New insight into active muscarinic receptors with the novel radioagonist [H-3]iperoxo

作者：Schrage Ramona; Holze Janine; Kloeckner Jessica; Balkow Aileen; Klause Anne S; Schmitz Anna Lena; De Amici Marco; Kostenis Evi; Traenkle Christian; Holzgrabe Ulrike; Mohr Klaus^*

来源：Biochemical Pharmacology, 2014, 90(3): 307-319.

DOI：10.1016/j.bcp.2014.05.012

• Muscarinic acetylcholine receptor
• Superagonism
• Iperoxo
• G protein-coupled receptor
• Allosteric modulation

Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M-2 receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [H-3]iperoxo - unlike the common radioagonists [H-3]acetylcholine and [H-3]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [H-3]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M-2 and M-4 receptor in the picomolar range. Probing ternary complex formation on the My receptor, [H-3]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M-2, the preferred G(i) protein acted as a positive allosteric modulator of [H-3]iperoxo binding, whereas G(s) and G(q) were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM(2) cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [H-3]iperoxo. Taken together, the muscarinic superagonist [H-3]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors.

• 出版日期2014-8-1