Alpha₂ adrenoceptors in rat brain

Summary

α₂-Adrenoceptors in rat brain membranes, identified with [³H] clonidine, have divalent cation sites. Nickel, manganese, magnesium, barium, and strontium are among the active cations which potentiate clonidine binding. The effects of magnesium were investigated in detail:

• 1. The potentiation of binding by magnesium is caused by an increase in the association constant (K_a) and by increased availability of sites to the agonist. Both, availability and affinity increase, are dependent on temperature. K_a increased with increasing temperature when free magnesium is present. However, whether the divalent cation is present or not, clonidine binding to α₂-adrenoceptors as well as the binding of the antagonist prazosin to α₂-adrenoceptors are mainly entropy driven.

• 2. The effects of magnesium on α₂-adrenoceptors can be reversed rapidly (t _1/2<1 min, 30°C) by chelation; the conversion of α₂-adrenoceptors into the divalent cation-liganded state is also rapid (t _1/2<1 min, 30°C). GTP, presumably via a guanyl nucleotide protein, is a potent inhibitor of agonist binding in the presence of magnesium.

• 3. The magnesium-induced affinity increase of clonidine to α₂-adrenoceptors is shared by other agonists but not by the antagonists phentolamine and yohimbine.

• 4. The α₁-adrenoceptors antagonist prazosin looses 30-fold in affinity for the α₂-adrenoceptors when the divalent cation requirement if fulfilled with magnesium.

• 5. Brain membrane α₂-adrenoceptors can be converted into an almost completely divalent cationdependent state by a brief and mild thermal denaturation.

We are able to discriminate four conformations of α₂-adrenoceptors in rat brain membranes: α₀ is a species of presumably very low affinity for clonidine (K_a<0.01 nM⁻¹) which cannot be detected by our current methodology, α_{L 1} has a K_a around 0.12 nM⁻¹ (at 30°C) and is predominant species in native membranes in the absence of free divalent cations, α_L2 is intermediate between α₀ and α_{L 1} in its affinity and is found after a brief heat treatment of membranes in EDTA containing buffers, α_H is the high affinity conformation of α₂-adrenoceptors. It can be formed in vitro by fulfilling the divalent cation requirement with magnesium. The divalent cation converts α₀₂, α_L and α_{2 z} into α_H.

Since the effects of magnesium are agonist-specific, they may relate to signal transfer.

It is proposed that the different conformations, identified by their affinity for agonists, represent free receptors and different complexes of receptors with effector and/or coupling proteins.