We investigated the mechanism of competition between Li+ and Mg2+ in Li(+)-loaded human red blood cells (RBCs) by making 7Li and 31P NMR and fluorescence measurements. We used 7Li NMR relaxation times to probe Li+ binding to the human RBC membrane and ATP; an increase in Mg2+ concentration caused an increase in both 7Li T1 and T2 values in packed Li(+)-loaded RBCs, in suspensions of Li(+)-loaded RBC ghosts, in suspensions of Li(+)-containing RBC membrane, and in aqueous solutions of ATP, indicating competition between Li+ and Mg2+ for binding sites in the membrane and ATP. We found that increasing concentrations of either Li+ or Mg2+ in the presence of human RBC membrane caused an increase in the 31P NMR chemical shift anisotropy parameter, which describes the observed axially symmetric powder pattern, indicating metal ion binding to the phosphate groups in the membrane. Competition between Li+ and Mg2+ for phosphate groups in ATP and in the RBC membrane was also observed by both fluorescence measurements and 31P NMR spectroscopy at low temperature. The ratio of the stoichiometric binding constants of Mg2+ to Li+ to the RBC membrane was approximately 20; the ratio of the conditional binding constants in the presence of a free intracellular ATP concentration of 0.2 mM was approximately 4, indicating that Li+ competes for approximately 20% of the Mg(2+)-binding sites in the RBC membrane. Our results indicate that, regardless of the spectroscopic method used, Li+ competes with Mg2+ for phosphate groups in both ATP and the RBC membrane; the extent of metal ion competition for the phosphate head groups of the phospholipids in the RBC membrane is enhanced by the presence of ATP. Competition between Li+ and Mg2+ for anionic phospholipids or Mg(2+)-activated proteins present in cell membranes may constitute the basis of a general molecular mechanism for Li+ action in human tissues.

Because Mg2+ and Li+ ions have similar chemical properties, we have hypothesized that Li+/Mg2+ competition for Mg2+ binding sites is the molecular basis for the therapeutic action of lithium in manic-depressive illness. By fluorescence spectroscopy with furaptra-loaded cells, the free intracellular Mg2+ concentration within the intact neuroblastoma cells was found to increase from 0. 39 +/- 0.04 mM to 0.60 +/- 0.04 mM during a 40-min Li+ incubation in which the total intracellular Li+ concentration increased from 0 to 5.5 mM. Our fluorescence microscopy observations of Li+-free and Li+-loaded cells also indicate an increase in free Mg2+ concentration upon Li+ incubation. By 31P NMR, the free intracellular Mg2+ concentrations for Li+-free cells was 0.35 +/- 0. 03 mM and 0.80 +/- 0.04 mM for Li+-loaded cells (final total intracellular Li+ concentration of 16 mM). If a Li+/Mg2+ competition mechanism is present in neuroblastoma cells, an increase in the total intracellular Li+ concentration is expected to result in an increase in the free intracellular Mg2+ concentration, because Li+ displaces Mg2+ from its binding sites within the nerve cell. The fluorescence spectroscopy, fluorescence microscopy, and 31P NMR spectroscopy studies presented here have shown this to be the case.

The biochemical action of lithium in the treatment of manic-depressive illness is still unknown. One hypothesis is that Li(+) competes for Mg(2+)-binding sites in biomolecules. We report here our studies on metal ion competition by three distinct methods: fluorescence, (31)P NMR, and (7)Li NMR spectroscopy, using ATP as a model ligand. By fluorescence spectroscopy, we used the dye, furaptra, by measuring the increases in Mg(2+) levels in an ATP solution as Li(+) levels were increased in the solution. This increase in Mg(2+) levels was indicated by increases in the fluorescence intensity ratio (335/370) of furaptra. By (31)P NMR spectroscopy, this competition was demonstrated by changes in the (31)P NMR spectrum of ATP. The Li(+)/Mg(2+) competition was indicated by predictable changes in the separation between the alpha and beta resonances of the phosphates of ATP. For (7)Li NMR spectroscopy, spin-lattice relaxation measurements were used, which provided free Li(+) concentrations that could be used for determining the free Mg(2+) values in ATP solutions. The values of the free Mg(2+) concentrations obtained by all three methods were in good agreement. The fluorescence and (7)Li NMR methods, however, proved to be more sensitive to low concentrations of Li(+) than the (31)P NMR method.

Liþ is the most effective drug used to treat bipolar disorder; however, its exact mechanism of action has yet to be elucidated. One hypothesis is that Li þ competes with Mg 2þ for the Mg 2þ binding sites on guanine-nucleotide binding proteins (G-proteins). Using 7 Li T 1 relaxation measurements and fluorescence spectroscopy with the Mg 2þ fluorophore furaptra, we detected Li þ /Mg 2þ competition in three preparations: the purified G-protein transducin (G t ), stripped rod outer segment membranes (SROS), and SROS with purified G t reattached (ROS-T). When purified ROS-T, SROS or transducin were titrated with Li þ in the presence of fixed amounts of Mg 2þ , the apparent Li þ binding constant decreased due to Li þ /Mg 2þ competition. Whereas for SROS the competition mechanism was monophasic, for G t , the competition was biphasic, suggesting that in G t , Li þ /Mg 2þ competition occurred with different affinities for Mg 2þ in two types of Mg 2þ binding sites. Moreover, as [Li þ ] increased, the fluorescence excitation spectra of both ROS-T and G t were blue shifted, indicating an increase in free [Mg 2þ ] compatible with Li þ displacement of Mg 2þ from two low affinity Mg 2þ binding sites of G t . G t release from ROS-T membrane was also inhibited by Li þ addition. In summary, we found evidence of Li þ /Mg 2þ competition in G t -containing preparations.

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