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Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors
Jonathan A Roberts1, Richard J Evans
1Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.
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.
Insights
Researchers identified key amino acid residues in human P2X1 receptors critical for binding extracellular ATP and gating the channel. This study advances understanding of purinergic signaling mechanisms.
Area of Science:
- Molecular biology
- Neuroscience
- Biochemistry
Background:
- P2X receptors are ligand-gated cation channels activated by extracellular ATP.
- Their precise agonist binding sites and gating mechanisms remain unclear due to a lack of common ATP-binding motifs.
Purpose of the Study:
- To identify specific amino acid residues involved in ATP binding and channel gating of the human P2X1 receptor.
- To elucidate the structural basis of ATP recognition and activation in P2X receptors.
Main Methods:
- Cysteine-scanning mutagenesis was employed to introduce specific amino acid substitutions.
- Radiolabeled 2-azido ATP binding assays were used to assess ligand interaction.
- Methanethiosulfonate (MTS) compounds were utilized to probe residue accessibility and function.
- MTSEA biotinylation mapped residue accessibility changes upon ATP addition.
Main Results:
- Mutations at N290, F291, R292, and K309 significantly reduced ATP potency and 2-azido ATP binding, indicating their direct involvement in ATP binding.
- MTS reagent modifications at K309 suggested direct interaction with ATP's phosphate group.
- Mutations at D316C, G321C, A323C, and I328C did not affect ATP potency but altered agonist-evoked responses, suggesting a role in channel gating.
Conclusions:
- Specific extracellular residues (N290, F291, R292, K309) are crucial for ATP binding to the human P2X1 receptor.
- Another region (D316, G321, A323, I328) appears to be involved in the gating mechanism of the P2X1 channel.
- These findings provide critical insights into the structure-function relationship of P2X receptors.
