport can be quantitatively investigated in the absence of interfering intracellular signaling pathways or transport by organelles.…”

“…This model provides well defined and controllable experimental conditions in which membrane transport can be quantitatively investigated in the absence of interfering intracellular signaling pathways or transport by organelles. Using sealed plasma membranes, we recently found that either extravesicular Na ϩ or Ca 2ϩ could mobilize intravesicular Mg 2ϩ (21). A major difference in liver plasma membrane vesicles is that they release intravesicular Mg 2ϩ in the presence of extravesicular Na ϩ or Ca 2ϩ alone, without the need of metabolic stimulation (21) necessary in intact hepatocytes (7,8).…”