Quartenary structure and sub-cellular localization of rat brain sodium ion/calcium ion+potassium ion-exchanger, NCKX2

Rat brain Na+/Ca2++K+-exchangers (NCKX2) are plasma membrane Ca2+ transporters that play a number of important roles in mediating intracellular Ca2+ signalling and homeostasis. They are thought to transport one Ca2+ and one K+ in exchange for four Na+, the direction of the flux determined by the relative electrochemical gradients of each ion. In this thesis, the quaternary structure and the subcellular localization of rat brain NCKX2 were explored in detail. The isolation of detergent-resistant membranes containing lipid rafts/caveolae microdomains from whole rat brain by density flotation and subsequent co-immunoprecipitation experiments demonstrated selective localization of rat brain NCKX2 within lipid raft microdomains likely to be distinct from caveolae. The dimerization of rat brain NCKX2 driven by non-covalent interactions between exchanger monomers was observed, in addition to the formation of higher order oligomeric species with an apparent molecular weight greater than that of the exchanger dimer. The precise oligomeric state and the nature of the higher order oligomers of rat brain NCKX2 remain unclear. Several potential interacting partners of rat brain NCKX2 were identified by mass spectrometric analyses. However, these apparent interactions could not be confirmed in co-immunoprecipitation and immunoblotting experiments, thereby making it difficult to draw convincing conclusions about the physiological relevance of the interactions observed by mass spectrometry. These findings present important new discoveries about the quaternary structure and the sub-cellular localization of rat brain NCKX2, and would be helpful for further studies regarding how the structure of the exchanger relates to its transport function.