P2X receptors exhibit at least three modes of allosteric antagonism.

P2X receptors are trimeric ion channels activated by adenosine triphosphate (ATP) that contribute to pathophysiological processes ranging from asthma to neuropathic pain and neurodegeneration. A number of small-molecule antagonists have been identified for these important pharmaceutical targets. However, the molecular pharmacology of P2X receptors is poorly understood because of the chemically disparate nature of antagonists and their differential actions on the seven constituent subtypes.

Cryo-EM structures of the human P2X1 receptor reveal subtype-specific architecture and antagonism by supramolecular ligand-binding.

P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states.

Human P2X4 receptor gating is modulated by a stable cytoplasmic cap and a unique allosteric pocket.

P2X receptors (P2XRs) are a family of ATP-gated ion channels comprising homomeric and heteromeric trimers of seven subunits (P2X - P2X ) that confer different rates of desensitization. The helical recoil model of P2XR desensitization proposes the stability of the cytoplasmic cap sets the rate of desensitization, but timing of its formation is unclear for slow-desensitizing P2XRs. We report cryo-EM structures of full-length, wild-type human P2X receptor in apo, antagonist-bound, and desensitized states.

High-affinity agonism at the P2X receptor is mediated by three residues outside the orthosteric pocket.

P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X is a potential experimental tool and therapeutic approach in cancer biology. However, mechanisms of high-affinity P2X activation have not been defined.

Molecular Pharmacology of P2X Receptors: Exploring Druggable Domains Revealed by Structural Biology.

Extracellular ATP is a critical signaling molecule that is found in a wide range of concentrations across cellular environments. The family of nonselective cation channels that sense extracellular ATP, termed P2X receptors (P2XRs), is composed of seven subtypes (P2X-P2X) that assemble as functional homotrimeric and heterotrimeric ion channels. Each P2XR is activated by a distinct concentration of extracellular ATP, spanning from high nanomolar to low millimolar.

How Structural Biology Has Directly Impacted Our Understanding of P2X Receptor Function and Gating.

P2X receptors are ATP-gated ion channels expressed in a wide variety of eukaryotic cells. They play key roles in diverse processes such as platelet activation, smooth muscle contraction, synaptic transmission, nociception, cell proliferation, and inflammation making this receptor family an important pharmacological target. Structures of P2X receptors solved by X-ray crystallography have been instrumental in helping to define mechanisms of molecular P2X receptor function.

Full-Length P2X Structures Reveal How Palmitoylation Prevents Channel Desensitization.

P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization.

X-ray structures define human P2X(3) receptor gating cycle and antagonist action.

P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear.

Distance mapping in proteins using fluorescence spectroscopy: the tryptophan-induced quenching (TrIQ) method.

Studying the interplay between protein structure and function remains a daunting task. Especially lacking are methods for measuring structural changes in real time. Here we report our most recent improvements to a method that can be used to address such challenges.

Rhodopsin self-associates in asolectin liposomes.

We show that the photoreceptor rhodopsin (Rh) can exist in the membrane as a dimer or multimer using luminescence resonance energy transfer and FRET methods. Our approach looked for interactions between Rh molecules reconstituted into asolectin liposomes. The low receptor density used in the measurements ensured minimal receptor crowding and artifactual association.

High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane.

We present a site-directed fluorescence labeling (SDFL) study of 25 different T4 lysozyme protein samples labeled with the thiol-cleavable fluorophore, (2-pyridyl)dithiobimane (PDT-Bimane). Our results demonstrate PDT-Bimane can be used in cysteine-scanning studies to detect protein secondary structure, and to map proximity between sites in proteins by monitoring tryptophan quenching of bimane fluorescence. In addition, the reducible nature of PDT-Bimane can be exploited to resolve problems often faced in SDFL studies: ensuring specific labeling of cysteine residues, determining the extent of free label contamination, and accurately determining labeling efficiency even at low concentrations.

Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence.

We present a novel method for mapping proximity within proteins. The method exploits the quenching of the fluorescent label bimane by nearby Trp residues. In studies of T4 lysozyme we show that this effect appears to be distance dependent and orientation specific.