| Summary: | 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 of === Biochemistry and Molecular Biology, Department of === Graduate
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