XB-ART-45896PLoS One January 1, 2012; 7 (8): e43766.
Engineering of an artificial light-modulated potassium channel.
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.
PubMed ID: 22928030
PMC ID: PMC3425490
Article link: PLoS One
Genes referenced: abcc8 gprc6a kcnj11 kcnj3 lrp5 rho
Article Images: [+] show captions
|Figure 2. PTX-sensitive activation of Kir3.1* by opsin and all-trans-retinal, or light-activated rhodopsin. (A) Xenopus oocytes were injected with opsin and Kir3.1* mRNAs. Current amplitude was recorded at −50 mV. Black bars represent the average current measured prior to all-trans-retinal application or in the dark (after 11-cis retinal incubation) in the case of light activation. White bars represent the average current induced by 5 µM all-trans-retinal or light stimulation, respectively. Numbers above bars denote the number of oocytes tested. (B) Percent change in current induced by application of either 5 µM all-trans-retinal or light (after 11-cis retinal incubation) in control (black bars) and in the presence of co-expressed catalytic subunit S1 of pertussis toxin (PTX-S1) (white bars). Changes in current were computed for each oocyte and then averaged (The resulting average changes are different from the changes in average current represented in panel A). (C) Concentration-dependent response to all-trans-retinal. Average data computed as in panel B. Line corresponds to Hill equation fit with h = 4 and EC50 = 2.5 µM. Each point represents the average of 7 to 40 measurements.|
|Figure 5. Possible mechanism linking channel gating to ligand binding.This sketch is a view of the opsin ICCR from the cytoplasmic side. The ICCR contains four Kir6.2 subunits (labeled Kir) that associate as a tetramer and four opsin receptors at the periphery. One hypothesis for the observed coupling between light activation of rhodopsin and channel gating could be that closing of the channel results from light-induced motion of receptor-channel linker (blue arrows) and rearrangement of opsin helices TM-V and TM-VI (brown arrows) (see text for details).|