title: Engineering of an artificial light-modulated potassium channel.
abstract: Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1(F137S) (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.
authors.affiliation: CNRS, Institut de Biologie Structurale, Grenoble, France.
authors.forename: Lydia N
authors.initials: LN
authors.lastname: Caro
authors.forename: Christophe J
authors.initials: CJ
authors.lastname: Moreau
authors.forename: Argel
authors.initials: A
authors.lastname: Estrada-Mondragón
authors.forename: Oliver P
authors.initials: OP
authors.lastname: Ernst
authors.forename: Michel
authors.initials: M
authors.lastname: Vivaudou
mesh_terms: D000818:Animals; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics* / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics; D008027:Light; D009865:Oocytes / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics; D014982:Xenopus laevis / Q000235:genetics; D000099161:Kcnj11 Channel
chemical_list: D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin; D000099161:Kcnj11 Channel