Identification, purification, and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors.
The cyclic GMP-dependent cation channel from bovine rod outer segments has been purified to greater than 90% homogeneity by a rapid two-step chromatographic procedure. The purified channel has an apparent molecular mass of 63 kDa as determined by NaDodSO4/gel electrophoresis. When incorporated into the membrane of liposomes, the purified protein mediates the cyclic GMP-dependent efflux of entrapped Ca2+. The reconstituted channel protein exhibits properties similar to the cyclic GMP-dependent channel observed in excised patches of the plasma membrane and in disk membranes. Cyclic GMP activated the channel cooperatively (Hill coefficient n = 3.1) with an apparent Michaelis constant of approximately 11 microM. After reconstitution of the purified protein into a planar lipid bilayer, we recorded cyclic GMP-stimulated single-channel activity. The single-channel conductance at physiological salt concentrations and in the absence of divalent cations was 26 pS. The drug l-cis-diltiazem, shown to block the cyclic GMP-dependent channel in excised patches of the plasma membrane and in isolated disks of rod outer segments, was ineffective against the purified channel.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006152:Cyclic GMP / Q000494:pharmacology*; D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D007700:Kinetics; D010786:Photoreceptor Cells / Q000502:physiology*; D012374:Rod Cell Outer Segment / Q000502:physiology*
D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins; D007473:Ion Channels; D006152:Cyclic GMP; D002118:Calcium