Cyclic GMP contact points within the 63-kDa subunit and a 240-kDa associated protein of retinal rod cGMP-activated channels

R L Brown; R Gramling; R J Bert; J W Karpen

doi:10.1021/bi00026a018

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Cyclic GMP contact points within the 63-kDa subunit and a 240-kDa associated protein of retinal rod cGMP-activated channels

Journal article

Peer reviewed

Cyclic GMP contact points within the 63-kDa subunit and a 240-kDa associated protein of retinal rod cGMP-activated channels

R L Brown, R Gramling, R J Bert and J W Karpen

Biochemistry (Easton), Vol.34(26), pp.8365-8370

07/04/1995

DOI: https://doi.org/10.1021/bi00026a018

Handle:

https://hdl.handle.net/2376/106302

PMID: 7541241

Abstract

Cyclic GMP - pharmacology

Molecular Weight

Humans

Molecular Sequence Data

Affinity Labels - metabolism

Retinal Rod Photoreceptor Cells - metabolism

Ion Channels - isolation & purification

Cyclic GMP - analogs & derivatives

Cattle

Azides - metabolism

Cyclic GMP - chemical synthesis

Azides - chemical synthesis

Ion Channels - chemistry

Amino Acid Sequence

Peptide Fragments - isolation & purification

Electrophoresis, Polyacrylamide Gel

Phosphorus Radioisotopes

Blotting, Western

Macromolecular Substances

Sequence Homology, Amino Acid

Peptide Fragments - chemistry

Animals

Cyclic GMP - metabolism

Ion Channels - metabolism

Cyanogen Bromide

Cyclic Nucleotide-Gated Cation 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.

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Title: Cyclic GMP contact points within the 63-kDa subunit and a 240-kDa associated protein of retinal rod cGMP-activated channels
Creators: R L Brown - Department of Physiology, University of Colorado School of Medicine, Denver 80262, USA
R Gramling
R J Bert
J W Karpen
Publication Details: Biochemistry (Easton), Vol.34(26), pp.8365-8370
Academic Unit: Integrative Physiology and Neuroscience, Department of
Publisher: United States
Grant note: EY06425 / NEI NIH HHS EY09275 / NEI NIH HHS T32NS07083 / NINDS NIH HHS
Identifiers: 99900547127001842
Language: English
Resource Type: Journal article

Cyclic GMP contact points within the 63-kDa subunit and a 240-kDa associated protein of retinal rod cGMP-activated channels

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