Table 2.
Summary of the colocalization indexes, homodimer lifetimes, fractions and PM residency times of molecules with internalizations detectable by the 35 min observations, and Ca2+ mobilization parameter, (FMax-Fb)/Fb, in the presence and absence of various modulators (0.2 µM agonists, ≈ 3 µM FAM-Xpep-TATs in the cytoplasm, and ≈ 6 µM mGFP-Xpeps in the cytoplasm)
| ORs | Additions | Colocalization Index (Mean ± SEM) | Homodimer Lifetime (ms) (Mean ± SEM) | Fraction of internalized molecules (%) | PM residency time of internalized molecules (min) | Ca2+ (FMax-Fb)/Fb |
|---|---|---|---|---|---|---|
| KOR | Control | 2.30 ± 0.2 | 178 ± 5 | 8.4 ± 4.1 | 32.1 ± 23.8 | 0.04 ± 0.01 |
| + FAM-pep-TAT | 1.53 ± 0.1a,Na | 62 ± 1a,Na | 6.9 ± 2.4 Nc,b | 26.2 ± 15.4Nc,b | 0.05 ± 0.01Nc,b | |
| + mGFP-pep | 1.41 ± 0.1a,Na | 61 ± 1a,Na | - | - | - | |
| + Agonist | 1.48 ± 0.1a | 59 ± 2a | 51.4 ± 2.7a | 11.9 ± 1.7a | 1.96 ± 0.32a | |
|
+ Agonist + FAM-pep-TAT |
1.23 ± 0.1a,b | 58 ± 1a,Na | 60.5 ± 2.5a,b | 9.6 ± 1.2a,Na | 2.46 ± 0.21a,b | |
|
+ Agonist + mGFP-pep |
1.17 ± 0.1a,b | 54 ± 2a,Na | - | - | 3.11 ± 0.17a,b | |
| MOR | Control | 1.82 ± 0.1 | 122 ± 3 | 6.4 ± 1.9 | 25.8 ± 12.6 | 0.03 ± 0.01 |
| + FAM-pep-TAT | 1.38 ± 0.1a,b | 59 ± 1a,b | 6.3 ± 1.0Nc,b | 22.1 ± 4.1Nc,b | 0.04 ± 0.01Nc,b | |
| + mGFP-pep | 1.32 ± 0.1a,b | 58 ± 2a,b | - | - | - | |
| + Agonist | 2.18 ± 0.2a | 137 ± 3a | 28.1 ± 1.7a | 11.3 ± 1.9a | 2.80 ± 0.21a | |
|
+ Agonist + FAM-pep-TAT |
1.38 ± 0.1a,b | 68 ± 1a,b | 28.9 ± 2.0a,Na | 10.7 ± 2.1a,Na | 2.78 ± 0.22a,Na | |
|
+ Agonist + mGFP-pep |
1.34 ± 0.1a,b | 67 ± 3a,b | - | - | 2.80 ± 0.24a,Na | |
| DOR | Control | 1.89 ± 0.1 | 120 ± 3 | 4.5 ± 3.5 | 29.8 ± 37.4 | 0.05 ± 0.01 |
| + FAM-pep-TAT | 1.36 ± 0.1a,b | 67 ± 1a,b | 4.9 ± 2.5Nc,b | 26.2 ± 22.6Nc,b | 0.05 ± 0.01Nc,b | |
| + mGFP-pep | 1.29 ± 0.1a,b | 64 ± 2a,b | - | - | - | |
| + Agonist | 3.00 ± 0.3a | 229 ± 5a | 61.6 ± 1.7a | 10.8 ± 0.9a | 3.85 ± 0.23a | |
|
+ Agonist + FAM-pep-TAT |
2.08 ± 0.2a,b | 171 ± 5a,b | 55.0 ± 1.0a,b | 10.1 ± 0.5a,Na | 2.84 ± 0.20a,b | |
|
+ Agonist + mGFP-pep |
2.00 ± 0.2a,b | 168 ± 5Nc,b | - | - | 2.65 ± 0.22a,b |
The agonists employed here were U-50488 for KOR, DAMGO for MOR, and SNC-80 for DOR.
a (Nc): Significant (insignificant) difference from the value for “Control”.
b (Na): Significant (insignificant) difference from the value for “+ Agonist”.
Significant and insignificant differences were determined by p < 0.05 and p ≥ 0.05, respectively. P values and statistical parameters for all results throughout this report are included in Supplementary Data 2. The p values were obtained by Tukey’s multiple comparison test, except the Brunner-Munzel test used for colocalization lifetimes. All data shown in this paper are the results of at least three independent replicates.