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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

Measuring the surface density of GIRK1/2 and Gβγ in Xenopus oocytes.(A) Immunochemical estimation of the amount of GIRK1 in manually separated plasma membranes of Xenopus oocytes injected with 1 or 2 ng of GIRK RNA. Shown is a Western blot of 20 manually separated plasma membranes and 4 cytosols, and variable known amounts of the GST-fused distal C-terminus of GIRK1 (the antibody's epitope) used for calibration of the antibody-produced signal. There was a non-specific band at ~75 KDa in cytosols but not PM of uninjected oocytes (“uninj”). (B) Summary of quantitative analysis of GIRK1 in PM from Western blots of 7 separate experiments. The fully glycosylated band was observed in 4 out of 7 blots. Molar amounts of protein and PM densities from Western blots were calculated as detailed in Methods. The dark red bar is the GIRK1/2 surface density in the high-density group estimated from Iβγ (see Table 1), shown for comparison. (C) Examples of confocal images of oocytes expressing YFP-GIRK1/2 (5 ng RNA) and Gβγ-YFP (5 ng RNA). (D) Estimating YFP molecules density in PM using YFP-GIRK1/2 as molecular ruler. A representative experiment is shown. The left plot shows the measured intensities of YFP-GIRK1/2 and YFP-Gβ coexpressed with wt Gγ in a separate group of oocytes (5:1 ng RNA). The right plot shows the PM densities of YFP in the YFP-GIRK1/2 oocytes, calculated as follows: Iβγ was 14.5±2.1 μA (n = 6), corresponding to 11.4±1.6 channels/μm2, or 22.8±3.3 YFP molecules/μm2. The density of YFP in the YFP-Gβγ expressing oocytes was calculated based on relative intensities from the left plot. (E) Estimating the amount of endogenous Gβ and expressed YFP-Gβ or YFP-Gβ-XL (5 ng RNA) coexpressed with wt-Gγ, in manually separated plasma membranes. Protocol was similar to Fig 4A; wt purified recombinant Gβγ was used for calibration. In parallel to biochemical measurements, we also measured GIRK currents and YFP intensity in 5–15 oocytes expressing either YFP-GIRK1/2-Gβγ or YFP-Gβγ, as explained in D. (F) Summary of YFP-Gβγ surface density measurements in 4 experiments by the two methods, quantitative Westerns and confocal imaging with YFP-GIRK1/2 as the molecular ruler.
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pcbi.1004598.g004: Measuring the surface density of GIRK1/2 and Gβγ in Xenopus oocytes.(A) Immunochemical estimation of the amount of GIRK1 in manually separated plasma membranes of Xenopus oocytes injected with 1 or 2 ng of GIRK RNA. Shown is a Western blot of 20 manually separated plasma membranes and 4 cytosols, and variable known amounts of the GST-fused distal C-terminus of GIRK1 (the antibody's epitope) used for calibration of the antibody-produced signal. There was a non-specific band at ~75 KDa in cytosols but not PM of uninjected oocytes (“uninj”). (B) Summary of quantitative analysis of GIRK1 in PM from Western blots of 7 separate experiments. The fully glycosylated band was observed in 4 out of 7 blots. Molar amounts of protein and PM densities from Western blots were calculated as detailed in Methods. The dark red bar is the GIRK1/2 surface density in the high-density group estimated from Iβγ (see Table 1), shown for comparison. (C) Examples of confocal images of oocytes expressing YFP-GIRK1/2 (5 ng RNA) and Gβγ-YFP (5 ng RNA). (D) Estimating YFP molecules density in PM using YFP-GIRK1/2 as molecular ruler. A representative experiment is shown. The left plot shows the measured intensities of YFP-GIRK1/2 and YFP-Gβ coexpressed with wt Gγ in a separate group of oocytes (5:1 ng RNA). The right plot shows the PM densities of YFP in the YFP-GIRK1/2 oocytes, calculated as follows: Iβγ was 14.5±2.1 μA (n = 6), corresponding to 11.4±1.6 channels/μm2, or 22.8±3.3 YFP molecules/μm2. The density of YFP in the YFP-Gβγ expressing oocytes was calculated based on relative intensities from the left plot. (E) Estimating the amount of endogenous Gβ and expressed YFP-Gβ or YFP-Gβ-XL (5 ng RNA) coexpressed with wt-Gγ, in manually separated plasma membranes. Protocol was similar to Fig 4A; wt purified recombinant Gβγ was used for calibration. In parallel to biochemical measurements, we also measured GIRK currents and YFP intensity in 5–15 oocytes expressing either YFP-GIRK1/2-Gβγ or YFP-Gβγ, as explained in D. (F) Summary of YFP-Gβγ surface density measurements in 4 experiments by the two methods, quantitative Westerns and confocal imaging with YFP-GIRK1/2 as the molecular ruler.

Mentions: To obtain an independent estimate of the density of GIRK1/2 in the PM, we used quantitative immunochemistry of GIRK1 in cytosol-free, manually separated plasma membranes of Xenopus oocytes (Fig 4A and 4B) [80,81]. GIRK1 was coexpressed with GIRK2 at high density, and GIRK1 was probed with a C-terminally directed antibody. Western blots of manually separated PM and of the rest of the cells without the nucleus ("cytosol") showed the presence of two forms of GIRK1. A double band of about 55–58 KDa was always observed, and an additional higher diffuse band was seen in four out of seven experiments (Fig 4A and 4B). These bands correspond to partly and fully glycosylated channels, respectively [82,83]. Three oocyte batches showed only partly glycosylated bands in the PM. Since oocytes injected with 2 ng RNA always had large GIRK1/2 currents, it is likely that both partly and fully glycosylated channels are functional at the PM, in agreement with [83]. Notably, the main fraction of the channel was found in the cytosolic fraction (most likely endoplasmic reticulum and Golgi), largely in a partly-glycosylated form (Fig 4A). This is not unexpected, because in Xenopus oocytes the PM constitutes only a very small fraction of the cell's total mass [81].


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)

Measuring the surface density of GIRK1/2 and Gβγ in Xenopus oocytes.(A) Immunochemical estimation of the amount of GIRK1 in manually separated plasma membranes of Xenopus oocytes injected with 1 or 2 ng of GIRK RNA. Shown is a Western blot of 20 manually separated plasma membranes and 4 cytosols, and variable known amounts of the GST-fused distal C-terminus of GIRK1 (the antibody's epitope) used for calibration of the antibody-produced signal. There was a non-specific band at ~75 KDa in cytosols but not PM of uninjected oocytes (“uninj”). (B) Summary of quantitative analysis of GIRK1 in PM from Western blots of 7 separate experiments. The fully glycosylated band was observed in 4 out of 7 blots. Molar amounts of protein and PM densities from Western blots were calculated as detailed in Methods. The dark red bar is the GIRK1/2 surface density in the high-density group estimated from Iβγ (see Table 1), shown for comparison. (C) Examples of confocal images of oocytes expressing YFP-GIRK1/2 (5 ng RNA) and Gβγ-YFP (5 ng RNA). (D) Estimating YFP molecules density in PM using YFP-GIRK1/2 as molecular ruler. A representative experiment is shown. The left plot shows the measured intensities of YFP-GIRK1/2 and YFP-Gβ coexpressed with wt Gγ in a separate group of oocytes (5:1 ng RNA). The right plot shows the PM densities of YFP in the YFP-GIRK1/2 oocytes, calculated as follows: Iβγ was 14.5±2.1 μA (n = 6), corresponding to 11.4±1.6 channels/μm2, or 22.8±3.3 YFP molecules/μm2. The density of YFP in the YFP-Gβγ expressing oocytes was calculated based on relative intensities from the left plot. (E) Estimating the amount of endogenous Gβ and expressed YFP-Gβ or YFP-Gβ-XL (5 ng RNA) coexpressed with wt-Gγ, in manually separated plasma membranes. Protocol was similar to Fig 4A; wt purified recombinant Gβγ was used for calibration. In parallel to biochemical measurements, we also measured GIRK currents and YFP intensity in 5–15 oocytes expressing either YFP-GIRK1/2-Gβγ or YFP-Gβγ, as explained in D. (F) Summary of YFP-Gβγ surface density measurements in 4 experiments by the two methods, quantitative Westerns and confocal imaging with YFP-GIRK1/2 as the molecular ruler.
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Related In: Results  -  Collection

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Show All Figures
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pcbi.1004598.g004: Measuring the surface density of GIRK1/2 and Gβγ in Xenopus oocytes.(A) Immunochemical estimation of the amount of GIRK1 in manually separated plasma membranes of Xenopus oocytes injected with 1 or 2 ng of GIRK RNA. Shown is a Western blot of 20 manually separated plasma membranes and 4 cytosols, and variable known amounts of the GST-fused distal C-terminus of GIRK1 (the antibody's epitope) used for calibration of the antibody-produced signal. There was a non-specific band at ~75 KDa in cytosols but not PM of uninjected oocytes (“uninj”). (B) Summary of quantitative analysis of GIRK1 in PM from Western blots of 7 separate experiments. The fully glycosylated band was observed in 4 out of 7 blots. Molar amounts of protein and PM densities from Western blots were calculated as detailed in Methods. The dark red bar is the GIRK1/2 surface density in the high-density group estimated from Iβγ (see Table 1), shown for comparison. (C) Examples of confocal images of oocytes expressing YFP-GIRK1/2 (5 ng RNA) and Gβγ-YFP (5 ng RNA). (D) Estimating YFP molecules density in PM using YFP-GIRK1/2 as molecular ruler. A representative experiment is shown. The left plot shows the measured intensities of YFP-GIRK1/2 and YFP-Gβ coexpressed with wt Gγ in a separate group of oocytes (5:1 ng RNA). The right plot shows the PM densities of YFP in the YFP-GIRK1/2 oocytes, calculated as follows: Iβγ was 14.5±2.1 μA (n = 6), corresponding to 11.4±1.6 channels/μm2, or 22.8±3.3 YFP molecules/μm2. The density of YFP in the YFP-Gβγ expressing oocytes was calculated based on relative intensities from the left plot. (E) Estimating the amount of endogenous Gβ and expressed YFP-Gβ or YFP-Gβ-XL (5 ng RNA) coexpressed with wt-Gγ, in manually separated plasma membranes. Protocol was similar to Fig 4A; wt purified recombinant Gβγ was used for calibration. In parallel to biochemical measurements, we also measured GIRK currents and YFP intensity in 5–15 oocytes expressing either YFP-GIRK1/2-Gβγ or YFP-Gβγ, as explained in D. (F) Summary of YFP-Gβγ surface density measurements in 4 experiments by the two methods, quantitative Westerns and confocal imaging with YFP-GIRK1/2 as the molecular ruler.
Mentions: To obtain an independent estimate of the density of GIRK1/2 in the PM, we used quantitative immunochemistry of GIRK1 in cytosol-free, manually separated plasma membranes of Xenopus oocytes (Fig 4A and 4B) [80,81]. GIRK1 was coexpressed with GIRK2 at high density, and GIRK1 was probed with a C-terminally directed antibody. Western blots of manually separated PM and of the rest of the cells without the nucleus ("cytosol") showed the presence of two forms of GIRK1. A double band of about 55–58 KDa was always observed, and an additional higher diffuse band was seen in four out of seven experiments (Fig 4A and 4B). These bands correspond to partly and fully glycosylated channels, respectively [82,83]. Three oocyte batches showed only partly glycosylated bands in the PM. Since oocytes injected with 2 ng RNA always had large GIRK1/2 currents, it is likely that both partly and fully glycosylated channels are functional at the PM, in agreement with [83]. Notably, the main fraction of the channel was found in the cytosolic fraction (most likely endoplasmic reticulum and Golgi), largely in a partly-glycosylated form (Fig 4A). This is not unexpected, because in Xenopus oocytes the PM constitutes only a very small fraction of the cell's total mass [81].

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