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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Kinetic characterization of tetrapropylammonium inhibition reveals how ATP and P_i alter access to the Na⁺-K⁺-ATPase transport site

¹Division of Biomedical Sciences, Cell Biology & Physiology Section, Department of Biological Sciences, Illinois State University, Normal, Illinois; and ²Department of Medical Pharmacology and Physiology, School of Medicine, and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri

Submitted 4 February 2005 ; accepted in final form 17 March 2005

Current models of the Na⁺-K⁺-ATPase reaction cycle have ATP binding with low affinity to the K⁺-occluded form and accelerating K⁺ deocclusion, presumably by opening the inside gate. Implicit in this situation is that ATP binds after closing the extracellular gate and thus predicts that ATP binding and extracellular cation binding to be mutually exclusive. We tested this hypothesis. Accordingly, we needed a cation that binds outside and not inside, and we determined that tetrapropylammonium (TPA) behaves as such. TPA competed with K⁺ (and not Na⁺) for ATPase, TPA was unable to prevent phosphoenzyme (EP) formation even at low Na⁺, and TPA decreased the rate of EP hydrolysis in a K⁺-competitive manner. Having established that TPA binding is a measurement of extracellular access, we next determined that TPA and inorganic phosphate (P_i) were not mutually exclusive inhibitors of para-nitrophenylphosphatase (pNPPase) activity, implying that when P_i is bound, the transport site has extracellular access. Surprisingly, we found that ATP and TPA also were not mutually exclusive inhibitors of pNPPase activity, implying that when the cation transport site has extracellular access, ATP can still bind. This is consistent with a model in which ATP speeds up the conformational changes that lead to intracellular or extracellular access, but that ATP binding is not, by itself, the trigger that causes opening of the cation site to the cytoplasm.

quaternary ammonium; Dixon plot; P-type adenosine triphosphatase; inorganic phosphate

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