Cyclic GMP contact points within the 63-kDa subunit and a 240-kDa associated protein of retinal rod cGMP-activated channels.
Ion channels from retinal rods and a variety of other cells are directly gated by cyclic nucleotides. The rod channel is known to contain a 63-kDa subunit, and there is molecular genetic evidence for the existence, in human retina, of a second subunit with a deduced molecular mass of about 100 kDa. When purified from bovine rods, the channel consists of the 63-kDa subunit and a 240-kDa associated protein that has been shown recently to contain a version of the cloned second subunit as part of a larger complex. We had previously shown that a photoaffinity analog of cGMP, 8-(p-azidophenacylthio)-[32P]cGMP, specifically labels both the 63- and 240-kDa proteins. Here the analog was used to identify cGMP-binding regions and amino acid contact points within these proteins. The specific labeling of the 63-kDa subunit was localized to a 66 amino acid fragment (Tyr-515-Met-580) that is contained entirely within a 110 amino acid region proposed to be the cGMP-binding site on the basis of homology with other cyclic nucleotide-binding proteins. Within this fragment, amino acid residues Val-524, Val-525, and Ala-526 were found to contain label. These residues are part of a larger hydrophobic cluster that appears to line the binding pocket. The results also indicate that the 240-kDa protein contains a similar cGMP-binding site. Sequencing of a specifically labeled 8-kDa fragment through 16 amino acid residues indicated that the fragment was derived from the portion of the 240-kDa complex that contains the second subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Brown
R L
RL
Department of Physiology, University of Colorado School of Medicine, Denver 80262, USA.
Gramling
R
R
Bert
R J
RJ
Karpen
J W
JW
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D003488:Cyanogen Bromide; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D054815:Cyclic Nucleotide-Gated Cation Channels; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000345:Affinity Labels; D001386:Azides; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; C081534:8-(4-azidophenacyl)thio-cyclic GMP; D006152:Cyclic GMP; D003488:Cyanogen Bromide