Data from: Comparing CB1 receptor GIRK channel responses to receptor internalization using a kinetic imaging assay
Data files
Oct 07, 2024 version files 257.25 KB
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Figure2RawData.xlsx
270.05 KB
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Figure4RawData.xlsx
88.58 KB
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Figure5RawData.xlsx
30.46 KB
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README.md
7.91 KB
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SupplementalFigure1RawData.xlsx
26.52 KB
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SupplementalFigure2RawData.xlsx
41.65 KB
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SupplementalFigure3RawData.xlsx
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SupplementalFigure4RawData.xlsx
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Abstract
The type 1 cannabinoid receptor (CB1R) mediates neurotransmitter release and synaptic plasticity in the central nervous system. Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (Gi) and the β-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The β-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2 > (±)CP55,940 > Δ9-THC > AEA. Efficacy was expressed relative to (±)CP55,940 with a rank order efficacy of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. Internalization efficacy was normalized to (±)CP55,940 with a rank order efficacy of WIN55,212-2 > AEA > (±)CP55,940 > Δ9-THC. (±)CP55,940 was significantly more potent and efficacious than AEA and Δ9-THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid’s GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential Gi signaling when compared to each other, this did not extend to CB1R internalization.
https://doi.org/10.5061/dryad.r4xgxd2nz
This Dryad submission includes excel files containing raw data and analyses related to the linked manuscript. Data are from:
(1) Kinetic G protein inwardly-rectifying potassium channel (GIRK) assays that have been analyzed for change in fluorescence, peak response, rate of response, and area under the curve following treatment of AtT20 SEP-CB1R cells with one of four cannabinoids (CP55,940, WIN55,212-2, d9-tetrahydrocannabiol, or anandamide). These data were also analyzed for concentration-response relationships using nonlinear regression to estimate the potency and efficacy of these four cannabinoids.
(2) Kinetic receptor internalization assays that have been analyzed for change in fluorescence, peak response, rate of response, and area under the curve following treatment of AtT20 SEP-CB1R cells with one of four cannabinoids (CP55,940, WIN55,212-2, d9-tetrahydrocannabiol [THC], or anandamide) or the CB1R inverse agonist SR141716. These data were also analyzed for concentration-response relationships using nonlinear regression to estimate the potency and efficacy of these four cannabinoids.
Description of the data and file structure
Data are organized into excel files such that each excel file directly corresponds to a figure appearing in the main manuscript or supplemental data files. Files are named according to the figure they correspond with. Within each excel file separate tabs are used for separate figure panels and named according to their corresponding panel.
Separate tabs within the excel files are also dedicated to analyses (e.g., nonlinear regression or analysis of variance). All such analyses have been copy/pasted from their original source files (GraphPad Prism version 9.0) into these excel files for ease of accessibility.
Specific notes - also present within each excel file - to help guide users of the data are as follows:
1) Figure2RawData.xlsx: GIRK channel responses in AtT20 cells
a) This file contains all data relating to figure 2 of our manuscript. Each figure panel (i.e., panels A, B, C, D, E, and F) appears as a separate tab within the excel file.
b) Tab "CRC Figure 2F": "n.d." refers to "not determined" as compounds were not tested at those corresponding concentrations and therefore otherwise empty cells are labelled as "n.d." to avoid ambiguity. Data values are presented for the number of replicate measurements taken in rows corresponding to each concentration of compound. Two excluded concentrations of compound for CP55,940 are indicated in blue text.
c) Analysis of variance (ANOVA) data are presented on the final tab of this file for Figure 2.
2) Figure4RawData.xlsx: CB1R internalization in AtT20 cells
a) This file contains all data relating to figure 4 of our manuscript. Each figure panel (i.e., panels A, B, C, D, E, and F) appears as a separate tab within the excel file.
b) Tab "Figure 4F": "n.d." refers to "not determined" as compounds were not tested at those corresponding concentrations and therefore otherwise empty cells are labelled as "n.d." to avoid ambiguity. Data values are presented for the number of replicate measurements taken in rows corresponding to each concentration of compound.
c) Concentration-response nonlinear regression curve fitting data are presented on the final tab "Table 1 ANOVA" along with ANOVA data a for Figure 4. As noted in the excel file, GraphPad Prism output of "???" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable.
3) Figure5RawData.xlsx: CB1R internalization in AtT20 cells with SR141716A
a) This file contains all data relating to figure 5A of our manuscript.
4) SupplementalFigure1RawData.xlsx: Rate of GIRK channel responses in AtT20 cells
a) This file contains all data relating to supplemental figure 1 of our manuscript. Figure panels A, B, C, and D appear on the first tab and figure panel E (ANOVA) as a separate tab within the excel file.
b) Tab "Rate Supp Fig1A_D": "n.d." refers to "not determined" as compounds were not tested at those corresponding concentrations and therefore otherwise empty cells are labelled as "n.d." to avoid ambiguity. Data values are presented for the number of replicate measurements taken in rows corresponding to each concentration of compound. As noted in the excel file, "Unstable" is denoted when GraphPad Prism was not able to calculate a rate value for those corresponding data.
5) SupplementalFigure2RawData.xlsx: Area Under the Curve analysis for GIRK channel responses in AtT20 cells
a) This file contains all data relating to supplementary figure 2 of our manuscript. Figure panels A and B (table with concentration-response nonlinear regression and ANOVA) appear as separate tabs within the excel file.
b) Tab "Suppl Fig 2": "n.d." refers to "not determined" as compounds were not tested at those corresponding concentrations and therefore otherwise empty cells are labelled as "n.d." to avoid ambiguity. Data values are presented for the number of replicate measurements taken in rows corresponding to each concentration of compound. Two excluded concentrations of compound for CP55,940 are indicated in blue text.
c) Nonlinear regression and ANOVA data are presented on the final tab of this file for supplementary Figure 2.
6) SupplementalFigure3RawData.xlsx: Rate of CB1R internalization in AtT20 cells
a) This file contains all data relating to supplemental figure 3 of our manuscript. Figure panels A, B, C, and D appear on the first tab and figure panel E (ANOVA) as a separate tab within the excel file.
b) Tab "Suppl Figure 3A_D": "n.d." refers to "not determined" as compounds were not tested at those corresponding concentrations and therefore otherwise empty cells are labelled as "n.d." to avoid ambiguity. Data values are presented for the number of replicate measurements taken in rows corresponding to each concentration of compound. As noted in the excel file, "Unstable" is denoted when GraphPad Prism was not able to calculate a rate value for those corresponding data.
7) SupplementalFigure4RawData.xlsx: Area Under the Curve analysis for CB1R internalization in AtT20 cells
a) This file contains all data relating to supplementary figure 4 of our manuscript. Figure panels A and B (table with concentration-response nonlinear regression and ANOVA) appear as separate tabs within the excel file.
b) Tab "Supply Fig 4A": "n.d." refers to "not determined" as compounds were not tested at those corresponding concentrations and therefore otherwise empty cells are labelled as "n.d." to avoid ambiguity. Data values are presented for the number of replicate measurements taken in rows corresponding to each concentration of compound. As noted in the excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, If GraphPad Prism is unable to out a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. For example, "Top" (i.e., Emax) is calculated as the mean of the highest observed response for the indicated compound.
c) Nonlinear regression and ANOVA data are presented on the final tab of this file for supplementary Figure 2.
Sharing/Access information
Data were derived from analyses in Gen5 software version 3.1 (Agilent), ImageJ/Fiji version 2.15.1 (National Institutes of Health), and GraphPad Prism version 9.0 and shared here copy/pasted into excel files for ease of accessibility.