Zebrafish P2X Receptor
Naomi Pang '19 and Clare Chou '19
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 . 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 . LGICs are
comprised of three families of receptors; among these are P2X
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 .
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
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 .
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
stabilize the triphosphate tail via hydrogen bonding interactions.
The adenine base is stabilized with
hydrogen bonding and hydrophobic interactions with
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
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