Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

Abstract

Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors--a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption.

Fig 1. Principle of time-resolved Scintillation Proximity Assay (SPA) to quantify stability of high-affinity radio-ligand binding to GPCRs.

A) Receptors are immobilized on the SPA beads. When radioligands bind to immobilized receptors, energy from the radioisotope decay is efficiently transferred to the scintillator contained in the beads, thus generating a detectable light signal. If the buffer, including detergent, causes the loss of receptor affinity to radiolabeled ligand, the signal will start to decrease irreversibly. The decay of high-affinity ligand binding to receptors can be measured when the same assay is counted repeatedly over time. B) Comparison of desalting gravity flow separation columns (blue circles) and SPA (black circles) to measure decrease in binding of [³H]-DPN to KOR over time at room temperature. KOR was solubilized in 1%/0.2% DDM/CHS (w/v) and diluted in the assay to 0.1%/0.02% DDM/CHS (w/v). Specific binding was normalized to initial binding. As control of assay stability, the signal of bead-immobilized [³H]-biotin was measured in parallel (red circles). Error bars are standard error of the mean from four (gravity flow columns) or three (SPA) independent experiments each done in triplicates. To quantify the stability of ligand binding, data was fitted to an exponential decay function (solid and dashed lines).

Fig 2. Reconstitution of KOR in Nanodiscs.

A) SEC chromatogram (Superdex200) representing UV_{280 nm} traces from resolution of empty Nanodiscs (black dotted line) and of KOR/Nanodisc complexes (black line). KOR/Nanodisc sample eluted as two peaks. The second peak on the chromatogram corresponded to Nanodiscs containing active KOR, confirmed by specific binding of [³H]-DPN (10 nM) to KOR in eluted fractions (red circles). The column was calibrated with standard proteins indicated by arrows. The Stokes diameters of empty Nanodiscs and KOR/Nanodisc complexes were calculated to be 11.9 nm and 14.1 nm respectively. B) SDS-PAGE detection of KOR reconstituted in Nanodiscs. Left: Coomassie brilliant blue staining, KOR (66 kDa) was visible around ≈ 75 kDa while the MSP1E3D1 protein (32.6 kDa) was visible at ≈ 30 kDa. Right: The presence of KOR was confirmed by western blot detection with streptavidin alkaline phosphatase binding to the C-terminal biotin-tag of KOR. Original uncropped and unadjusted gel and blot can be seen in S2 Fig.

Fig 3. Time-resolved stability of KOR in detergent or when reconstituted in Nanodiscs.

Stability of high-affinity ligand binding (10 nM [³H]-DPN ≈ 10 x K_D) to KOR in 0.1%/0.02% DDM/CHS (w/v) micelles (open diamonds) or to KOR reconstituted in Nanodiscs (black diamonds) at 23°C. Specific [³H]-DPN binding was normalized to initial ligand binding at time zero where incubation at RT was initiated. Data are average of at least three experiments each performed in triplicates. Error bars represents standard error of the mean. Data were fitted to a double exponential decay curve to calculate the half-life of high-affinity ligand binding to KOR (see Table 1).

Fig 4. Pharmacology of KOR in isolated P. pastoris membrane and when reconstituted in Nanodiscs.

A) Saturation binding assay of [³H]-DPN binding to KOR in membrane (black triangles) or KOR reconstituted in Nanodiscs (open triangles). From the specific binding the affinity constant, K_D, of [³H]-DPN binding to KOR was calculated by fitting to a one-site binding model (see Table 2) B) Competition binding experiments with KOR antagonist naloxone and C) KOR agonist dynorphin A (1–17). Increasing concentrations of ligand competed the binding of 0.8 nM [³H]-DPN binding to KOR in P. pastoris cell membrane (black marks) or reconstituted in Nanodiscs (open marks). The calculated binding constants (K_i) are shown in Table 2. Data shown are representative experiments of specific binding normalized to initial binding of [³H]-DPN binding to KOR. Data points are shown with standard deviation of triplicate measurements in one assay. All binding experiments were repeated at least three times.

Fig 5. Isolated KOR reconstituted in Nanodiscs activated G_i protein.

BODIPY FL GTPγS was used to confirm Gα_i protein activation by KOR after isolation and reconstitution into Nanodiscs. The intrinsic activity of G proteins was measured in the presence of empty Nanodiscs (light grey, Empty ND). Compared to KOR/Nanodisc sample without agonist stimulation (KOR/ND, dark grey), the fluorescence from BODIPY FL GTPγS binding to Gα_i increased by 56% when the KOR/Nanodisc sample was stimulated with 1 μM dynorphin A (1–17) (KOR/ND + Dyn A, dark grey). Error bars represent standard error of the mean between two independent experiments.