Table 1.
Summary of derm A594 and Ntx A594 affinity measurements. [3H] diprenorphine competition performed in crude membrane preparations in the presence and absence of Na+, Mg2+, and GTPγS (Ki ±sd, Fig 2A) revealed low and high affinity states of derm A594 binding. Kinetic affinity measurements from live cells were calculated from initial apparent binding kinetics measured for a range of derm A594 concentrations performed in live cells untreated and pretreated with ME 2 hrs (Kd ±error range Fig 2C,D). Steady state binding of derm A594 to live cells untreated and pretreated with ME 2 hrs (EC50 ±95% confidence interval, Fig 2B, 3B) are shown demonstrating increased affinity after ME treatment. Affinity of Ntx A594 was based on measurement of apparent on rate and off rate of ntx A594 (100nM, 5 min) (Kd± error range, Fig 6B). Ntx A594 shows similar affinity to derm A594 despite different kinetics.
| Ligand | Assay | Affinity (nM) |
|---|---|---|
| Derm A594 | Radioligand competition | 2.9 +/− 0.8 (Ki) |
| +Na+, Mg2+, GTPγS | 120 +/− 40nM (Ki) | |
| Association kinetics | 111 (85 – 149) (Kd) | |
| Association kinetics post ME 2hrs | 32 (24 – 42( Kd) | |
| Steady state imaging | 85 (50 – 142) (EC50) | |
| Steady state post ME 2hrs | 11.6 (8.4 – 16.2) (EC50) | |
| Ntx A594 | Kinetics | 53 (44 – 65) (Kd) |