Table 1.

Summary of derm A594 and ntx A594 affinity measurements^{^a}

Ligand	Assay	Affinity (nm)
derm A594	Radioligand competition	2.9 ± 0.8 (K_i)
	+Na⁺, Mg²⁺, GTPγS	120 ± 40 nm (K_i)
	Association kinetics	111 (85–149) (K_d)
	Association kinetics after ME 2 h	32 (24–42 (K_d)
	Steady-state imaging	85 (50–142) (EC₅₀)
	Steady-state after ME 2 h	11.6 (8.4–16.2) (EC₅₀)
ntx A594	Kinetics	53 (44–65) (K_d)

↵^a [³H]Diprenorphine competition performed in crude membrane preparations in the presence and absence of Na⁺, Mg²⁺, and GTPγS (K_i ± 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 h (K_d ± error range; Fig. 2C,D). Steady-state binding of derm A594 to live cells untreated and pretreated with ME 2 h (EC₅₀ ± 95% CI; Figs. 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 (100 nm, 5 min) (K_d ± error range; Fig. 6B). ntx A594 shows similar affinity to derm A594 despite different kinetics.