In the past year our work has focused on two proteins, vcINDY, a succinate transproter which has been implicated in longevity and obesity and NCX_Mj, a Na/Ca exchanger critical for Ca homeostasis in cells. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. In the past year, we have been establishing a system for the functional analysis of the Na/Ca exchanger NCX_Mj. We have successfully reconstituted purified NCX_Mj into proteoliposomes and established functional assays for the protein. Initial work establishes kinetic parameters of transport and corroborates previous work demonstrating that this protein utilizes a ping-pong mechanism for transport. We have also used the stoichiometry methods reported last year to establish the functional stoichiometry of this system, revealing for the first time that in this protein 3 Na+ ions are exchanged for each Ca++ ion taken up. This work was published this year in the Journal of General Physiology.
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