Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2011 Mar 09;63:e18226. doi: 10.1371/journal.pone.0018226.
Show Gene links
Show Anatomy links
β2-Adrenergic ion-channel coupled receptors as conformational motion detectors.
Caro LN
,
Moreau CJ
,
Revilloud J
,
Vivaudou M
.
???displayArticle.abstract???
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.
???displayArticle.pubmedLink???
21464970
???displayArticle.pmcLink???PMC3064670 ???displayArticle.link???PLoS One
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-K0â25 and D2-K0â25 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-K0â25 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 2. TMD0 of SUR boosts expression of β2-based ICCRs.Basal currents are the whole-cell currents measured in the first minute of TEVC recording from unstimulated Xenopus oocytes. E122 W is a mutation of residue 122 of β2AR from Glu to Trp reported to increase β2 surface expression.TMD0 is the first transmembrane domain of the sulfonylurea receptor SUR1, a physiological partner of Kir6.2. *P<0.05 and **P<0.00001 represent significant differences from the basal current measured in non-injected oocytes.
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 â50 mV. Dashed lines indicate the baseline of Ba2+-sensitive currents. (B) Concentration-effect curves for isoproterenol measured in oocytes co-expressing the indicated proteins. Kir6.2ÎC36 is deleted of its last 36 residues to allow surface expression of the channel alone. Values are average of 5â14 measurements. Smooth lines correspond to Hill equations fits with EC50 in parentheses and hâ=â1.07 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-K0â25, 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 â=â 452 nM and hâ=â0.6. 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 5â14 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.
Andersson,
Membrane assembly of the cannabinoid receptor 1: impact of a long N-terminal tail.
2003, Pubmed
Andersson,
Membrane assembly of the cannabinoid receptor 1: impact of a long N-terminal tail.
2003,
Pubmed
Antcliff,
Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit.
2005,
Pubmed
Baker,
The selectivity of beta-adrenoceptor agonists at human beta1-, beta2- and beta3-adrenoceptors.
2010,
Pubmed
Chan,
N-terminal transmembrane domain of the SUR controls trafficking and gating of Kir6 channel subunits.
2003,
Pubmed
,
Xenbase
Chelikani,
Role of group-conserved residues in the helical core of beta2-adrenergic receptor.
2007,
Pubmed
Cherezov,
High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.
2007,
Pubmed
Corringer,
Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues.
2010,
Pubmed
Davare,
A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2.
2001,
Pubmed
Doupnik,
GPCR-Kir channel signaling complexes: defining rules of engagement.
2008,
Pubmed
Dupuis,
Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the K(ATP) channel subunits SUR2A and Kir6.2.
2008,
Pubmed
,
Xenbase
Frishman,
beta-Adrenergic blockers: a 50-year historical perspective.
2008,
Pubmed
Granier,
Structure and conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from fluorescence resonance energy transfer studies.
2007,
Pubmed
Hague,
The N terminus of the human alpha1D-adrenergic receptor prevents cell surface expression.
2004,
Pubmed
Hosy,
Remodelling of the SUR-Kir6.2 interface of the KATP channel upon ATP binding revealed by the conformational blocker rhodamine 123.
2007,
Pubmed
,
Xenbase
Hosy,
Impact of disease-causing SUR1 mutations on the KATP channel subunit interface probed with a rhodamine protection assay.
2010,
Pubmed
,
Xenbase
Inagaki,
Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor.
1995,
Pubmed
Kisilevsky,
D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry.
2008,
Pubmed
Kobilka,
Conformational complexity of G-protein-coupled receptors.
2007,
Pubmed
Lavine,
G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase.
2002,
Pubmed
,
Xenbase
Lee,
Dual regulation of NMDA receptor functions by direct protein-protein interactions with the dopamine D1 receptor.
2002,
Pubmed
Liu,
Direct protein-protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors.
2000,
Pubmed
Makarova,
Generation of deletion and point mutations with one primer in a single cloning step.
2000,
Pubmed
Mikhailov,
3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.
2005,
Pubmed
Minneman,
Beta-adrenergic receptor subtypes: properties, distribution, and regulation.
1981,
Pubmed
Moreau,
SUR, ABC proteins targeted by KATP channel openers.
2005,
Pubmed
Moreau,
The molecular basis of the specificity of action of K(ATP) channel openers.
2000,
Pubmed
Moreau,
Coupling ion channels to receptors for biomolecule sensing.
2008,
Pubmed
Nichols,
KATP channels as molecular sensors of cellular metabolism.
2006,
Pubmed
Rosenbaum,
The structure and function of G-protein-coupled receptors.
2009,
Pubmed
Roth,
Stabilization of the human beta2-adrenergic receptor TM4-TM3-TM5 helix interface by mutagenesis of Glu122(3.41), a critical residue in GPCR structure.
2008,
Pubmed
Taira,
Measurement of inverse agonism in β-adrenoceptors.
2010,
Pubmed
Thompson,
The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
1997,
Pubmed
Tucker,
Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor.
1997,
Pubmed
,
Xenbase
Wacker,
Conserved binding mode of human beta2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography.
2010,
Pubmed
Yao,
Coupling ligand structure to specific conformational switches in the beta2-adrenoceptor.
2006,
Pubmed
Zerangue,
A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels.
1999,
Pubmed
,
Xenbase
