Relative affinities of various opioid agonists and antagonists for the wild-type and third intracellular loop deletion mutant receptors
| Competition binding studies were carried out with membranes isolated from HEK293 cells stably expressing the WT and mutant receptors as described under Materials and Methods. For i3-1, the HEK293cells expressing this mutant receptor were treated with 10 μM naloxone for 48 h before membrane preparation. Binding data were analyzed using the GraphPad Prism program. Results were reported for two affinity states in cases when the two-site fit were significantly better than the one-site fit as defined by the program, p < 0.05. For two-site fits, KH and KL represented the Ki values of the high- and low-affinity states, respectively. The values in parentheses represent the percentage of the total receptor in the high-affinity state. Data represent mean ± S.E.M. of two (when Ki values were similar to WT) to four (when Ki values were different from WT) experiments in duplicates. | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Receptor | CTOP Ki | Naloxone Ki | DAMGO | Morphine | PL017 | |||||||||||
| KH | KL | KH | KL | KH | KL | |||||||||||
| nM | nM | nM | nM | |||||||||||||
| WT | 9.7 ± 1.4 | 4.4 ± 0.63 | 1.4 ± 0.44 (48 ± 4) | 140 ± 30 | 2.5 ± 0.48 (43 ± 4) | 170 ± 6 | 11 ± 1.5 (36 ± 7) | 330 ± 0 | ||||||||
| i3-1 | N.D. | N.D. | 300 ± 80* | N.D. | N.D. | N.D. | N.D. | |||||||||
| i3-2 | 10 ± 2.7 | 7.1 ± 0.23* | 2.3 ± 1.7 (23 ± 1)* | 100 ± 10 | 5.7 ± 1.3* (49 ± 9) | 390 ± 170 | 7.9 ± 5.8 (23 ± 4) | 540 ± 50 | ||||||||
| i3-3 | 12 ± 7.7 | 1.3 ± 0.52* | 1.1 ± 0.56 (51 ± 3) | 100 ± 10 | 0.93 ± 0.31 (56 ± 2) | 110 ± 70 | 15 ± 9.1 (47 ± 12) | 410 ± 270 | ||||||||
| i3-4 | 5.2 ± 0.13* | 4.9 ± 0.41 | 0.27 ± 0.1* (20 ± 5)* | 80 ± 30 | 7.4 ± 3.9 (47 ± 3) | 330 ± 90 | 2.4 ± 1.6* (29 ± 8) | 390 ± 60 | ||||||||
| i3-5 | 5.2 ± 1.1* | 13 ± 2.0* | 390 ± 60* | 550 ± 30* | N.D. | N.D. | ||||||||||
| 5A | 22 ± 4.9* | 15 ± 6.5* |
| 60 ± 20* |
| 180 ± 40 | N.D. | N.D. | ||||||||
N.D., not determined; WT, wild-type.
↵* Signifies statistical significant different (p < 0.05) when comparing the mutants with the wild-type receptor using student unpaired t test.