THE NA CA-K EXCHANGER OF ROD PHOTORECEPTOR EXISTS AS DIMER IN THE PLASMA-MEMBRANE/

The oligomeric state of the Na/Ca-K exchanger in the plasma membrane o f bovine photoreceptors was investigated using chemical cross-linking techniques. In the natural membrane, virtually all Na/Ca-K exchanger c ould be cross-linked mainly to a complex having an apparent molecular mass of 490 kDa by cupric phenanthroline catalyzed disulfide bonding a s evidenced by Western blotting. Stable cross-links of the exchanger w ere also obtained with the thiol-specific reagent N,N'-p-phenylidenedi rnaleimide. Neuraminidase treatment seduced the apparent molecular mas s of the highly glycosylated Na/Ca-K exchanger and of the 490 kDa cros s-link product by 50 and 85 kDa, respectively. DL-1,4-Bismaleimido-2,3 -butanediol (BMBD), a novel cleavable dimaleimide, was synthesized in order to produce cross-links that were stable to reductive conditions. Purification of the BMBD cross-linked exchanger followed by two-dimen sional SDS polyacrylamide electrophoresis identified the cross-linked homodimers of the exchanger. There was no indication of higher oligome rs, suggesting that the exchanger exists as a dimer in the plasma memb rane. Hydrodynamic properties of the detergent-solubilized exchanger w ere determined by velocity sedimentation and gel filtration chromatogr aphy. The Triton X-100-solubilized exchanger ran as a single species h aving a Stokes radius of 10.0 nm, a sedimentation coefficient of 5.4 S , and a partial specific volume of 0.74 mL/g in Triton X-100. Similar results were obtained for the CHAPS-solubilized exchanger. A molecular mass of 236 and 205 kDa was calculated for the exchanger-detergent co mplex and the detergent-free protein, respectively. Neuraminidase trea tment further reduced the molecular mass of the exchanger indicating t hat glycosylation contributes significantly to the mass of the exchang er. Cross-Links of the exchanger were not detected if cross-linking wa s attempted after solubilization in 10 mM CHAPS. However, after recons titution of the purified exchanger into Soybean phosphatidylcholine ve sicles, chemical cross-linking yielded again dimers. On the basis of t hese crosslinking and hydrodynamic studies, we conclude that the excha nger exists as a homodimer in the rod outer segment plasma membrane bu t dissociates into a monomer when solubilized in detergent.