January 1, 2012;
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
, 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
. 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.
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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 3. Design strategy of rhodopsin-based ICCRs and functional coupling.(A) Sequence alignment of the fusion area between GPCRs and Kir6.2ΔN25. Alignment of GPCR C-terminal sequences were based on the presence of the H8 Helix. (B) Percent change in current for each construct in response to 10 µM all-trans-retinal or light (after 11-cis retinal incubation). Oocytes were co-injected with the specified Ops-Kir6.2 and TMD0, a SUR transmembrane domain. Numbers below bars indicate the number of oocytes tested. The changes induced by all-trans-retinal and light were statistically significant for Ops-K−16–25 (Student t-test; P<0.04 & P<0.0001, respectively), but not for Ops-K0–25 (P>0.4 & P>0.05, respectively). (C) Representative TEVC recordings for each construct in the case of photoactivation. Yellow bar represents oocyte illumination. Blue bar corresponds to Ba2+ application at 3 mM. Dashed line indicates the Ba2+-sensitive current baseline. (D) Opsin ability to activate G proteins within the fusion Ops-Kir6.2. Both constructs were co-expressed with Kir3.1* and change in current was measured in response to 10 µM all-trans-retinal. All measurements were done at −50 mV. Numbers above bars indicate the number of oocytes tested.
Figure 4. ICCRs report conformational changes of receptors deficient in G protein coupling.(A) Representative TEVC traces. Yellow and blue bars correspond to oocyte illumination and Ba2+ (3 mM) application, respectively. PTX (Pertussis Toxin) ADP-ribosylates Gαi/o proteins, thus preventing Gα-Gβγ dissociation. OpsΔicl3 is a rhodopsin mutant with a deletion in the third intracellular loop (Δ244–249) known for its inability to activate G proteins. Dashed line indicates the Ba2+-sensitive current baseline. Measurements were done at −50 mV. (B) Percent change in current induced by light after 11-cis retinal incubation in the dark. Numbers below bars indicate the number of oocytes tested. The responses of OpsΔicl3-K-16–25 and Ops-K-16–25+ PTX were not statistically different (Student t-test; P>0.5) while those of OpsΔicl3-K-16–25 and Ops-K-16–25 were different (P<0.002). (C) Oocytes were co-injected with Ops-K-16–25+ Kir3.1* or OpsΔicl3-K−16–25+ Kir3.1*. Ability of opsin to activate G proteins was determined by the addition of all-trans-retinal at 10 µM and measurement of Kir3.1* activation as a percent change in current. Numbers above bars indicate the number of oocytes tested.
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).