β2-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors

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 M2 or dopaminergic D2 receptors. Here, we extend the concept to the distinct, longer β2-adrenergic receptor which, unlike M2 and D2 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 β2 constructs. We then demonstrate how engineering of both receptor and channel can produce β2-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 β2 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 β2 receptor.

Photolithographic Fabrication of Microapertures with Well-Defined, Three-Dimensional Geometries for Suspended Lipid Membrane Studies

“…9,10 Additionally, artificial lipid membranes are increasingly utilized in a range of biomimetic chemical sensors, 11–13 with much focus on-hemolysin nanopores for the detection and characterization of biopolymers and other biological analytes. 14–21 The recent advent of engineered transmembrane proteins that combine the high sensitivity of ion channel conductance measurements with the specificity of molecular recognition, 22,23 presents a number of opportunities for novel sensor development. However, producing BLMs in a high density device format amenable to long-term monitoring applications, or to being integrated into robust and deployable analytical systems, presents a significant challenge.…”

Functional Assay for T4 Lysozyme-Engineered G Protein-Coupled Receptors with an Ion Channel Reporter

“…In ICCRs, the open probability of Kir6.2 is similar to that of the unfused channel (KΔ) and the channel is partly open at rest in our experimental conditions. This basal activity offers the advantage of sensing either agonist-evoked channel inhibition (shortened M2, rhodopsin and D2 L ICCRs) (Caro et al , 2012; Moreau et al , 2008) or activation (full-length M2, β2 ICCRs) (Caro et al , 2011; Moreau et al , 2008). In the case of the M2(T4L) ICCR, the physiological agonist acetylcholine (ACh) causes an inhibition of Kir6.2 comparable to that observed with the wt M2 ICCR (Fig.…”

“…In order to validate this concept with another GPCR, we used the previously created adrenergic β 2 -K ICCR (Caro et al , 2011) and inserted the T4L domain in the i3 loop. The position of the T4L domain was the same as in the first crystallized β 2 (T4L) receptor (Rosenbaum et al , 2007), but the glycosylation sites were left intact.…”

“…Building on the concept of Ion Channel-Coupled Receptors (ICCRs) (Caro et al , 2011; Caro et al , 2012; Moreau et al , 2008), we developed a functional assay for GPCRs by genetically fusing a potassium channel to the receptor C-terminus. The channel acts as a direct reporter of the agonists- and antagonists-induced conformational changes of the GPCRs.…”

Computational Study on the Different Ligands Induced Conformation Change of β2 Adrenergic Receptor-Gs Protein Complex

“…The analysis of β 2 AR-Gs complex could provide some information about the essential mechanism of structural events linking GPCR-Gs protein complex formation by using peptide amide hydrogen-deuterium exchange mass spectrometry [13]. Engineering and characterization of β 2 AR-based on ion-channel coupled receptors gave new insights into the conformational dynamics of β 2 AR [14]. All these studies also indicated that it was difficult to obtain the crystal structure of the agonist-bound to active conformation of β 2 AR if the G protein did not bind to β 2 AR.…”