The long term goal of this project remains a detailed understanding in physicochemical terms, of the transport mechanism of the Na/K pump. This is important because the Na/K pump maintains the electrochemical gradients for Na and K ions that underlie electrical signalling, essential coupled transport processes, and cell volume regulation; the Na/K pump is also thought to mediate the therapeutic action of the cardiotonic steroid and specific inhibitor of the pump, digoxin, still one of the most widely prescribed cardiac drugs. Charge translocation by the pump during the ion transport cycle, or during its partial reactions, is a fundamental feature of pump activity and not only provides a readily accessible, reproducible, and exquisitely sensitive signal for assaying turnover rates and rates of conformational transitions, but also sheds light on the mechanisms of ion transport.
The specific aims are (i) to further characterize the charge translocating steps in the transport cycle by pursuing quantitative analysis of the dependence of pump charge movements on membrane potential, [Na]o, [K]o, and temperature, and (ii) to address the questions of whether, under what conditions, and by which mechanisms, pump activity may be modulated by cellular regulatory processes like protein kinase-mediated phosphorylation of the pump (or closely associated regulatory molecule). Steady-state, and voltage jump induced transient (pre-steady-state), pump currents are measured in entire guinea-pig ventricular myocytes, in giant excised patches of myocyte membrane, or in squid giant axons (in which unidirectional tracer fluxes are also measured under voltage clam), all preparations with a relatively high pump site density (about 1200 um-2). Myocytes and axons are voltage clamped and internally dialyzed with solutions that can be exchanged during recordings. Access to the cytoplasmic surface of the membrane is further improved in excised inside-out patches, a particular advantage for investigating modulation of the Na/K pump. Recent improvements in hardware and software now permit ultra high-speed measurements of pump-mediated charge movements in squid axons and in giant patches, allowing resolution of processes with relaxation rates as fast as 10-5 s-1, some 3 orders of magnitude faster than the Na/K pump's maximum turnover rate. Explicit kinetic models of the Na/K transport mechanism are developed to account rigorously and economically for experimental observations: fits to the data are used to refine the models, and then simulations based on them explore predicted pump behavior and suggest new experiments.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG2-LBPA (01))
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Rockefeller University
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New York
United States
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Vedovato, Natascia; Gadsby, David C (2014) Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps. J Gen Physiol 143:449-64
Gadsby, David C; Bezanilla, Francisco; Rakowski, Robert F et al. (2012) The dynamic relationships between the three events that release individual Na? ions from the Na?/K?-ATPase. Nat Commun 3:669
Castillo, Juan P; De Giorgis, Daniela; Basilio, Daniel et al. (2011) Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid. Proc Natl Acad Sci U S A 108:20556-61
Vedovato, Natascia; Gadsby, David C (2010) The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions. J Gen Physiol 136:63-82
Takeuchi, Ayako; Reyes, Nicolás; Artigas, Pablo et al. (2009) Visualizing the mapped ion pathway through the Na,K-ATPase pump. Channels (Austin) 3:383-6
Gadsby, David C (2009) Ion channels versus ion pumps: the principal difference, in principle. Nat Rev Mol Cell Biol 10:344-52
Takeuchi, Ayako; Reyes, Nicolas; Artigas, Pablo et al. (2008) The ion pathway through the opened Na(+),K(+)-ATPase pump. Nature 456:413-6
Rakowski, R F; Artigas, Pablo; Palma, Francisco et al. (2007) Sodium flux ratio in Na/K pump-channels opened by palytoxin. J Gen Physiol 130:41-54
Artigas, Pablo; Gadsby, David C (2006) Ouabain affinity determining residues lie close to the Na/K pump ion pathway. Proc Natl Acad Sci U S A 103:12613-8
Artigas, Pablo; Al'aref, Subhi J; Hobart, E Ashley et al. (2006) 2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms: the role of bilayer material properties. Mol Pharmacol 70:2015-26

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