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. 2018 Dec 10;7:e42908. doi: 10.7554/eLife.42908

Figure 1. Gβγ specificity between GPCRs and GIRK channels.

(A) A schematic representation of GPCR signal transduction and GIRK channel activation. Agonist binding promotes the formation of a GPCR-Gα(GDP)βγ complex. The activated GPCR then triggers the exchange of GDP to GTP on the Gα subunit. Gα(GTP) and Gβγ subunits subsequently dissociate from the GPCR. Dissociated Gβγ directly binds to and activates GIRK channels. Dissociated Gα(GTP) hydrolyzes GTP to GDP, which then reassociates with Gβγ to form Gα(GDP)βγ. (B) A representative current-clamp recording of spontaneous action potentials from an acutely isolated murine sinoatrial node (SAN) cell. 1 µM isoproterenol (Iso) or acetylcholine (ACh) was applied as indicated. (C) A representative voltage-clamp recording from the same SAN cell in (B). The membrane potential was held at −80 mV, and 1 µM Iso or ACh was applied as indicated. (D) Representative voltage-clamp recordings of HEK-293T cells transiently co-transfected with GIRK channels, and either M2Rs, β2ARs or β1ARs. The membrane potential was held at −80 mV. 10 µM ACh or Iso was applied as indicated. (E) Validation of the function of βARs. HEK-293T cells expressing βARs or untransfected HEK-293T cells (Ctrl) were treated with 10 µM propranolol (Pro) or isoprennaline (Iso), and intracellular cAMP levels were quantified (N = 3, ±SD). See also Figure 1—figure supplement 1.

Figure 1.

Figure 1—figure supplement 1. Gβγ specificity in heterologously expressed GIRK channels.

Figure 1—figure supplement 1.

(A) Endogenous M2Rs do not activate GIRK. HEK-293T cells were transiently transfected with GIRK channels. The membrane potential was held at −80 mV. 10 µM ACh was applied as indicated. (B) (C) Confocal images of HEK-293T cells expressing M2Rs or β2ARs. HEK2-93T cells were transiently transfected with (B) SNAP-M2Rs or (C) SNAP-β2ARs. Receptors were stained using Alexa Fluor 488 conjugate to a SNAP-ligand. Three representative images are shown for each receptor. (D) Representative voltage-clamp recordings of CHO cells transiently co-transfected with GIRK channels, and either M2Rs, β2ARs or β1ARs. The membrane potential was held at −80 mV. 10 µM ACh or Iso was applied as indicated. The function of βARs was validated by quantifying intracellular cAMP (N = 3, ±SD). (E) Representative voltage-clamp recordings of Sf9 cells co-infected with GIRK channels, and either M2Rs, β2ARs or β1ARs. The membrane potential was held at −80 mV. 10 µM ACh or Iso was applied as indicated. The function of βARs was validated by quantifying intracellular cAMP (N = 3, ±SD).