Zebrafish P2X Receptor

Naomi Pang '19 and Clare Chou '19


I. Introduction

Of the various membrane proteins involved in ion transport, ligand-gated ion channels (LGICs) form a crucial superfamily of membrane proteins, which are integral in driving a plethora of physiological processes including neuronal signaling [1]. These membrane proteins contain a pore, which allows for the ligand-mediated flow of specific ions across the plasma membrane along their respective electrochemical gradients [2]. LGICs are comprised of three families of receptors; among these are P2X receptors.

P2X receptors form a family of ATP-gated cation channel proteins characterized by their uncommon trimeric structure. There are seven widely recognized subunits of P2X (P2X1-7), which assemble together as homomers or heteromers to form various subtypes of P2X. Found in a variety of tissues, P2X receptors are implicated in many different physiological and pathological processes such as pain sensation, inflammation, and neurotransmitter release. Thus, P2X receptors have become increasingly popular therapeutic targets, making structural and functional characterization particularly critical. Here we will explore the structure of the zebrafish P2X4 receptor (zfP2X4) as a general model for P2X receptors. Due to the lack of amino acid sequence homology with other known LGICs or ATP-binding proteins, structural modeling of P2X is needed to understand the mechanism of its functional role in ion transport. The recently characterized apo and ATP-bound conformations of zfP2X4 have marked a significant milestone in understanding the mechanisms of P2X channel activation [1].

II. General Structure

The P2X receptors possesses that form a trimeric ion channel. Each subunit possesses , which are linked to the (TM1 and TM2). The overall shape of a P2X subunit is defined via the in which each domain is named according to a homology comparison to a dolphin’s body plan. The extracellular domain is depicted by the dolphin’s body, which is composed of highly conserved residues that make up a rigid beta sheet structure. Also part of the extracellular domain are four structurally flexible domains: the head, dorsal fin, and left/right flippers, which extend from the body and are thought to facilitate ATP binding.


The transmembrane domains, TM1 and TM2, are comprised of alpha-helices and retain structural homology to the tail (flukes) of the dolphin model. TM1 is positioned peripherally to the TM2 ion-conducting pore.


III. ATP Binding

Upon ATP activation, P2X receptors undergo a conformational change from their closed resting state to their open activated state, allowing for the influx of Na+, K+, and Ca2+ ions across the plasma membrane [1]. binds to an intersubunit pocket comprised of the head and left flipper domains on one subunit and the dorsal fin domain on another subunit. Thus, in the P2X trimer, there are three .

Systematic site-directed mutagenesis suggests that within the binding site, the residues , , , , and stabilize the triphosphate tail via hydrogen bonding interactions. The adenine base is stabilized with and hydrogen bonding and hydrophobic interactions with [4].


IV. Gating Mechanism

Functional studies have provided a glimpse into the P2X gating mechanism. This process can be defined in 4 steps: ATP binding, tightening of the binding “jaw,” flexing of the lower body domains, and ion gate opening. As suggested by structural modeling, each of these steps occurs in a sequential manner, though macroscopic observation shows an almost simultaneous conformational change in P2X upon ATP binding.

In the absence of ATP, the ion gate is in remains in a . To better stabilize the ATP, the head and dorsal fin domains of adjacent subunits move closer together, tightening the ATP-binding intersubunit cavity. The intersubunit cavity that forms the ATP binding site is often referred to as the “binding jaw” due to its ability to “open” and “close” depending on the presence of ATP. When this “binding jaw” closes, the inflexible beta sheets comprising much of the lower body domains (adjacent to TM2) of both subunits move away from each other . Subsequently, the P2X receptor ion gate, which is defined by the hydrophobic region of the TM2 domains of the adjacent subunits also expand , opening the pore to facilitate ion flow.


Interestingly, prolonged ATP exposure causes P2X receptors to be subjected to a desensitized state in which cations cannot flow through the protein’s transmembrane domain as the receptor experiences temporary inactivation despite the binding of ATP. Eventually ATP dissociates from the desensitized P2X receptor, allowing the channel to re-enter its resting state, and enabling its reactivation. However, the exact mechanism of resensitization is unclear and remains the center of current P2X functional studies [1].

V. References

[1] Habermacher C, Dunning K, Chataigneau T, Grutter T, Molecular structure and function of P2X receptors, Neuropharmacology, Volume 104, 2016, Pages 18-30, ISSN 0028-3908, https://doi.org/10.1016/j.neuropharm.2015.07.032.

[2] “Ligand-Gated Ion Channels.” British Journal of Pharmacology 164.Suppl 1 (2011): S115–S135. PMC. Web. 8 Dec. 2017.

[3] Kawate, Toshimitsu et al. “Crystal Structure of the ATP-Gated P2X4 Ion Channel in the Closed State.” Nature 460.7255 (2009): 592–598. PMC. Web. 8 Dec. 2017.

[4] Hattori M, Gouaux E. Molecular mechanism of ATP binding and ion channel activation in P2X receptors. Nature. 2012;485(7397):207-212. doi:10.1038/nature11010.

Back to Top