Competition between Na+ and Li+ for Unsealed and Cytoskeleton-Depleted Human Red Blood Cell Membrane: A 23Na Multiple Quantum Filtered and 7Li NMR Relaxation Study

Abstract

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.

Biomedical applications of7Li NMR

“…35 The strongly quadrupolar 23 Na exhibits MQ behavior as a result of motional restriction upon binding. 4 We had previously shown that Li þ quenches the 23 Na double-quantum signal in erythrocytes.…”

“…36 That observation was confirmed and extended to both isotropic and anisotropic binding sites. 35 Lithium-7 T 1 measurements showed that cytoskeleton depletion favors Li þ to Na þ binding, increasing the affinity of Li þ for the membrane by 50%.…”

Effects of Li+ transport and Li+ immobilization on Li+/Mg2+ competition in cells: implications for bipolar disorder

“…These are reasonable assumptions, considering the lability and low association constants associated with Li þ binding (even for a tridentate ligand-like ATP) [2,3], the lack of sensitivity of the chemical shift and relaxation times of the 7 Li nucleus to the type of coordinating atoms present in its binding site(s) and its small quadrupolar coupling constant relative to 23 Na [30], which lead to much longer relaxation times than for 23 Na. Based on these assumption, it can then be shown that T 1obs and T 2obs are given by the expressions [21,25,26]:…”

“…The lymphoblastoma [23], neuroblastoma [24], and RBC [25] PMs were prepared, and the final PM pellet was washed twice in 5 mmol/L Tris-Cl buffer (pH 7.3) and stored at À80.08 for less than 2 weeks before use. Because viscosity affects relaxation rates [26], the viscosity was adjusted to 5 cP with polyvinyl pyrrolidone (MW-360,000); 5 cP is a reasonable estimate of intracellular viscosity [27].…”

Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5 ′ -diphosphate bound conformation

“…(1) using at least five 7 Li T 1 values [27,28]. The calculation of K app from 7 Li T 1 measurements assumes a two-state (free, f, and bound, b, metal ions) model undergoing fast exchange that uses a total binding site concentration, [B], and thus, [27][28][29][30][31]:…”