P2X receptors overview

There are three families of membrane proteins that recognize extracellular nucleotides: ligand-gated P2X receptor ion channels and G-protein coupled P2Y and P1 receptors. Found throughout eukaryotes, P2X receptors are expressed in a variety of cells and modulate processes such as platelet activation, smooth muscle contraction, nociception, synaptic transmission, and inflammation, making this receptor family an active pharmacological target.

Functional P2X receptors are composed of assemblies of receptor subtypes (P2X₁ – P2X₇) to form homo- and hetero-trimeric complexes. All subunits share a common topology with intracellular N- and C-termini, two transmembrane (TM) helices that form the ion channel, and an extracellular domain containing the orthosteric ATP binding site. Although all P2X receptors are ATP-gated, cation channels permeable to Na⁺, K⁺, and Ca²⁺ ions, the pharmacology of receptor subtypes varies with respect to sensitivity to agonists and to small molecule antagonists. For example, extracellular ATP activates P2X₁ and P2X₃ receptors at concentrations of only 1 nM while it takes 50-100 mM concentrations of extracellular ATP to activate P2X₇ receptors. Understanding the molecular structure of different subtypes has important implications for pharmacology and design of subtype-specific antagonists for more targeted and specific therapeutic intervention.

In addition, the kinetics of ion channel gating vary by receptor subtype, with P2X₂, P2X₄, and P2X₅ receptors showing slow rates of desensitization (seconds), P2X₁ and P2X₃ receptors undergoing rapid desensitization (milliseconds), and P2X₇ receptors showing no desensitization. What is the structural mechanism by which different P2X receptor subtypes undergo desensitization at different rates? Why does P2X₇ receptor not undergo desensitization? Our structural work on the P2X₃ receptor and the P2X₇ receptor support a novel mechanism of desensitization we term the “helical recoil model” of receptor desensitization.

Structural studies of P2X receptors

The first full-length structure of the P2X7 receptor using cryo-EM reveals how, unlike other P2X receptor subtypes, it remains active without undergoing desensitization by post-translational palmitoylation modifications to a cluster of cysteine residues to stabilize the “open” pore of the receptor. The palmitoylation moieties prevent P2X7 receptor from undergoing the conformational changes necessary for channel desensitization. The unique intracellular structure of the P2X7 receptor also revealed the unpredicted presence of both a guanosine nucleotide-binding site and a Zinc ion cluster which may be involved in downstream signaling and apoptosis. Our current structural studies are further probing these aspects of P2X receptors in addition to exploring the binding mechanisms of new agonists and antagonists.

P2X electrophysiology

Findings from the structures are complemented and verified by functional studies. We use electrophysiology techniques to test how these ion channel receptors behave in living cells. By expressing the receptors in oocytes and HEK cells and using techniques called two-electrode voltage clamp and whole cell recordings, we measure the effects of both well-known and novel agonists and antagonists. Our recent electrophysiology studies have provided further confirmation of our helical-recoil model, showing that by removing or mutating the palmitoylated cysteine residues, P2X₇ regains the ability to desensitize like other P2X receptors. Electrophysiology experiments allow us to study the action of novel, potential agonists and antagonists on receptors in a living cell, which can inform decisions about what new molecules might be worth pursuing as new drugs.

P2X ligand binding

In addition to testing for ligand efficacy using electrophysiology, ligands are tested for affinity using ligand binding assays. This typically involves measuring a radioactively labeled ligand bind to the receptor in a saturation binding experiment (K_d) or watching bound radioactive ligand get displaced by a ligand that is not radioactively labeled in an inhibition binding assay (K_i). These types of radioactive binding assays can be performed to directly monitor binding of ligands to P2X receptors.