XB-ART-43046PLoS One March 2, 2011; 6 (3): e18226.
β2-Adrenergic ion-channel coupled receptors as conformational motion detectors.
Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K(+)-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K(+) channel Kir6.2 and muscarinic M(2) or dopaminergic D(2) receptors. Here, we extend the concept to the distinct, longer β(2)-adrenergic receptor which, unlike M(2) and D(2) receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of β(2) constructs. We then demonstrate how engineering of both receptor and channel can produce β(2)-Kir6.2 ICCRs. Specifically, removal of 62-72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the β(2) ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the β(2) receptor.
PubMed ID: 21464970
PMC ID: PMC3064670
Article link: PLoS One
Genes referenced: abcc8 kcnj11 kcnj3
Article Images: [+] show captions
|Figure 1. Design strategy of β2-based Ion Channel-Coupled Receptors.ICCRs were formed by covalent linkage of GPCRs C-termini to Kir6.2 channel N-terminus. Helix H8 and β-bridge β1 are predicted from the β2AR (PDB code: 2RH1) and chimeric Kir3.1 (PDB code: 2QKS) structures, respectively. M2-K0–25 and D2-K0–25 are the ICCRs previously shown to be functional with 25 residues deleted from the Kir6.2 N-terminus. We used the same Kir6.2 deletion to build β2 ICCRs, with additional deletions in the receptor C-terminus. β2-K0–25 ICCR contains the full-length receptor, β2-K-62-25 and β2-K-72-25 are based on the β2AR deleted of 62 and 72 residues in its C-terminal domain to match the lengths of M2 and D2, respectively.|
|Figure 3. Receptor-channel coupling in β2 ICCRs: response to the agonist isoproterenol.(A) Representative TEVC recordings from Xenopus oocytes expressing each β2 ICCR and TMD0. Membrane potential was −50 mV. Dashed lines indicate the baseline of Ba2+-sensitive currents. (B) Concentration-effect curves for isoproterenol measured in oocytes co-expressing the indicated proteins. Kir6.2ΔC36 is deleted of its last 36 residues to allow surface expression of the channel alone. Values are average of 5–14 measurements. Smooth lines correspond to Hill equations fits with EC50 in parentheses and h = 1.07 for β2-K-62-25 and 1 for β2-K-72-25.|
|Figure 4. Effect of a β-adrenergic antagonist on β2 ICCRs.(A) TEVC recordings showing antagonist effect of 5 µM alprenolol during addition of 0.5 µM isoproterenol on β2-K0–25, β2-K-62-25 and β2-K-72-25. (B) Change in whole-cell currents evoked by isoproterenol before and after addition of 5 µM alprenolol. **P<0.00075 indicates a significant inhibition induced by alprenolol.|
|Figure 5. Effect of a β2AR partial agonist on β2-K-62-25.Concentration-effect curves for salbutamol measured in oocytes co-expressing the indicated proteins. Values are average of 3-7 measurements. The smooth line is a Hill equation fit to the β2-K-62-25+TMD0 data with EC50 = 452 nM and h = 0.6. Data obtained with the unfused Kir6.2 as a control could not be fitted.|
|Figure 6. The stabilizing mutation E122 W weakens agonist-induced channel responses.(A) Location of Glu122 (in red) in the β2-adrenergic receptor structure. (B) Concentration-effect curves of isoproterenol on β2-K-62-25 and β2-K-72-25, unmodified (WT) and harboring mutation E122 W (all co-expressed with TMD0). Values are average of 5–14 measurements. Hill equation fits, represented as smooth lines, yielded EC50 of 149 nM, 247 nM, and 288 nM for β2-K-62-25, β2(E122 W)-K-62-25, and β2-K-72-25, respectively. h was 1.07, 1, and 1.18.|