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.

Evidence for competition between Li(+) and Na(+) for binding sites of human unsealed and cytoskeleton-depleted human red blood cell (csdRBC) membranes was obtained from the effect of added Li(+) upon the (23)Na double quantum filtered (DQF) and triple quantum filtered (TQF) NMR signals of Na(+)-containing red blood cell (RBC) membrane suspensions. We found that, at low ionic strength, the observed quenching effect of Li(+) on the (23)Na TQF and DQF signal intensity probed Li(+)/Na(+) competition for isotropic binding sites only. Membrane cytoskeleton depletion significantly decreased the isotropic signal intensity, strongly affecting the binding of Na(+) to isotropic membrane sites, but had no effect on Li(+)/Na(+) competition for those sites. Through the observed (23)Na DQF NMR spectra, which allow probing of both isotropic and anisotropic Na(+) motion, we found anisotropic membrane binding sites for Na(+) when the total ionic strength was higher than 40 mM. This is a consequence of ionic strength effects on the conformation of the cytoskeleton, in particular on the dimer-tetramer equilibrium of spectrin. The determinant involvement of the cytoskeleton in the anisotropy of Na(+) motion at the membrane surface was demonstrated by the isotropy of the DQF spectra of csdRBC membranes even at high ionic strength. Li(+) addition initially quenched the isotropic signal the most, indicating preferential Li(+)/Na(+) competition for the isotropic membrane sites. High ionic strength also increased the intensity of the anisotropic signal, due to its effect on the restructuring of the membrane cytoskeleton. Further Li(+) addition competed with Na(+) for those sites, quenching the anisotropic signal. (7)Li T(1) relaxation data for Li(+)-containing suspensions of unsealed and csdRBC membranes, in the absence and presence of Na(+) at low ionic strength, showed that cytoskeleton depletion does not affect the affinity of Na(+) for the RBC membrane, but increases the affinity of Li(+) by 50%. This clearly indicates that cytoskeleton depletion favors Li(+) relative to Na(+) binding, and thus Li(+)/Na(+) competition for its isotropic sites. Thus, this relaxation technique proves to be very sensitive to alkali metal binding to the membrane, detecting a more pronounced steric hindrance effect of the cytoskeleton network to binding of the larger hydrated Li(+) ion to the membrane phosphate groups.