Co2+ as a shift reagent for 35Cl NMR of chloride with vesicles and cells

Applications of high-resolution 35Cl NMR to the study of chloride in vivo and in vesicles have hitherto been limited by problems of NMR detectability and of resolving internal from external signals. We have characterized the effects of Co2+ on the 35Cl resonance of Cl- in solution and have shown that when added to suspensions of lipid vesicles, Co2+ shifts the 35Cl signal of the extravesicular Cl-, allowing clear resolution and quantitation of two peaks. We have assigned these signals to chloride inside and outside the vesicles. The spectra do not change over a 90-min period, demonstrating the stability of the vesicles in the presence of Co2+. This technique is shown to be applicable to red blood cell ghosts, where intravesicular and extravesicular chloride signals were separated and measured and chloride/sulfate exchange through the band 3 anion transport protein A was followed. In two plant species (an alga and a higher plant), an intracellular Cl- signal can be observed and resolved from the extracellular signal. The intracellular transportable chloride was found to be fully NMR-visible (+/- 5%) in the algal cells. The high steady-state levels of Cl- seen in the alga were consistent with previous work using 36Cl- labeling on a related species [Doblinger, R., & Tromballa, H.W. (1982) Planta 156, 10-15]. Successive spectra acquired after adding Co2+ to Chlorella cells under deenergizing conditions allow us to follow the time course of movement of Cl- out of the cells.