Determination of membrane potential and cell volume by 19F NMR using trifluoroacetate and trifluoroacetamide probes.
The distribution of ionic species between intra- and extracellular compartments forms one basis for the determination of cell membrane potential. It is shown that fluorine-19 NMR studies of erythrocytes in the presence of trifluoroacetate, a stable, relatively nontoxic anion with pK = -0.3, provide a sensitive probe of membrane potential. Since such measurements are based on ion concentrations, the parallel use of the neutral analogue trifluoroacetamide to provide information on intra/extracellular volume ratios was also explored. In both cases, separate 19F resonances corresponding to intra- and extracellular ions were observed, with the intracellular resonance shifted downfield by approximately 0.2 ppm and the intracellular peak typically somewhat broader than the extracellular resonance. Studies with the band 3 anion-exchange inhibitor 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) indicate that both transmembrane diffusion and flux involving the band 3 anion exchanger contribute to the observed transport of the trifluoroacetate anion. Intra/extracellular volume ratios determined on the basis of trifluoroacetamide intensity ratios were in good agreement with determinations based on measured hematocrits. On the basis of the high sensitivity of 19F NMR and the capability of monitoring volume changes simultaneously, the time resolution for these measurements can approach the lifetime of intracellular trifluoroacetate ions and hence be limited by the trifluoroacetate flux rate.
London
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Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Gabel
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D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000081:Acetamides; D004910:Erythrocyte Membrane / Q000502:physiology*; D004912:Erythrocytes / Q000502:physiology*; D005461:Fluorine; D005463:Fluoroacetates / Q000097:blood*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008564:Membrane Potentials; D014269:Trifluoroacetic Acid / Q000097:blood*
D000081:Acetamides; D005463:Fluoroacetates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D005461:Fluorine; C030688:trifluoroacetamide; D014269:Trifluoroacetic Acid