ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α
1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of
3H-prazosin or
125I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α
1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α
1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α
1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of
3H-prazosin or
125I-HEAT, and the IC
50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca
2+ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α
1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106
3.32A and the S188
5.42A/S192
5.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86
2.64A, F288
6.51A and F312
7.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86
2.64 was identified as a key interaction point for
125I-HEAT, as the variant F86
2.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α
1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86
2.64, F288
6.51 and F312
7.39) and agonist (F288
6.51 and F312
7.39) ligands. Its selectivity for the α
1A-adrenoceptor may result, at least partly, from its interaction with the residue F86
2.64, which appears to be important also for HEAT binding.
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