Table 5.

List of binding kinetic variables (association rate, dissociation rate, residence time (RT) and kinetic K_D) for CCPA, NECA and LUF5834 in the absence or presence of an allosteric modulator (PD81,723 or BC-1) at CHO-hA₁R membranes, derived from competition association assays

Cmpd	kon (M−1·min−1)a	koff (min−1)a	RT (min)b	KD (nM)c
CCPA	9.6 ± 1.8 × 106	1.2 ± 0.2	0.86 ± 0.17	131 ± 21
+1 μM PD81,723	1.9 ± 0.5 × 107	0.77 ± 0.03	1.3 ± 0.1	43 ± 7*
+10 μM PD81,723	1.0 ± 0.1 × 107	0.32 ± 0.03*	3.1 ± 0.3*	32 ± 3**
+33 μM PD81,723	3.4 ± 0.8 × 107*	0.20 ± 0.02**	5.0 ± 0.4***	5.9 ± 1.9**
+1 μM BC-1	1.9 ± 0.1 × 107*	0.063 ± 0.005**	16 ± 1***	3.3 ± 0.2**
+10 μM BC-1	3.5 ± 0.2 × 107***	0.011 ± 0.003**	91 ± 14**	0.31 ± 0.05**
+33 μM BC-1	3.5 ± 0.3 × 107**	0.0058 ± 0.0029**	172 ± 50*	0.17 ± 0.05**
NECA	0.9 ± 0.4 × 106	0.47 ± 0.09	2.1 ± 0.5	522 ± 146
+10 μM PD81,723	3.0 ± 0.3 × 106*	0.21 ± 0.02*	4.8 ± 0.3**	70 ± 6*
+10 μM BC-1	1.6 ± 0.2 × 106	0.025 ± 0.005**	40 ± 5**	16 ± 2*
LUF5834	2.0 ± 0.2 × 108	0.92 ± 0.09	1.1 ± 0.1	4.6 ± 0.4
+10 μM PD81,723	2.6 ± 0.2 × 108	0.78 ± 0.08	1.3 ± 0.1	3.0 ± 0.2
+10 μM BC-1	4.2 ± 0.4 × 107**	0.034 ± 0.007***	29 ± 3***	0.81 ± 0.11***

Data are shown as mean ± SEM of three separate experiments each performed in duplicate.

^*P < 0.05,

^**P < 0.01,

^***P < 0.001 compared with the values in the absence of an allosteric modulator; Student's t-test.

^ak_on (k₃) and k_off (k₄) of unlabelled A₁ receptor agonists in the absence or presence of an allosteric modulator (PD81,723 or BC-1) were determined in [³H]-DPCPX (2.6 nM) competition association assays.

^bRT (residence time) = 1/k_off.

^cKinetic K_D = k_off/k_on; k_on (k₃) and k_off (k₄) values of unlabeled antagonists were generated from [³H]-DPCPX (2.6 nM) competition association assays at 25°C.