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Nanoscale Resolution of Self-assembled Liposomes Containing Functional Sodium Pumps

Beatrice M. Maier*, dagger

Department of Physiology and Pharmacology, Loma Linda University Medical School,
Loma Linda, CA 92350 USA

This is an abstract for a presentation given at the
Ninth Foresight Conference on Molecular Nanotechnology.
There will be a link from here to the full article when it is available on the web.

The sodium pump (SP; Na,K-ATPase) is the most abundant and vital membrane protein of the body; it replaces the intracellular Na ions by K ions; it is at the basis of, e.g., brain, muscle and kidney activity; it is an energy conversion and an electrogenic system; it contains powerful receptors for cardiotonic drugs, toxins and for putative natriuretic hormones (digitalis, bufotoxins, palytoxin, carbon suboxide and endogenous inhibitors). To study the still unknown molecular mechanism of the pump and receptor functions, it is necessary to insert the purified pump protein into artificial membranes.

Artificial membranes containing functional SP were formed as described (1) by adding 1 mg phosphatidylcholine (PC), dissolved in 50 µl buffered Mg solution (with varying Na and K concentrations) containing 1% cholate, to 50 µl of the same solution without PC but with 100-200 µg dissolved SP protein. Upon removal of the detergent by dialysis, the PC and SP-containing micelles bend to join their hydrophobic core to form SP containing liposomes (SPL) reaching a stable minimal energy state at a 100 nm vesicle diameter.

Quantitative analysis of electron micrographs of freeze-fractured SPL reveal non-interacting SP molecules in symmetrical orientation (2).

Right-side-out or inside-out oriented SPs were activated selectively or in sequence by adding ATP (the fuel) either inside, outside or sequentially on both sides of the SPL membrane (two-sided bifunctional SPL). The specific radioactivity of Na and K (Rb) isotopes used for the ion-transport measurement was maximized so that the SPL volume required for a single experimental point could be downsized to 1-2 µl. In the miniaturized test-system, fmoles of an endogenous SP inhibitory factor from bovine hypothalamus (HIF) could be detected and the physicochemical properties of HIF characterized in comparison to known inhibitors using less than 2 µl of SPL suspension (3).

Ultrathin sections of negatively stained SPL at nanometer resolution showed the presence of transmembrane SP protein (1). The SP protein appeared to establish membrane-to-membrane contact points and to induce (thereby?) formation of larger and some multilayered SPL as compared to SP-free liposomes (4). SP-induced membrane structuring has not been reported before and may be relevant for better understanding the formation of biological membranes.

  1. Anner, B.M. Transposing results from an artificial minicell to a real cell : experimental evidence for a working hypothesis linking Na,K-ATPase permeability states to specific alterations of cell life. Ann. New York Sci. 834, 367-371, 1997.
  2. Anner, B.M., Robertson, J.D., and Ting-Beall, H.P. Characterization of (Na + K)-ATPase liposomes. II. Effect of alpha-subunit digestion on intramembrane particle formation and Na,K-transport. Biochim. Biophys. Acta 773, 262-270, 1984.
  3. Anner, B.M., Rey, H.G., Moosmayer, M., Meszoely, I., and Haupert, G.T. Hypothalamic Na,K-ATPase inhibitor characterized in two-sided liposomes containing pure renal Na,K-ATPase. Am. J. Physiol., 258, F144-F153, 1990.
  4. Volet, B., Lacotte, D., Moosmayer, M. and Anner, B. M. Na,K-ATPase or carboxyfluorescein alter vesicle formation in vitro. Biochim. Biophys. Acta, 1191, 1-6 1994.

Abstract in RTF format 6,143 bytes

*Corresponding Address:
Beatrice M. Maier
Department of Physiology and Pharmacology, Loma Linda University Medical School
Risley Hall, Room 219, Loma Linda, CA 92350 USA
phone: (909) 558-4564
fax: (909) 558-0119

daggerFormer name and address:
Beatrice M. Anner,
Department of Pharmacology, Geneva University Medical School, Switzerland.

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