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 made major progress in developing methods to accurately determine transporter stoichiometry. We have developed a new and widely applicable method for determining transporter stoichiometry for Na-coupled transporters that will be especially useful for characterizing transporters amenable to structure determination. We applied this method to vcINDY and established that the transporter couples 3 Na ions to the transport of each succinate molecule, and shows that the transporter can concentrate succinate to high levels in the cell. We also confirmed the validity of the method using a sugar transporter of known stoichiometry, vSGLT. We are now applying the new method to other transporters for which accurate understanding of stoichiometry is important. THis work was published in eLife this year. In addition, 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. In the coming year we seek to utilize the stoichiometry methods reported above to establish the functional stoichiometry of this system.
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