The long term objective of this new application is to perform detailed characterization of the membrane locus of ATP that fuels red cell's Na/K and Ca++ pumps and the role of this pool in regulating membrane function. The applicant has previously shown that within the cytoskeletal/membrane complex of red cell exists a compartment of ATP that is the proximate energy source for both the Na/K pumps and Ca pumps. The objective of the proposed studies is to find out where this ATP is located by identifying the cytoskeletal proteins involved in the compartmentalization process. To achieve this goal the following four specific aims are outlined: 1. Identify in human red blood cell ghosts candidate proteins that are directly involved with the ATP pool by photolabelling these proteins with an [a-32P] ATP derivative that is specifically incorporated into the pool; 2. Determine the reassemblage of the ATP pool in ghosts in which, by pretreatment, cytoskeletal/membrane proteins have been removed with concomitant loss of the ability to form the membrane pool. Study the ATP pool characteristics of ghosts prepared from red cell variants with defective membrane/cytoskeletal interactions;3. Determine the membrane topography of ATP pools by confocal microscopy after filling the pools with a fluorescent derivative of ATP; and 4. Determine, using inside-out vesicles, the properties of the membrane ATP pool driven Ca and Na uptake by Ca and Na/K pumps. These studies will provide information regarding the physiologic significance of this ATP pool in Ca and Na transport. It is anticipated that the successful accomplishment of these goals will provide new insights into the role of membrane ATP pool serving both as an energy reserve capable of sustaining ion pumping during periods of transient stress, as well as a putative transduction mechanism by which cell's cytoskeleton may sense cellular energy stores.
| Stengelin, M K; Hoffman, J F (1997) Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma. Proc Natl Acad Sci U S A 94:5943-8 |
| Stengelin, M; Hoffman, J F (1997) Differences in uncoupled sodium efflux between red blood cells and kidney Na,K-ATPase are not based on differences in the cDNA for the alpha subunit. Ann N Y Acad Sci 834:119-22 |
