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

Figure 7. Kinetic model of Gβγ specificity.

(A) Reaction scheme used to model GPCR activation of GIRK. kxy are the rate constants of the reactions between two G protein states. Rate, equilibrium and cooperativity constants are summarized in Table 2. (B) ACh-stimulated GIRK currents from two different SAN cells are shown in grey. Calculated GIRK-βγ4 concentration as a function of time for two different k12 magnitudes are shown in black solid and dashed curves. (C) Calculated steady state GIRK-βγ4 concentration as a function of k12 magnitude. (D) Steady state two-dimensional Gβγ concentration profile (molecules µm−2; one molecule µm−2 = 0.2 µM in a layer 80 Å thick below the membrane surface) surrounding a single GPCR generating 1 Gβγ sec−1 with mean Gβγ lifetime 1 s and diffusion coefficient 0.2 µm2 sec−1. (E) Steady state two-dimensional concentration profile of Gβγ in and surrounding a hotspot of radius 0.3 µm with a density of 50 GPCR µm−2. Gβγ lifetime and diffusion coefficient are the same as in (D). (F) Two dimensional cross sections of concentration profiles in (D) and (E). (G) Steady state Gβγ concentration at the center of hotspot as a function of hotspot radius. See also Figure 7—figure supplement 1, and Table 2.

Figure 7.

Figure 7—figure supplement 1. Simulation of GPCR-activation of GIRK.

Figure 7—figure supplement 1.

(A) Calculated receptor and G protein concentrations as a function of time (k12 = 1 µM−1 sec−1). (B) Muscarinic partial agonists activate GIRK channels to a limited extent. Whole-cell voltage-clamp recordings were performed on stable HEK-293T cells expressing M2Rs and GIRK channels. The membrane potential was held at −80 mV. 10 µM ACh, 100 µM Oxotremorine (Oxo), or 100 µM Pilocarpine (Pilo) was applied as indicated. (C) Partial agonist-activated GIRK currents were normalized to ACh-activated GIRK currents (N = 5–6, ±SEM). (D) Representative changes in BRET signal upon stimulation of M2Rs with different agonists. HEK-293T cells were transfected with M2Rs, Gβγ-Venus, GIRK-NLuc, and increasing amounts of Gαi1. 5 µM acetylcholine (ACh), 50 µM oxotremorine (Oxo), or 50 µM pilocarpine (Pilo) was applied at t = 5 s.