The objective of this project is to obtain a greater understanding of the voltage dependent step(s) in the reaction cycle of the Na/K pump.
Four specific aims are: 1) measure steady-state Na/K pump current-voltage relationships (I-Vs) for various electrogenic modes of pump operation, 2) test whether the stoichiometry of the Na/K pump is fixed at 3Na/2K and is voltage-independent over wide-ranging conditions, 3) investigate transient Na/K pump currents (pump charge movement), and 4) test whether pump- mediated Na/Na exchange, though electroneutral, is voltage-dependent. To achieve these aims, simultaneous measurements of changes in pump-mediated current and flux, produced by exposure to the reversible pump inhibitor dihydrodigitoxigenin (H2DTG), will be made in voltage-clamped, internally- dialyzed squid giant axons. Control experiments have already demonstrated that: 1) H2DTG has no direct effect on ionic conductances; 2) current and 22Na efflux derive from the same membrane area; 3) changes in passive K current, due to changes of extracellular [K] on stopping the pump, are largely prevented by K channel-blocking agents; 4) pump-mediated Na/Na exchange has been minimized by adding high [ATP] and the phosphagens phosphoenolpyruvate and phosphoarginine, to the dialysis fluid. Steady- state Na/K pump I-Vs for the forward and reverse modes of pump operation will be obtained under various conditions as the difference between membrane I-Vs measured before and during exposure to H2DTG. The stoichiometry of the Na/K pump can be investigated since appropriate control experiments have shown that H2DTG-sensitive flux and current can be equated with Na/K pump flux and current, and thus used to calculate the ratio of Na to K transport, provided the pump transports no other ion. Preliminary measurements of transient pump currents have been made in voltage-clamped Xenopus oocytes in the absence of external K, which presumably limits the pump to Na/Na exchange. A prediction from such measurements, that pump-mediated electroneutral Na/Na exchange is voltage- dependent, can be tested in squid axons by assessing the voltage dependence of H2DTG-sensitive Na efflux from high-[ADP] axons into K-free seawater. Further understanding of the Na/K pump of animal cells is important since it maintains the Na electrochemical gradient which is essential for excitability and for Na-coupled transport processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS022979-06
Application #
3405877
Study Section
Physiology Study Section (PHY)
Project Start
1985-05-01
Project End
1994-04-30
Budget Start
1990-05-01
Budget End
1991-04-30
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Rosalind Franklin University
Department
Type
Schools of Medicine
DUNS #
069501252
City
North Chicago
State
IL
Country
United States
Zip Code
60064
Potvin, Olivier; Dieumegarde, Louis; Duchesne, Simon et al. (2017) Freesurfer cortical normative data for adults using Desikan-Killiany-Tourville and ex vivo protocols. Neuroimage 156:43-64
Ding, Yanli; Hao, Jingping; Rakowski, Robert F (2011) Effects of oligomycin on transient currents carried by Na+ translocation of Bufo Na+/K(+)-ATPase expressed in Xenopus oocytes. J Membr Biol 243:35-46
Ding, Yanli; Rakowski, Robert F (2010) The effect of holding potential on charge translocation by the Na+/K +-ATPase in the absence of potassium. J Membr Biol 236:203-14
Guennoun-Lehmann, Saida; Fonseca, James E; Horisberger, Jean-Daniel et al. (2007) Palytoxin acts on Na(+),K (+)-ATPase but not nongastric H(+),K (+)-ATPase. J Membr Biol 216:107-16
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
Fonseca, J; Kaya, S; Guennoun, S et al. (2007) Temporal Analysis of Valence &Electrostatics in Ion-Motive Sodium Pump. J Comput Electron 6:381-385
Holmgren, Miguel; Rakowski, Robert F (2006) Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence. Biophys J 90:1607-16
Rakowski, Robert F; Kaya, Savas; Fonseca, James (2005) Electro-Chemical Modeling Challenges of Biological Ion Pumps. J Comput Electron 4:189-193
Vasilyev, A; Khater, K; Rakowski, R F (2004) Effect of extracellular pH on presteady-state and steady-state current mediated by the Na+/K+ pump. J Membr Biol 198:65-76
Vasilyev, A; Indyk, E; Rakowski, R F (2002) Properties of a sodium channel (Na(x)) activated by strong depolarization of Xenopus oocytes. J Membr Biol 185:237-47

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