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A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gβγ.

Yakubovich D, Berlin S, Kahanovitch U, Rubinstein M, Farhy-Tselnicker I, Styr B, Keren-Raifman T, Dessauer CW, Dascal N - PLoS Comput. Biol. (2015)

Bottom Line: Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter.In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases.The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
G protein-gated K+ channels (GIRK; Kir3), activated by Gβγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.

No MeSH data available.


Related in: MedlinePlus

Dose-dependent activation of GIRK1/2 by coexpressed Gβγ: experiment and simulation.GIRK1/2 was expressed at 0.2 ng RNA. All data are mean ± SEM from one experiment. (A) Confocal images of Gβγ in giant excised plasma membranes stained with the anti-Gβ antibody. The intensity of all images was increased equally for a better viewing in this figure, but not in the process of image analysis. (B) Dose-dependence of Gβγ levels and Iβγ in oocytes injected with incrementing amounts of wt Gβγ RNA (0.05–30 ng per oocyte). Gβγ expression in the PM (grey bars) was measured from images shown in A, in 4–8 oocyte membranes, and Iβγ currents (red circles; right Y-axis) were measured in 12–16 oocytes. The dashed line shows the basal level of fluorescence, arising from the endogenous Gβγ. Note that, unlike in Western blots, in immunocytochemistry the antibody poorly recognized the endogenous Gβγ compared to the expressed bovine Gβγ. (C) Comparison of measured Iβγ and Rβγ (red circles) and simulated currents (curves). The relative Gβγ levels (from grey bars in B) have been converted into surface densities assuming that 5 ng Gβγ gives 30 molecules Gβγ/μm2. The blue line presents the simulation using graded contribution model and amounts of Gα and Gβγ (prior to coexpression of Gβγ) calculated using the methods described above: channel density was calculated from Iβγ (13.75 channels/μm2 with 5 ng Gβγ RNA in this experiment), and Gβγ and Gα were estimated from Itotal and Ibasal, giving 3.16 and 0.73 Gβγ:GIRK and Gα:GIRK ratios, respectively. For simulation with endogenous G proteins only and no Gβγ recruitment allowed (red, black and green lines), the channel density was the same and 1, 10 or 24 endogenous Gαβγ were assumed to be available for GIRK1/2.
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pcbi.1004598.g006: Dose-dependent activation of GIRK1/2 by coexpressed Gβγ: experiment and simulation.GIRK1/2 was expressed at 0.2 ng RNA. All data are mean ± SEM from one experiment. (A) Confocal images of Gβγ in giant excised plasma membranes stained with the anti-Gβ antibody. The intensity of all images was increased equally for a better viewing in this figure, but not in the process of image analysis. (B) Dose-dependence of Gβγ levels and Iβγ in oocytes injected with incrementing amounts of wt Gβγ RNA (0.05–30 ng per oocyte). Gβγ expression in the PM (grey bars) was measured from images shown in A, in 4–8 oocyte membranes, and Iβγ currents (red circles; right Y-axis) were measured in 12–16 oocytes. The dashed line shows the basal level of fluorescence, arising from the endogenous Gβγ. Note that, unlike in Western blots, in immunocytochemistry the antibody poorly recognized the endogenous Gβγ compared to the expressed bovine Gβγ. (C) Comparison of measured Iβγ and Rβγ (red circles) and simulated currents (curves). The relative Gβγ levels (from grey bars in B) have been converted into surface densities assuming that 5 ng Gβγ gives 30 molecules Gβγ/μm2. The blue line presents the simulation using graded contribution model and amounts of Gα and Gβγ (prior to coexpression of Gβγ) calculated using the methods described above: channel density was calculated from Iβγ (13.75 channels/μm2 with 5 ng Gβγ RNA in this experiment), and Gβγ and Gα were estimated from Itotal and Ibasal, giving 3.16 and 0.73 Gβγ:GIRK and Gα:GIRK ratios, respectively. For simulation with endogenous G proteins only and no Gβγ recruitment allowed (red, black and green lines), the channel density was the same and 1, 10 or 24 endogenous Gαβγ were assumed to be available for GIRK1/2.

Mentions: Relative levels of Gβγ in the PM were directly measured in giant membrane patches of the oocytes [30,95] (Fig 6A) using the anti-Gβ antibody. Absolute surface densities of the expressed Gβγ (X axis in Fig 6C) were calculated assuming that 5 ng mRNA of Gβγ gives 30±4 molecules/μm2 (n = 47 oocytes). This density was calculated based on 3 experiments performed during the same period as the experiments of Fig 6 and S8 Fig, with wt-Gβ and YFP-Gγ, and using YFP-GIRK1/2 as the molecular ruler.


A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gβγ.

Yakubovich D, Berlin S, Kahanovitch U, Rubinstein M, Farhy-Tselnicker I, Styr B, Keren-Raifman T, Dessauer CW, Dascal N - PLoS Comput. Biol. (2015)

Dose-dependent activation of GIRK1/2 by coexpressed Gβγ: experiment and simulation.GIRK1/2 was expressed at 0.2 ng RNA. All data are mean ± SEM from one experiment. (A) Confocal images of Gβγ in giant excised plasma membranes stained with the anti-Gβ antibody. The intensity of all images was increased equally for a better viewing in this figure, but not in the process of image analysis. (B) Dose-dependence of Gβγ levels and Iβγ in oocytes injected with incrementing amounts of wt Gβγ RNA (0.05–30 ng per oocyte). Gβγ expression in the PM (grey bars) was measured from images shown in A, in 4–8 oocyte membranes, and Iβγ currents (red circles; right Y-axis) were measured in 12–16 oocytes. The dashed line shows the basal level of fluorescence, arising from the endogenous Gβγ. Note that, unlike in Western blots, in immunocytochemistry the antibody poorly recognized the endogenous Gβγ compared to the expressed bovine Gβγ. (C) Comparison of measured Iβγ and Rβγ (red circles) and simulated currents (curves). The relative Gβγ levels (from grey bars in B) have been converted into surface densities assuming that 5 ng Gβγ gives 30 molecules Gβγ/μm2. The blue line presents the simulation using graded contribution model and amounts of Gα and Gβγ (prior to coexpression of Gβγ) calculated using the methods described above: channel density was calculated from Iβγ (13.75 channels/μm2 with 5 ng Gβγ RNA in this experiment), and Gβγ and Gα were estimated from Itotal and Ibasal, giving 3.16 and 0.73 Gβγ:GIRK and Gα:GIRK ratios, respectively. For simulation with endogenous G proteins only and no Gβγ recruitment allowed (red, black and green lines), the channel density was the same and 1, 10 or 24 endogenous Gαβγ were assumed to be available for GIRK1/2.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636287&req=5

pcbi.1004598.g006: Dose-dependent activation of GIRK1/2 by coexpressed Gβγ: experiment and simulation.GIRK1/2 was expressed at 0.2 ng RNA. All data are mean ± SEM from one experiment. (A) Confocal images of Gβγ in giant excised plasma membranes stained with the anti-Gβ antibody. The intensity of all images was increased equally for a better viewing in this figure, but not in the process of image analysis. (B) Dose-dependence of Gβγ levels and Iβγ in oocytes injected with incrementing amounts of wt Gβγ RNA (0.05–30 ng per oocyte). Gβγ expression in the PM (grey bars) was measured from images shown in A, in 4–8 oocyte membranes, and Iβγ currents (red circles; right Y-axis) were measured in 12–16 oocytes. The dashed line shows the basal level of fluorescence, arising from the endogenous Gβγ. Note that, unlike in Western blots, in immunocytochemistry the antibody poorly recognized the endogenous Gβγ compared to the expressed bovine Gβγ. (C) Comparison of measured Iβγ and Rβγ (red circles) and simulated currents (curves). The relative Gβγ levels (from grey bars in B) have been converted into surface densities assuming that 5 ng Gβγ gives 30 molecules Gβγ/μm2. The blue line presents the simulation using graded contribution model and amounts of Gα and Gβγ (prior to coexpression of Gβγ) calculated using the methods described above: channel density was calculated from Iβγ (13.75 channels/μm2 with 5 ng Gβγ RNA in this experiment), and Gβγ and Gα were estimated from Itotal and Ibasal, giving 3.16 and 0.73 Gβγ:GIRK and Gα:GIRK ratios, respectively. For simulation with endogenous G proteins only and no Gβγ recruitment allowed (red, black and green lines), the channel density was the same and 1, 10 or 24 endogenous Gαβγ were assumed to be available for GIRK1/2.
Mentions: Relative levels of Gβγ in the PM were directly measured in giant membrane patches of the oocytes [30,95] (Fig 6A) using the anti-Gβ antibody. Absolute surface densities of the expressed Gβγ (X axis in Fig 6C) were calculated assuming that 5 ng mRNA of Gβγ gives 30±4 molecules/μm2 (n = 47 oocytes). This density was calculated based on 3 experiments performed during the same period as the experiments of Fig 6 and S8 Fig, with wt-Gβ and YFP-Gγ, and using YFP-GIRK1/2 as the molecular ruler.

Bottom Line: Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter.In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases.The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
G protein-gated K+ channels (GIRK; Kir3), activated by Gβγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.

No MeSH data available.


Related in: MedlinePlus