Role of histidine residues in agonist and antagonist binding sites of A1 adenosine receptor.

The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N6-[adenine-2,8-3H, ethyl-2-3H]phenylisopropyladenosine ([3H]R-PIA) and antagonist 8-cyclopentyl-1,3-[3H]-dipropylxanthine ([3H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [3H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [3H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A1 adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (KD) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand.(ABSTRACT TRUNCATED AT 250 WORDS)