Ca2+ and proton transport in chromaffin granule membranes: a proton NMR study.
High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000313:Adrenal Medulla / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002458:Cell Fractionation; D002837:Chromaffin Granules / Q000378:metabolism* / Q000648:ultrastructure; D002838:Chromaffin System / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods
D000255:Adenosine Triphosphate; D004492:Edetic Acid; D002118:Calcium