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PubMed: 26088141

Title
Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors.
Journal
The Journal of biological chemistry
Volume
290
Issue
None
Pages
20060-70
Date
2015-08-14
Authors
Bridges SP | Kabbani N | King JR | Lin MK | Nordman JC

Evidence 265d111b02
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In α7345–348A nAChR expressing cells, nifedipine had no effect on the peak or the duration of the calcium transient (peak: 957.00% ΔF/Fθ ± 252.2%; AUC: 333.33% ΔF/Fθ2 × s ± 91.53%) relative to choline treatment alone (Fig. 5, A–C). The findings suggest that choline-induced calcium responses in PC12 cells involve the activity of VGCC (37, 38).

a(CHEBI:"calcium(2+)") association act(a(MESH:"Calcium Channels")) View Subject | View Object

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act(a(CHEBI:choline)) association act(a(MESH:"Calcium Channels")) View Subject | View Object

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a(CHEBI:nifedipine) causesNoChange bp(GO:"calcium-mediated signaling") View Subject | View Object

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a(CHEBI:nifedipine) causesNoChange act(p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) View Subject | View Object

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act(a(MESH:"Calcium Channels")) association act(a(CHEBI:choline)) View Subject | View Object

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act(a(MESH:"Calcium Channels")) association a(CHEBI:"calcium(2+)") View Subject | View Object

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Evidence b464408d37
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PC12 cells transfected with α7345–348A showed a reduction in choline-mediated calcium responses.

act(a(CHEBI:choline)) increases a(CHEBI:"calcium(2+)") View Subject | View Object

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Annotations
MeSH
PC12 Cells

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) decreases act(a(CHEBI:choline)) View Subject | View Object

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Annotations
MeSH
PC12 Cells

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) decreases a(CHEBI:"calcium(2+)") View Subject | View Object

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MeSH
PC12 Cells

Evidence b8123ae644
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Treatment of PC12 cells with 10 mM choline was associated with a translocation of PH-mCherry from the cell surface as determined by the presence of the fluorescence signal within 1 μm of the edge of the cell into the cytosol of the GC (Fig. 6, A and B). Pre-treatment of cells with SP abolished this translocation (Fig. 6B).

a(CHEBI:choline) association bp(MESH:"Pleckstrin Homology Domains") View Subject | View Object

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Annotations
MeSH
Cytosol
MeSH
PC12 Cells

a(MESH:"GP Antagonist-2A peptide") decreases bp(MESH:"Pleckstrin Homology Domains") View Subject | View Object

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MeSH
Cytosol
MeSH
PC12 Cells

bp(MESH:"Pleckstrin Homology Domains") association a(CHEBI:choline) View Subject | View Object

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Annotations
MeSH
Cytosol
MeSH
PC12 Cells

Evidence 78e1783378
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Sequential imaging of PH-mCherry and GCaMP5G confirms that choline promotes a rise in intracellular calcium and PH-mCherry translocation in the same cellular compartment (Fig. 6, B and C). Cytoplasmic translocation of PH-mCherry occurred on a slower time scale (40 s after choline application) than peak calcium responses (∼1 s after choline application). These kinetics are consistent with the translocation of the PH domain sensor in the cell (20, 29).

a(CHEBI:choline) increases bp(MESH:"Pleckstrin Homology Domains") View Subject | View Object

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MeSH
Cytosol

Evidence 0ce964c17d
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As shown in Fig. 8, A and B, IP of the α7 using the C-20 antibody suggests that choline application attenuates G protein binding with the nAChR. Choline treatment resulted in a 56% reduction in Gαq and 47% reduction in Gβ association within the α7 nAChR complex (Fig. 8B).

a(CHEBI:choline) decreases complex(p(FPLX:"G_protein"), p(HGNC:CHRNA7)) View Subject | View Object

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a(CHEBI:choline) decreases complex(p(HGNC:CHRNA7), p(HGNC:GNAQ)) View Subject | View Object

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a(CHEBI:choline) decreases complex(p(HGNC:CHRNA7), p(HGNCGENEFAMILY:"G protein subunits beta")) View Subject | View Object

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Evidence 0d84125a06
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As shown in Fig. 5, A–C, nifedipine was found to decrease the peak calcium response to choline in PC12 cells (peak: 795.00% ΔF/Fθ ± 107.1%) by 56.94% (p = 0.003), whereas prolonging the duration of the choline-induced calcium transient (AUC: 749.50% ΔF/Fθ2 × s ± 64.02%) in the same cell.

a(CHEBI:nifedipine) decreases a(CHEBI:"calcium(2+)") View Subject | View Object

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MeSH
PC12 Cells

a(CHEBI:nifedipine) decreases act(a(CHEBI:choline)) View Subject | View Object

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Annotations
MeSH
PC12 Cells

a(CHEBI:nifedipine) increases bp(GO:"calcium-mediated signaling") View Subject | View Object

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Annotations
MeSH
PC12 Cells

Evidence 3c8d819aaf
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SP pretreatment did not significantly alter calcium peaks in α7345–348A nAChR expressing cells, showing a small (−31.24%) reduction in calcium responses relative to the α7345–348A baseline measure (p > 0.05).

a(MESH:"GP Antagonist-2A peptide") causesNoChange a(CHEBI:"calcium(2+)") View Subject | View Object

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a(MESH:"GP Antagonist-2A peptide") causesNoChange act(p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) View Subject | View Object

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Evidence 039e34e390
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We confirmed the involvement of IP3Rs in choline-induced calcium transients at the GC of PC12 cells. Cells were preincubated with the IP3R blocker xestospongin C (1 μM) prior to imaging. As shown in Fig. 7, A–C, pretreatment with xestospongin C reduced the α7 nAChR calcium response peak by 46.82% in α7 cells (peak: 1308.43% ΔF/Fθ ± −238.13%; + xestospongin C = 695.80% ΔF/Fθ ± 101.46%).

a(MESH:"xestospongin C") decreases act(p(HGNC:CHRNA7)) View Subject | View Object

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MeSH
PC12 Cells

a(MESH:"xestospongin C") decreases bp(GO:"calcium-mediated signaling") View Subject | View Object

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Annotations
MeSH
PC12 Cells

Evidence f43db8ee20
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Xestospongin C treatment did not impact the calcium peak (628.87% ΔF/Fθ ± 69.43%) or total calcium transient (AUC: 443.86% ΔF/Fθ2 × s ± 54.72%; + xestospongin C = 340.39% ΔF/Fθ2 × s ± 96.99%) in α7345–348A expressing cells (α7 to α7 + xestospongin C, p = 0.017; α7 to α7345–348A, p = 0.013; α7 to α7345-348A + xestospongin C, p = 0.017) (Fig. 7, B and C). The data underscores the inability of α7345–348A nAChRs to activate intracellular calcium via IP3Rs (18).

a(MESH:"xestospongin C") decreases act(p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) View Subject | View Object

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p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) decreases act(a(CHEBI:"calcium(2+)", loc(GO:intracellular))) View Subject | View Object

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Evidence 504b620002
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As shown in Fig. 5, A–C, barium replacement had little to no effect on the peak and total calcium transient observed in α7 (peak: 1474.83% ΔF/Fθ ± 162.00%; AUC: 693.5% ΔF/Fθ2 × s ± 154.15%) and α7345–348A expressing cells (peak: 794% ΔF/Fθ ± 81.36%; AUC: 543.5% ΔF/Fθ2 × s ± 89.59%).

a(MESH:Barium) causesNoChange bp(GO:"calcium-mediated signaling") View Subject | View Object

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a(MESH:Barium) causesNoChange act(p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) View Subject | View Object

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a(MESH:Barium) causesNoChange act(p(HGNC:CHRNA7)) View Subject | View Object

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Evidence f54af86ef9
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The findings confirm that the mutation in α7345–348A does not interfere with the trafficking or expression of the nAChR.

p(HGNC:CHRNA7) increases bp(HBP:"nAChR assembly") View Subject | View Object

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p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) increases bp(HBP:"nAChR assembly") View Subject | View Object

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Evidence 1d7ce778ae
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Western blot analysis confirms that transfection of α7 nAChRs augments total α7 subunit expression in PC12 cells, which express endogenous α7 nAChRs. Transfection with α7 (α7+) increased the immunoreactive α7 signal by over 60% from endogenous mock-transfected control cells, whereas transfection with the mutant α7345–348A increased the total α7 signal by 48% over the endogenous α7 from control cells (Fig. 3A).

p(HGNC:CHRNA7) increases p(HGNC:CHRNA7) View Subject | View Object

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Annotations
MeSH
PC12 Cells

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) increases p(HGNC:CHRNA7) View Subject | View Object

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Annotations
MeSH
PC12 Cells

Evidence f05d34d96d
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An increase in G protein association within the α7 complex was observed in α7+ cells (Gαq +16.71%; Gβγ +19.90%) (Fig. 3A). Similar findings in transfected N2a cells indicate a loss in G protein association within the α7 complex when α7345–348A nAChRs are expressed (Fig. 3B).

p(HGNC:CHRNA7) increases complex(p(HGNC:CHRNA7), p(HGNC:GNAQ)) View Subject | View Object

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p(HGNC:CHRNA7) increases complex(p(FPLX:"G_beta"), p(FPLX:"G_gamma"), p(HGNC:CHRNA7)) View Subject | View Object

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p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) directlyDecreases complex(p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")), p(HGNC:GNAQ)) View Subject | View Object

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p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) directlyDecreases complex(p(FPLX:"G_beta"), p(FPLX:"G_gamma"), p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) View Subject | View Object

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Evidence ee029b5a7f
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As shown in Fig. 3A, coupling between Gαq and α7 nAChRs was virtually abolished by expression of α7345–348A. A noticeable loss in Gαq (−62.18%) and Gβγ (−20.03%) expression within the α7 nAChR complex IP was seen in cells transfected with α7345–348A (Fig. 3A).

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) directlyDecreases complex(p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")), p(HGNC:GNAQ)) View Subject | View Object

Appears in Networks:

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) directlyDecreases complex(p(FPLX:"G_beta"), p(FPLX:"G_gamma"), p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) View Subject | View Object

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Evidence 88d2cc218b
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The data complements earlier findings on the ability of α7345–348A to function as a dominant negative blocker of G protein coupling in PC12 cells, and suggests that the GPBC directs nAChR association with Gαq and Gβγ.

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) directlyDecreases complex(p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")), p(HGNC:GNAQ)) View Subject | View Object

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p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) directlyDecreases complex(p(FPLX:"G_beta"), p(FPLX:"G_gamma"), p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) View Subject | View Object

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Evidence 867fcbf0a0
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Because α7345–348A- transfected cells did not show any responsiveness to SP, these findings suggest this receptor mutant is not functionally coupled to Gαq.

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) directlyDecreases complex(p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")), p(HGNC:GNAQ)) View Subject | View Object

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Evidence c850c7d9ea
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These findings in N2a cells are consistent with data from PC12 cells and suggest that α7345–348A nAChR expression impairs nAChR calcium signaling.

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) decreases bp(GO:"calcium-mediated signaling") View Subject | View Object

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Evidence 55c565791b
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In PC12 cells expressing α7345–348A nAChRs choline had a weak effect on PH-mCherry translocation relative to empty plasmid-transfected controls. Expression α7345–348A nAChRs was surprisingly associated with strong levels of PH-mCherry at the cell surface in the absence of drug treatment (Fig. 6B).

p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) decreases bp(MESH:"Pleckstrin Homology Domains") View Subject | View Object

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Annotations
MeSH
Cytosol
MeSH
PC12 Cells

Evidence 37080747bc
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As shown in Fig. 1A, α7 nAChRs were found in association with Gαs, Gαq, and Gαi proteins within the hippocampus and prefrontal cortex. Striatal fractions suggest α7 interaction with Gαq and Gαi subunits (Fig. 1A).

p(HGNC:GNAI1) increases complex(p(HGNC:CHRNA7), p(HGNC:GNAI1)) View Subject | View Object

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Hippocampus
MeSH
Prefrontal Cortex

p(HGNC:GNAI2) increases complex(p(HGNC:CHRNA7), p(HGNC:GNAI2)) View Subject | View Object

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Hippocampus
MeSH
Prefrontal Cortex

p(HGNC:GNAQ) increases complex(p(HGNC:CHRNA7), p(HGNC:GNAQ)) View Subject | View Object

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MeSH
Hippocampus
MeSH
Prefrontal Cortex

p(HGNC:GNAS) increases complex(p(HGNC:CHRNA7), p(HGNC:GNAS)) View Subject | View Object

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MeSH
Hippocampus
MeSH
Prefrontal Cortex

Evidence 7593bc6837
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An association between α7 nAChRs and Gβ was observed in the prefrontal cortex and hippocampus, whereas little to no detection of Gαo was seen in the adult brain (Fig. 1A).

p(HGNCGENEFAMILY:"G protein subunits beta") increases complex(p(FPLX:"G_protein"), p(HGNC:CHRNA7)) View Subject | View Object

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Annotations
MeSH
Hippocampus
MeSH
Prefrontal Cortex

Evidence a10399ee46
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In α7+ cells, choline application resulted in a calcium signal that peaked at 1050% ΔF/Fθ (±176.4%) in the neurite. Calcium transients in the GC were found to last for ∼1.6 s in both α7 and α7+ cells peaking at 1396% (±154.4%) and 1316% (±146.9%) ΔF/Fθ, relatively (Fig. 4, A and B).

composite(a(CHEBI:choline), p(HGNC:CHRNA7)) increases a(CHEBI:"calcium(2+)") View Subject | View Object

Appears in Networks:
Annotations
MeSH
Neurites

Evidence 80755d8d8b
JSON

In N2a cells α7345–348A nAChR expression fostered a weak calcium response to (10 mM) choline, whereas expression of α7 nAChRs correlated with noticeable choline-induced calcium transient. Imaging studies in N2a cells expressing α7345–348A nAChRs indicate a significantly lowered calcium signal peak to choline (333.4% ΔF/Fθ ± 35.7%) when compared with N2a cells that express the α7nAChR (peak: 581% ΔF/Fθ ± 122.7%; p = 0.04) (Fig. 4, D and E).

composite(a(CHEBI:choline), p(HGNC:CHRNA7)) increases a(CHEBI:"calcium(2+)") View Subject | View Object

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Annotations
Experimental Factor Ontology (EFO)
Neuro-2a cell

composite(a(CHEBI:choline), p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) decreases a(CHEBI:"calcium(2+)") View Subject | View Object

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Annotations
Experimental Factor Ontology (EFO)
Neuro-2a cell

Evidence 5fc668f7ec
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Most notably, in the GC, the calcium peak values were significantly lower in α7345–348A-transfected cells compared with α7 cells after Tukey's HSD post hoc comparisons (peak: 741 ± 159.8% ΔF/Fθ, p = 0.006). This represents a 46.92% reduction from the α7 baseline calcium response (Fig. 4, A and B). This reduction approached significance in the neurite of α7345–348A cells (peak: 561 ± 124.9% ΔF/Fθ) (Fig. 4, A and B)

composite(a(CHEBI:choline), p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A"))) decreases a(CHEBI:"calcium(2+)") View Subject | View Object

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Annotations
MeSH
Neurites

Evidence f9a48648de
JSON

Gel lanes of the BgtxC were divided into 3 molecular mass fractions: F1, 190-90 kDa; F2, 89-45 kDa; and F3, 44-15 kDa (Fig. 1C). Proteomic analysis was performed on the individual fractions using tryptic in-gel digestion followed by high-performance LC-ESI. A cohort of G proteins was identified in the F2 and F3 fractions (Fig. 1D). These G proteins were not detected in BgtxC from α7−/− mice (data not shown).

composite(p(HBP:"alpha-Bungarotoxin"), p(HGNC:CHRNA7), p(MESH:"Heterotrimeric GTP-Binding Proteins")) increases complex(a(HBP:"alpha-Bungarotoxin"), p(MESH:"Heterotrimeric GTP-Binding Proteins")) View Subject | View Object

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Evidence 7f167b197a
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Studies in N2a cells indicate that transfection with α7345–348A yields a similar α7 nAChR expression as the wild-type (α7) and supports the finding that mutation at the GPBC does not impact the synthesis of the nAChR in cells.

g(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) causesNoChange p(HGNC:CHRNA7, var("p.345A"), var("p.346A"), var("p.347A"), var("p.348A")) View Subject | View Object

Appears in Networks:
Annotations
Experimental Factor Ontology (EFO)
Neuro-2a cell

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