The Na/Ca-K exchanger of rod photoreceptor exists as dimer in the plasma membrane

Biochemistry. 1997 Nov 4;36(44):13667-76. doi: 10.1021/bi9710232.

Abstract

The oligomeric state of the Na/Ca-K exchanger in the plasma membrane of bovine photoreceptors was investigated using chemical cross-linking techniques. In the natural membrane, virtually all Na/Ca-K exchanger could be cross-linked mainly to a complex having an apparent molecular mass of 490 kDa by cupric phenanthroline catalyzed disulfide bonding as evidenced by Western blotting. Stable cross-links of the exchanger were also obtained with the thiol-specific reagent N,N'-p-phenylidenedimaleimide. Neuraminidase treatment reduced the apparent molecular mass of the highly glycosylated Na/Ca-K exchanger and of the 490 kDa cross-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-dimensional SDS polyacrylamide electrophoresis identified the cross-linked homodimers of the exchanger. There was no indication of higher oligomers, suggesting that the exchanger exists as a dimer in the plasma membrane. Hydrodynamic properties of the detergent-solubilized exchanger were determined by velocity sedimentation and gel filtration chromatography. The Triton X-100-solubilized exchanger ran as a single species having 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 complex and the detergent-free protein, respectively. Neuraminidase treatment further reduced the molecular mass of the exchanger indicating that glycosylation contributes significantly to the mass of the exchanger. Cross-links of the exchanger were not detected if cross-linking was attempted after solubilization in 10 mM CHAPS. However, after reconstitution of the purified exchanger into soybean phosphatidylcholine vesicles, chemical cross-linking yielded again dimers. On the basis of these cross-linking and hydrodynamic studies, we conclude that the exchanger exists as a homodimer in the rod outer segment plasma membrane but dissociates into a monomer when solubilized in detergent.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carrier Proteins / chemistry*
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism
  • Cattle
  • Cell Membrane / chemistry
  • Centrifugation, Density Gradient
  • Chromatography, Gel
  • Cross-Linking Reagents
  • Dimerization
  • Membrane Proteins / chemistry*
  • Molecular Weight
  • Neuraminidase
  • Octoxynol
  • Rod Cell Outer Segment / chemistry*
  • Sodium-Calcium Exchanger*
  • Solubility
  • Sulfhydryl Compounds

Substances

  • Carrier Proteins
  • Cross-Linking Reagents
  • Membrane Proteins
  • Sodium-Calcium Exchanger
  • Sulfhydryl Compounds
  • potassium-dependent sodium-calcium exchanger
  • Octoxynol
  • Neuraminidase