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An Allosteric Inhibitory Site Conserved in the Ectodomain of P2X Receptor Channels
Ariel R Ase1, Éric Therrien2, Philippe Séguéla1
1Alan Edwards Centre for Research on Pain, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada.
Abstract:
P2X receptors constitute a gene family of cation channels gated by extracellular ATP. They mediate fast ionotropic purinergic signaling in neurons and non-excitable cell types in vertebrates. The highly calcium-permeable P2X4 subtype has been shown to play a significant role in cardiovascular physiology, inflammatory responses and neuro-immune communication. We previously reported the discovery of a P2X4-selective antagonist, the small organic compound BX430, with submicromolar potency for human P2X4 receptors and marked species-dependence (Ase et al., 2015). The present study investigates the molecular basis of P2X4 inhibition by the non-competitive blocker BX430 using a structural and functional approach relying on mutagenesis and electrophysiology. We provide evidence for the critical contribution of a single hydrophobic residue located in the ectodomain of P2X4 channel subunits, Ile312 in human P2X4, which determines blockade by BX430. We also show that the nature of this extracellular residue in various vertebrate P2X4 orthologs underlies their specific sensitivity or resistance to the inhibitory effects of BX430. Taking advantage of high-resolution crystallographic data available on zebrafish P2X4, we used molecular dynamics simulation to model the docking of BX430 on an allosteric binding site around Ile315 (zebrafish numbering) in the ectodomain of P2X4. We also observed that the only substitution I312D (human numbering) that renders P2X4 silent by itself has also a profound silencing effect on all other P2X subtypes tested when introduced at homologous positions. The generic impact of this aspartate mutation on P2X function indicates that the pre-TM2 subregion involved is conserved functionally and defines a novel allosteric inhibitory site present in all P2X receptor channels. This conserved structure-channel activity relationship might be exploited for the rational design of potent P2X subtype-selective antagonists of therapeutic value.
Insights
Researchers identified a specific amino acid in P2X4 receptors, Ile312, crucial for blocking by the drug BX430. This finding reveals a conserved inhibitory site across all P2X channels, aiding the design of new therapeutics.
Area of Science:
- Molecular pharmacology
- Ion channel biophysics
- Structural biology
Background:
- P2X receptors are ATP-gated cation channels involved in vital physiological processes.
- The P2X4 subtype plays key roles in cardiovascular and neuro-immune functions.
- BX430 is a known P2X4-selective antagonist with species-dependent activity.
Purpose of the Study:
- To elucidate the molecular mechanism underlying P2X4 receptor inhibition by BX430.
- To identify key residues responsible for BX430's potency and species-selectivity.
- To explore conserved structural features for P2X channel modulation.
Main Methods:
- Site-directed mutagenesis of P2X4 receptor subunits.
- Electrophysiological recordings to assess channel function.
- Molecular dynamics simulations using crystallographic data.
Main Results:
- A single hydrophobic residue, Ile312 in human P2X4, is critical for BX430 blockade.
- Variations in this residue across P2X4 orthologs explain differential sensitivity to BX430.
- A conserved allosteric inhibitory site in the pre-TM2 region was identified, modulated by an Ile312Asp mutation.
Conclusions:
- The study defines Ile312 as a key determinant for BX430 interaction with P2X4 receptors.
- A conserved functional site in the P2X channel family offers a target for novel drug development.
- Rational design of P2X subtype-selective antagonists with therapeutic potential is feasible.
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