Gating of heteromeric retinal rod channels by cyclic AMP: role of the C-terminal and pore domains.
Cyclic nucleotide-gated channels are tetramers composed of homologous alpha and beta subunits. C-terminal truncation mutants of the alpha and beta subunits of the retinal rod channel were expressed in Xenopus oocytes, and analyzed for cGMP- and cAMP-induced currents (single-channel records and macroscopic currents). When the alpha subunit truncated downstream of the cGMP-binding site (alpha D608stop) is co-injected with truncated beta subunits, the heteromeric channels present a drastic increase of cAMP sensitivity. A partial effect is observed with heteromeric alpha R656stop-containing channels, while alpha K665stop-containing channels behave like alpha wt/beta wt. The three truncated alpha subunits have wild-type activity when expressed alone. Heteromeric channels composed of alpha wt or truncated alpha subunits and chimeric beta subunits containing the pore domain of the alpha subunit have the same cAMP sensitivity as alpha-only channels. The results disclose the key role of two domains distinct from the nucleotide binding site in the gating of heteromeric channels by cAMP: the pore of the beta subunit, which has an activating effect, and a conserved domain situated downstream of the cGMP-binding site in the alpha subunit (I609-K665), which inhibits this effect.
Bennett
Nelly
N
Département de Biologie Moléculaire et Structurale, Laboratoire BMC, UMR CNRS 5090, CEA-Grenoble, Grenoble, France. nbennett@cea.fr
Ildefonse
Michèle
M
Pagès
Frédérique
F
Ragno
Michel
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014981:Xenopus / Q000378:metabolism
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D006152:Cyclic GMP