Objective
Active ion transport through ion pumps in biological membranes is a process, which is much less understood than passive transport through ion channels. The reasons for the lack of knowledge have to be searched in a more complex structure of the pump proteins as well as in a more complex transport process in which the energizing enzymatic process and the movement of the ion(s) through the intra-protein pathway are coupled. In the presented research project a number of biophysical and biochemical techniques will be applied to obtain detailed information on the transport processes in two P-type ATPases and to reveal the underlying mechanisms. Both ion pumps, the Na,K-ATPase and the Ca-ATPase of the sarcoplasmatic reticulum, are vital proteins in the cells of all animals. Together with the published data base and with the scarce structural details available on the P-type ATPases, the concepts which model the transport processes will be either refined or precise constraints will be formulated which have to be met by structure-function relations.
The experimental goals of the joint effort are
(1) the identification and characterization of essential structural elements of the proteins crucial for ion transport;
(2) determination of the spatial position of the ion binding sites within the Na,K-ATPase, and;
(3) analysis of the kinetics of charge movements when the pumps run through their cycle executing the ion transport.
To approach these aims experiments will be performed in a first step with membrane preparations that contain ion pumps in a high concentration. Such preparations are already available. In a next step the proteins will be reconstituted in vesicles and then in planar lipid bilayers to get an easy access to both aqueous surfaces of the proteins. And in parallel, transmembrane sections of the proteins will be prepared by specific tryptic digestions, they will be characterized and investigated either in their native membranes or in reconstituted membranes. Reconstitution of different transmembrane fragments will allow an identification of the crucial components, which form the ion pathway inside the protein.
To detect ion transport, e.g. charge movements within the proteins, a number of experimental techniques will be applied which were introduced and optimised during recent years by the teams involved in this proposal or which will be developed as part of the tasks. Direct electrical measurements will be performed with tree methods: with protein-containing membrane fragments adsorbed to planar lipid bilayers, which is a well established technique for investigations of the Na,K-ATPase, with protein-containing membrane fragments adsorbed to supported lipid bilayers, and with ion pumps reconstituted in planar lipid bilayers. These methods are excellently suitable to investigate kinetic properties of the ion pumps. A different, partially complementary technique is the use of electrochromic fluorescent styryl dyes, which indicate charge movements by a change of their spectroscopic properties, which can be easily detected. To gain information on the steric localization of the ions when bound inside the protein it is planned to study the effect of the ion-pump action on a number of styryl dyes with chromophores located in different positions in the membrane dielectric. A completely new strategy to learn about the ion movement in the Na,K-ATPase is the determination of local electric fields by electrostriction phenomena. So far this technique was applied only to less complex charge-carrying systems in membranes.
The joint efforts of the different expertises and techniques provided by the participants and a discussion of the results obtained from the different approaches will be a solid base to make progress in the understanding of molecular mechanisms of ion transport in P-type ATPases und to develop concepts and methods which my be used widely by the scientific community dealing with active ion transport in biological membranes.
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Coordinator
78457 Konstanz
Germany
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