Characterization of [35S]‐ATPαS and [3H]‐α,β‐MeATP binding sites in rat brain cortical synaptosomes: regulation of ligand binding by divalent cations
Authors: Schäfer Rainer , Reiser Georg 
Published: August 2006

We made a comparative analysis of the binding characteristics of the radioligands [35S]‐ATPαS and [3H]‐α,β‐MeATP in order to test whether these ligands can be used to analyse P2‐purinoceptors in synaptosomal membranes from rat brain cortex.

Synaptosomes possess sites with high affinity for [35S]‐ATPαS (Kd=22.2±9.1 nM, Bmax=14.8 pmol mg−1 protein). The rank order of the competition potency of the different compounds (ATPαS, ATP, ATPγS>ADPβS, 2‐MeSATP>deoxyATP, ADP>>UTP, α,β‐MeATP, AMP, Reactive Blue‐2, suramin, isoPPADS) is consistent with pharmacological properties of P2Y‐purinoceptors.

Under identical conditions [35S]‐ATPαS and [3H]‐α,β‐MeATP bind to different binding sites at synaptosomal membranes from rat brain cortex. The affinity of the [3H]‐α,β‐MeATP binding sites (Kd=13.7±1.8 nM, Bmax=6.34±0.28 pmol mg−1 protein) was 38 fold higher than the potency of α,β‐MeATP to displace [35S]‐ATPαS binding (Ki=0.52 μM). ATP and ADPβS competed at both binding sites with different affinities, 60 fold and 175 fold, respectively. The other agonists tested (2‐MeSATP, UTP, GTP) did not affect specific [35H]‐α,β‐MeATP binding at concentrations up to 100 μM. The antagonists (suramin, isoPPADS, Evan's Blue) showed completely different affinities for both binding sites.

Binding of [35S]‐ATPαS on synaptosomes was regulated by GTP, which is indicative for G‐protein coupled receptors. The Kd value for the high affinity binding site was reduced in the presence of GTP about 5 fold (from 1.8 nM to 8.6 nM). In the presence of Mg2+ the affinity was increased (Kd 1.8 nM versus 22 nM in the absence of Mg2+).

The binding of both radioligands was regulated in an opposite manner by physiological concentrations of Ca2+ and Mg2+. Binding of [3H]‐α,β‐MeATP to synaptosomal membranes was increased 3 fold by raising the Ca2+ concentration from 10 μM to 1 mM, whereas the addition of Mg2+ in the same concentration range resulted in an 80% reduction of the binding. In contrast, [35S]‐ATPαS binding was not influenced at the same range of Ca2+ or Mg2+ concentrations (10 μM to 1 mM). The addition of Mg2+ (5 mM) increased the affinity of [35S]‐ATPαS for the high affinity site 10 fold.

Diadenosine polyphosphates had a bimodal effect on [35S]‐ATPαS binding to synaptosomal membranes. AP5A and Ap6A enhanced binding of [35S]‐ATPαS 1.6 fold in a concentration range between 0.1 and 50 μM. Ap3A was a weak inhibitor with a Ki value of 7.2 μM. Ap4A, AP5A and Ap6A inhibited with Ki values>100 μM. These data support the concept that diadenosine polyphosphates do not directly interact with ATPαS binding sites.

In conclusion, on the basis of present knowledge of the interaction of P2‐purinoceptor active compounds with P2X‐ and/or P2Y‐purinoceptors, our data strongly suggest that [35S]‐ATPαS is a useful tool to study P2Y‐purinoceptors. Thus, the [35S]‐ATPαS binding site might to a large extent represent P2Y‐purinoceptors in synaptosomes from rat brain cortex. The nucleotide binding is regulated by G proteins, indicated by the effects of GTP/Mg2+ on binding.