Topological organization, functional characterization and localization of the bovine rod photoreceptor na/ca-k exchanger

The Na/Ca-K exchanger in vertebrate photoreceptors plays an essential role in regulating the calcium concentration in photoreceptor outer segments. The structural and functional properties of the bovine rod photoreceptor Na/Ca-K exchanger were examined in this study using eight, anti-exchanger monoclonal antibodies. Experimental evidence for the topological organization of the exchanger was provided by mapping the epitopes of the antibodies using glutathione S-transferase fusion proteins containing specific regions of the exchanger. Five antibodies were mapped to the large N-terminal hydrophilic domain and shown to label the extracellular surface of the rod outer segment (ROS) plasma membrane by immunogold labeling for electron microscopy. Three antibodies were mapped to the hydrophilic domain connecting the two putative transmembrane domains and shown to label the cytoplasmic surface. The antibodies were also used to determine subcellular localization of the exchanger and to purify the exchanger by immunoaffinity chromatography. The exchanger was localized to the plasma membrane of rod photoreceptor outer segments. When the ROS membrane proteins were solubilized in CHAPS detergent, the exchanger did not appear to co-purify with other ROS membrane proteins. Treatment of the immunoaffinity-purified exchanger with deglycosylating enzymes indicated that the exchanger possesses a large amount of O-linked oligosaccharides in the large N-terminal hydrophilic domain. Limited proteolysis was used to determine the importance of the large extracellular and intracellular domains for ion transport. Trypsin degraded the extracellular domain, and kallikrein degraded a large part of the cytoplasmic domain. Following the removal of either of these hydrophilic domains, the exchanger exhibited Na⁺ and K⁺-dependent Ca²⁺ efflux activity similar to that of the untreated exchanger. The Vmax for the protease treated exchangers, however, increased ~2 fold. These results indicate that the ion binding and transport sites are located within or near the transmembrane domains and the removal of the bulky hydrophilic domain enables the exchanger to undergo a faster conformational change during ion transport. The oligomeric nature of the exchanger was determined using velocity sedimentation and size exclusion chromatography. Hydrodynamic properties of the detergent solubilized exchanger suggested that the exchanger is a monomer. Chemical crosslinking and velocity sedimentation studies, however, indicated that the exchanger exists as a dimer within the plasma membrane. These studies provide insight into the structural and functional properties of the exchanger. The O-glycosylated exchanger is exclusively localized in the ROS plasma membrane as a dimer, and the large hydrophilic domains of the exchanger do not appear to play a role in ion transport.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat