The cGMP-gated cation channel of bovine rod photoreceptor cells is associated with a 240-kDa protein exhibiting immunochemical cross-reactivity with spectrin.
A 240-kDa protein exhibiting immunochemical cross-reactivity with red blood cell spectrin has been shown to be directly associated with the 63-kDa cGMP-gated channel of bovine rod outer segments. When detergent-solubilized, chromatographically purified channel preparations were treated with Sepharose beads coupled to either an anti-240-kDa monoclonal antibody (PMs 4B2) or an anti-63-kDa channel monoclonal antibody (PMc 1D1), both the 240-kDa protein and the 63-kDa channel protein were concomitantly immunoprecipitated as analyzed by Western blotting of sodium dodecyl sulfate gels. Both of these antibody-Sepharose matrices also removed cGMP-gated channel activity as measured by functional reconstitution. In control studies anti-rhodopsin monoclonal antibody (Rho 1D4)-Sepharose beads removed residual rhodopsin, but not the 63/240-kDa complex or channel activity. Western blotting of purified rod outer segment disk and plasma membrane fractions and immunogold-dextran labeling of lysed rod outer segments indicated that the 240-kDa polypeptide, like the 63-kDa channel, is preferentially localized to the plasma membrane as visualized by electron microscopy. The 240-kDa protein does not appear to be directly involved in the cGMP-gated channel activity, but it may be part of a cytoskeletal system that serves to maintain the organization of the 63-kDa channel complex within the rod outer segment plasma membrane.
Molday
L L
LL
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Cook
N J
NJ
Kaupp
U B
UB
Molday
R S
RS
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D015153:Blotting, Western; D002417:Cattle; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D006152:Cyclic GMP / Q000502:physiology; D007150:Immunohistochemistry; D015640:Ion Channel Gating; D007473:Ion Channels / Q000648:ultrastructure*; D046911:Macromolecular Substances; D011233:Precipitin Tests; D012374:Rod Cell Outer Segment / Q000502:physiology / Q000648:ultrastructure*; D013049:Spectrin / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007473:Ion Channels; D046911:Macromolecular Substances; D013049:Spectrin; D006152:Cyclic GMP