Accessibility of cysteines in the native bovine rod cGMP-gated channel.
Cyclic nucleotide-gated channels of photoreceptors and olfactory sensory neurons are tetramers consisting of A and B subunits. Here, the accessibility of the cysteines of the bovine rod cyclic nucleotide-gated channel is examined as a function of ligand binding. N-Ethylmaleimide-modified cysteines of both subunits were identified by mass spectrometry after trypsin digestion. In the absence of ligand, the intracellular carboxy-terminal cysteines of both subunits were accessible to N-ethylmaleimide. Activation of the channel abolished the accessibility of Cys505 of the A subunit and Cys1104 of the B subunit, with both being conserved cysteines of the cyclic nucleotide-binding sites. The cysteine of the pore loop of the B subunit was also found to be modified by this reagent in the absence of ligand. The total number of accessible cysteines of each subunit was determined by mass shifting upon modification with polyethylene glycol maleimide. In the absence of cyclic nucleotides, this hydrophilic reagent only weakly labeled cysteines of the A subunit but readily labeled at least three cysteines of the B subunit. Ligand binding exposed two cysteines of the A subunit and one cysteine of the B subunit to chemical modification. Double-modification experiments suggest that some of these cysteines are in or close to membrane-spanning domains. However, these cysteines could not yet be identified. Together, the cysteine accessibility of the native rod cyclic nucleotide-gated channel varies markedly upon ligand binding, thus indicating major structural rearrangements, which are of functional importance for channel activation.
Bauer
Paul J
PJ
Institute of Biological Information Processing, Research Center, D-52425 Juelich, Germany. p.j.bauer@fz-juelich.de
Krause
Eberhard
E
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D017124:Conserved Sequence; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry*; D054815:Cyclic Nucleotide-Gated Cation Channels; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D005136:Eye Proteins / Q000737:chemistry* / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008301:Maleimides / Q000737:chemistry; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins; D007473:Ion Channels; D008301:Maleimides; D021122:Protein Subunits; C043592:maleimide; C016276:8-bromocyclic GMP; D011092:Polyethylene Glycols; D006152:Cyclic GMP; D003545:Cysteine; D005033:Ethylmaleimide