Competition between Na+ and Li+ for unsealed and cytoskeleton-depleted human red blood cell membrane: A Na-23 multiple quantum filtered and Li-7 NMR relaxation study

Evidence for competition between Li+ and Na+ for binding sites of human uns ealed and cytoskeleton-depleted human red blood cell (csdRBC) membranes was obtained from the effect of added Li+ upon the Na-23 double quantum filter ed (DQF) and triple quantum filtered (TQF) NMR signals of Na+-containing re d blood cell (RBC) membrane suspensions. We found that, at low ionic streng th, the observed quenching effect of Li+ an the Na-23 TQF and DQF signal in tensity probed Li+/Na+ competition for isotropic binding sites only. Membra ne cytoskeleton depletion significantly decreased the isotropic signal inte nsity, strongly affecting the binding of Na+ to isotropic membrane sites, b ut had no effect on Li+/Na+ competition for those sites. Through the observ ed Na-23 DQF NMR spectra, which allow probing of both isotropic and anisotr opic Na+ motion, we found anisotropic membrane binding sites for Na+ when t he total ionic strength was higher than 40 mM. This is a consequence of ion ic 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 ev en at high ionic strength. Li+ addition initially quenched the isotropic si gnal the most, indicating preferential Li+/Na+ competition for the isotropi c membrane sites. High ionic strength also increased the intensity of the a nisotropic signal, due to its effect on the restructuring of the membrane c ytoskeleton, Further Li+ addition competed with Na+ for those sites, quench ing the anisotropic signal. Li-7 T-1 relaxation data for Li+-containing suspensions of unsealed and csd RBC membranes, in the absence and presence of Na+ at low ionic strength, sh owed that cytoskeleton depletion does not affect the affinity of Na+ for th e RBC membrane, but increases the affinity of Li+ by 50%. This clearly indi cates that cytoskeleton depletion favors Li+ relative to Na+ binding, and t hus Li+/Na+ competition for its isotropic sites. Thus, this relaxation tech nique proves to be very sensitive to alkali metal binding to the membrane, detecting a more pronounced steric hindrance effect of the cytoskeleton net work to binding of the larger hydrated Li+ ion to the membrane phosphate gr oups. (C) 1999 Academic Press.