Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors.
ABSTRACT P2X receptors for extracellular ATP are a distinct family of ligand-gated cation channels involved in physiological processes ranging from synaptic transmission to muscle contraction. Common ATP binding motifs are absent from P2X receptors, and the extent of the agonist binding site is unclear. We used cysteine-scanning mutagenesis, radiolabeled 2-azido ATP binding, and methanethiosulfonate (MTS) compounds to identify amino acid residues involved in ATP binding and gating of the human P2X1 receptor. The pattern of MTSEA [(2-aminoethyl)methanethiosulfonate hydrobromide] biotinylation was also used to determine the accessibility of substituted cysteine residues and whether this changed on addition of ATP. Analysis of cysteine-substituted mutants of the last 44 amino acid residues (S286-I329) in the extracellular loop before the second transmembrane segment showed that N290, F291, R292, and K309 mutants had reduced ATP potency and 2-azido ATP binding. MTS reagents produced additional shifts in ATP potency at these residues, suggesting that they are directly involved in ATP binding; the effects were dependent on the charge of the MTS reagent at K309C; one explanation for this is that K309 interacts directly with the negatively charged phosphate of ATP. The remainder of the cysteine substitutions had little or no effect on ATP potency. However, at the mutants D316C, G321C, A323C, and I328C, MTS reagents did not change ATP potency but modified agonist-evoked responses, suggesting that this region may contribute to the gating of the channel.
Article: Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP.[show abstract] [hide abstract]
ABSTRACT: The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor.Journal of Neurochemistry 03/2006; 96(3):843-52. · 4.06 Impact Factor