Plots of the average normalized ratios of membrane-to-intracellular (M/I) Gβ₁γ_2/11-V fluorescence intensity from cells transfected with C-TM-Gα_s and either 0.1 μg (n = 8) or 0.8 μg (n = 7) of A2AR plasmid DNA; thinner lines represent the average ± s.e.m. The agonist sensitivity was enhanced by transfecting more plasmid DNA, suggesting more A2ARs were expressed. The maximal Gβ₁γ_2/11-V translocation was the same in both cases, suggesting that A2AR expression was not a limiting factor.

A, confocal images of live cells expressing C-TM-Gα_oA and Gβ₁γ_2/11-V stained using Alexa 647-conjugated anti-GFP. Alexa 647 signal (633 nm excitation) is present only on the cell surface, whereas C-TM-Gα_oA signal (458 nm excitation) is present at the plasma membrane and in intracellular compartments. B, scatter plots of Alexa 647 and Gβ₁γ_2/11-V fluorescence intensity from samples of 10⁴ cells. Untransfected cells showed no fluorescence in either channel, unstained cells showed Gβ₁γ_2/11-V fluorescence only, and cells transfected with C-TM-Gα_oA and C-TM-Gα_s showed comparable levels of Alexa 647 fluorescence. The quadrants indicate threshold (above background) fluorescence levels. C, an immunoblot probed with a polyclonal anti-GFP antibody (top) and reprobed with an anti-β-actin antibody (bottom). Protein samples were prepared from cells transfected with C-TM-Gα_oA or C-TM-Gα_s (together with unlabelled Gβ and Gγ subunits) and EGFP alone as well as untransfected controls (UT). Both C-TM-Gα subunits migrate as a single predominant species; the larger size of C-TM-Gα_s is predicted by the greater length (40 amino acids) of this Gα isoform.

A, exemplary traces from cells expressing GIRK1/2 heteromers, Gβ₁γ_2/11-V, and either C-TM-Gα_oA and A1Rs or C-TM-Gα_s and A2ARs. High (30 mm) K⁺ solution reveals basal inward current in both cases, and additional current in the presence of adenosine (10 μm); Ba²⁺ refers to 0.2 mm barium. The slow reversal of adenosine-activated current in the cell expressing C-TM-Gα_s was characteristic of these cells. B, current responses to voltage ramps from –90 mV to 0 mV recorded from the same cells shown in panel A. C, summary of basal and evoked GIRK currents in cells expressing the indicated combinations of adenosine receptors, C-TM-Gα subunits and Gβ₁γ_2/11-V dimers or adenosine receptors only. C-TM-Gα_oA subunits harboured a mutation (C351G) rendering them insensitive to PTX, and all cells were treated overnight with PTX. D, a confocal image showing plasma membrane fluorescence in cells expressing metabolically biotinylated GIRK1/2 channels stained with Alexa 647-conjugated streptavidin. E, scatter plots of Alexa 647 and Gβ₁γ_2/11-V fluorescence intensity from samples of 10⁴ cells expressing metabolically biotinylated GIRK1/2 channels and either C-TM-Gα_oA or C-TM-Gα_s. Live cells were stained with Alexa 647-conjugated streptavidin (SA). Comparable levels of Alexa 647 fluorescence are evident above background levels (indicated by the quadrants; representative data from 3 similar experiments).

A, images of venus fluorescence in a cell expressing C-TM-Gα_oA, Gβ₁γ_2/11-V and A1Rs before (control) and after (adenosine) application of 10 μm adenosine; the lookup table is inverted for clarity. Gβ₁γ_2/11-V fluorescence translocates from the plasma membrane to the cell interior. This is shown more clearly in the difference image (right), where regions that lost fluorescence after adenosine are pseudocoloured blue, and regions that gained fluorescence are pseudocoloured red. Regions of interest (ROI) typical of membrane and intracellular regions are show in blue and red, respectively. B, plots of the average normalized ratios of membrane-to-intracellular (M/I) Gβ₁γ_2/11-V fluorescence intensity from cells expressing C-TM-Gα_oA and A1Rs (n = 50) or C-TM-Gα_s and A2ARs (n = 52); thinner lines represent the average ± s.e.m. C, CFP (left) and venus (right) fluorescence intensity in a narrow region of interest including the plasma membrane in cells expressing C-TM-Gα_oA (n = 15) or C-TM-Gα_s (n = 18) together with Gβ₁γ_2/11-V and cognate adenosine receptors.

A, images of HEK 293 cells bathed in 0.1 mm BODIPY FL GTPγS. Intact cells exclude green fluorescence, whereas cells permeabilized with α-toxin accumulate the labelled nucleotide. B, recovery of Gβ₁γ₂-V fluorescence into photobleached regions of the plasma membrane in cells expressing immobile C-TM-Gα_oA or C-TM-Gα_s. Cells were permeabilized in the presence of 0.1 mm GTPγS and 10 μm adenosine or 1 mm GDPβS. Traces are the average of all experiments summarized in panels C and D. C, summary of fluorescence recovery 180 s after photobleaching in cells expressing C-TM-Gα_oA (left) together with Gβ₁γ₂-V (right). Both were mobile in cells that were not antibody crosslinked (no Ab; n = 6), as indicated by complete fluorescence recovery. Both became less mobile in crosslinked cells loaded with GDPβS (n = 11). C-TM-Gα_oA mobility was not significantly increased in crosslinked cells loaded with GTPγS, whereas Gβ₁γ₂-V mobility was significantly increased in these cells (n = 14), indicating a GTPγS-induced decrease in the affinity of C-TM-Gα_oA for Gβ₁γ₂-V. D, summary of fluorescence recovery in cells expressing C-TM-Gα_s (left) and Gβ₁γ₂-V (right). Both were mobile without antibody crosslinking (no Ab; n = 6), and both became less mobile in crosslinked cells loaded with GDPβS (n = 10). C-TM-Gα_s mobility was not increased in cells loaded with GTPγS, whereas Gβ₁γ₂-V mobility was significantly increased in these cells (n = 14), indicating a GTPγS-induced decrease in the affinity of C-TM-Gα_s for Gβ₁γ₂-V. Comparison of cells expressing C-TM-Gα_oA and C-TM-Gα_s indicated that Gβ₁γ₂-V mobility was greater both in cells loaded with GDPβS (P < 0.0005) and in cells loaded with GTPγS (P < 0.00001), indicating that in both cases the affinity of immobile C-TM-Gα_oA for Gβ₁γ₂-V was lower.