The sodium potassium (Na+/K+) pump is an important membrane protein that uses the energy of ATP to transport Na+ and K+ against their electrochemical gradients to maintain cell homeostasis. This study is aimed at the molecular understanding of the ionic events in the Na+/K+ pump transport cycle. Specifically, we aim at the description and quantification of the movements of Na+ and K+ in and out from the solution to the binding sites located within the pump protein. The experiments consist of measuring the charge movement that results from voltage perturbations using a fast voltage clamp in the squid giant axon. These experiments allow the measurement of the time course of the movement of each of the three Na+ ions released from the pump into the external solution. This proposal is to be carried out at the Laboratory of Cellular Physiology in Montemar, Chile, where 1 mm axons from the squid Dosidicus gigas are available. With these axons, the signal-to-noise ratio of the charge movement is much higher than in the Loligo pealeii axons, which will allow measurements that are not feasible with Loligo axons.
The specific aims are 1) understand the thermodynamics of extracellular Na+ translocation transitions.
We aim at the study of the entropic and enthalpic changes that occur during the binding and release of the Na+ ions in and out of the external access channel of the pump. This will be done by measuring the temperature dependence of all three components of the charge movement associated with the Na+ translocation. 2) Understand the biophysical properties of K+ translocation mechanism of the Na+/K+ pump.
This specific aim requires a comprehensive study of transitions involving the movement of K+ as a function of external concentration of K+, voltage, intracellular ATP and inorganic phospate. These studies are expected to give a mechanistic and molecular description of the electrical events mediated by the Na+/K+ pump, which is a ubiquitous membrane protein of fundamental importance in health and disease. This research will be done primarily in Montemar, Chile in collaboration with Ramon Latorre, Professor, Universidad de Valparaiso, as an extension of NIH grant No R37GM30376-29.

Public Health Relevance

The experiments proposed here are expected to give direct measurements of the ion movements in and out of the Na+/K+ pump and provide the biophysical mechanisms of the ion binding and unbinding during the pump operation. The activity of the Na+/K+ pump is vital for cell homeostasis because it helps maintaining the Na+ and K+ gradients. It is especially important in excitable cells where the loads change during the action potential. Clinically, the Na+/K+ pump is important because it is the receptor of digoxin, digitoxin and other cardiotonic steroids.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
5R03TW008351-03
Application #
8085913
Study Section
International and Cooperative Projects - 1 Study Section (ICP1)
Program Officer
Michels, Kathleen M
Project Start
2009-08-10
Project End
2013-05-31
Budget Start
2011-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2011
Total Cost
$52,895
Indirect Cost
Name
University of Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Castillo, Juan P; Rui, Huan; Basilio, Daniel et al. (2015) Mechanism of potassium ion uptake by the Na(+)/K(+)-ATPase. Nat Commun 6:7622
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