Figure 1.
Pharmacological profile of ρ-Da1a binding to various human AR subtypes expressed in eukaryotic cells.
Binding inhibition curves for 3H-prazosin (2 nM), 3H-rauwolscine (2 nM) and 3H-CGP-12177 (6 nM) on hα1A- (1 µg, ○), hα1B- (3 µg, •), hα1D- (29 µg, □), hα2A- (140 µg, ◊), hα2B- (100 µg, Δ), hα2C- (3 µg, x), β1- (3 µg,▾) and β2-AR (1.5 µg, ▪) with recombinant ρ-Da1a. n = 4.
Figure 2.
Inhibition of 3H-prazosin (2 nM, 1 µg, open symbols) by HEAT (□), and ρ-Da1a (circle), and inhibition of 125I-HEAT (0.2 nM, 0.2 µg, full symbols) binding by prazosin (♦) and ρ-Da1a (circle) to α1A-AR.
n = 3.
Figure 3.
Influence of various ligands on 3H-prazosin and 125I-HEAT dissociation.
Panel A: Dissociation of 3H-prazosin (2 nM) binding to α1A-AR (1 µg) in the presence of prazosin (10 µM, black), prazosin plus ρ-Da1a (2.5 µM, blue), prazosin plus adrenaline (2 mM, red) and prazosin plus EPA (150 µM, green). Panel B : dissociation of 125I-HEAT (0.4 nM) binding to α1A-AR (0.2 µg) in the presence of HEAT (5 µM, black), HEAT plus ρ-Da1a (2.5 µM, blue), HEAT plus prazosin (10 µM, red) and HEAT plus EPA (150 µM, green). n = 2.
Figure 4.
Inhibition of the binding of a series of concentrations of 3H-prazosin and 125I-HEAT to α1A-AR by ρ-Da1a.
Panel A 3H-prazosin binding (from 0.2 to 16 nM) inhibited by ρ-Da1a. Panel B 125I-HEAT binding (from 0.1 to 1.25 nM) inhibited by ρ-Da1a. Panel C and D: Fitting, by the Cheng and Prusoff equation IC50 = Ki+Ki(L/Kd), of IC50 values as a function of the radiotracer concentrations.
Figure 5.
Concentration-response curves for stimulation of Ca2+ release by the α1A-AR.
Agonist responses represent the difference between basal fluorescence and the peak [Ca2+]i (reached within 20 sec of agonist addition), expressed as a percentage of the response to the Ca2+ ionophore A23187 (1 µM). Concentration-dependent Ca2+ release was stimulated by noradrenaline (panel A), phenylephrine (panel B), A61603 (panel C) or oxymetazoline (panel D). Concentration response curves were performed in the presence or absence of differing concentrations of ρ-Da1a (• control, ▪ 1 nM, ▴ 3 nM, ▾ 10 nM, ♦ 30 nM, ◊ 100 nM, ○ 300 nM). Values are means ± SEM of 3–4 independent experiments.
Figure 6.
Saturation experiments with 125I-HEAT on receptor variants.
Table 1.
Effect of human α1A-AR mutations on receptor expression and affinity for HEAT and ρ-Da1a.
Figure 7.
Receptor affinities for ρ-Da1a (dash lines) and HEAT (solid lines) on mutated α1A-ARs.
Binding inhibition curves for 125I-HEAT binding to WT (200 pM, 0.2 µg, ○), D1063.32A (200 pM, 1 µg, □) and F862.64A (1.3 nM, 0.8 µg, •) receptor variants. n = 3–4.
Figure 8.
Receptor affinities for ρ-Da1a on mutated α1A-ARs.
Binding inhibition curves for 125I-HEAT (200 pM) binding to WT (0.2 µg, black), F1875.41A (0.15 µg, light blue), the double S1885.42,S1925.46-AA (0.3 µg, dark blue), F1935.47A (0.25 µg, green), F2816.44A (0.15 µg, orange), F2886.51A (0.2 µg, red), M2926.55A (0.2 µg, purple), F3087.35A (0.1 µg, brown), F3127.39A (0.8 µg, grey), n = 3–4.
Figure 9.
Homology modelling of the ρ-Da1a binding site in the α1A-AR and the MT7 toxin.
Views from the side of the TM bundle (Panel A), and from the top of the extracellular space (Panel B). F1875.41, F1935.47, F2816.44, M2926.55, F3087.35 in green. D1063.32 and the double S1885.42/S1925.46 in orange. F862.64, F2886.51 and F3127.39 in red. Panel C :3D structure of the three-finger fold MT7 toxin (2vlw) with the four conserved disulfide bridges in red.