Flux of calcium ions, potent signals, controls many if not all cellular processes from fertilization and cell division to eventual cell death. Sodium/calcium exchangers (NCX), high-capacity integral membrane transporters, are the principal Ca2+ pumps in the plasma membranes of most cells. The unmatched ability of NCX to rapidly extrude Ca2+ using preexisting Na+-gradient potentials is critical to the normal function of excitable cells such as cardiac myocytes and neurons. These cells undergo quick, recurrent oscillations in levels of intracellular Ca2+ to achieve cardiac muscle contraction and neuronal signaling, respectively;and perturbations in NCX activity have been implicated in cardiac dysfunction and neuronal cell death. At present, an atomic-resolution structure of NCX has not been determined. Lack of 3D structure impedes the elucidation of structure-function relationships that are key to understanding NCX's mechanism of action. Our long-term goal is to describe at the atomic level the mechanism of transport and regulation of Ca2+/cation exchange in cardiac and neuronal cells. Toward this goal, the specific aims proposed here are as follows: 1. Heterologous overexpression of membrane proteins using a novel method. Optimized expression methods developed in my laboratory to increase membrane protein yields and reduce their host toxicity will enable us to produce enough protein for biochemical, biophysical, and crystallographic analyses. Initial expression targets will be prokaryotic NCX homologs selected for their significant sequence identity to eukaryotic sodium-calcium exchangers. Subsequently we will demonstrate our method's applicability to a large panel of membrane protein targets and investigate the molecular basis of overexpression toxicity. 2. Demonstration that prokaryotic NCX is an appropriate model for studies of Na+/Ca2+ exchange. This will be achieved by determining the oligomerization state and topology of the transporter, followed by characterization of structure-function and transport properties of prokaryotic NCX homologs by reconstitution into liposomes. Wild-type ion transport kinetics will be determined and inhibitors of transport identified. 3. Structure. We will crystallize a prokaryotic NCX homolog(s) alone and/or in complex with antibody fragments to determine and analyze structure(s). Combining biochemical and biophysical data with the crystal structure of an NCX homolog will lead to development of structure-based mechanistic models for Na+-Ca2+ exchange, spur new structure-based hypotheses to understand the mechanism of cardiac NCX, and may inspire structure-based drug design to find pharmacologically active agents.
The proposed research is relevant to public health and to the mission of NIH because it will deepen our understanding of calcium transport and signaling in normal and diseased cardiac myocytes and neuronal cells. Upon completion of the proposed goals, tractable targets potentially useful in drug design for future therapies for heart disease and ischemia-induced neuronal cell death will be available.