The P2X receptor is the baby brother of the ligand-gated ion channel super-family. An understanding of its role in human physiology is still developing, and no one truly knows how it works to transport ions across the membrane. In this study, we review some aspects of P2X channel biophysics, concentrating on ion permeation and gating. P2X channels transport both small and large cations and anions across cell membranes in a manner that depends on both the subunit composition of the receptor and the experimental conditions. We describe the pore properties of wild-type receptors and use the altered phenotypes of mutant receptors to point the way towards a structural model of the pore.
P2X receptors are trimeric cation channels with widespread roles in health and disease. The recent crystal structure of a P2X4 receptor provides a 3D view of their topology and architecture. A key unresolved issue is how ions gain access to the pore, because the structure reveals two different pathways within the extracellular domain. One of these is the central pathway spanning the entire length of the extracellular domain and covering a distance of ≈70 Å. The second consists of three lateral portals, adjacent to the membrane and connected to the transmembrane pore by short tunnels. Here, we demonstrate the preferential use of the lateral portals. Owing to their favorable diameters and equivalent spacing, the lateral portals split the task of ion supply threefold and minimize an ion's diffusive path before it succumbs to transmembrane electrochemical gradients.fenestration | fractional calcium current | ligand-gated ion channel | cysteine scanning mutagenesis P 2X receptors are a family of ligand-gated ion channels that open when bound to extracellular ATP, providing a regulated path for depolarizing cation current across cell surface membranes. In mammals, the resulting entry of Ca 2+ and Na + triggers muscle contraction, release of neurotransmitters, hormones, and inflammatory mediators, as well as activation of downstream signaling cascades (1, 2). In combination with a ubiquitous distribution, the widespread actions of ATP suggest that P2X receptors play essential roles in the body. Indeed, advances with mouse genetics and pharmacology show that P2X receptors are involved in a variety of important physiological responses and disease states (3), making them attractive targets for drug discovery. A key goal is to understand how P2X receptors work at a molecular level.The P2X receptor family is made of seven gene products, designated P2X1-P2X7, that can form homomeric and/or heteromeric assemblies (4). One family member, the PX4 receptor, is particularly prevalent in the central nervous system (5). This receptor is involved in microglia-neuron interactions, and mounting evidence suggests it plays a critical role in neuropathic pain, a persistent and prevalent form of pain whose molecular and cellular neurobiology is poorly understood and for which there is no effective treatment (6).Each P2X4 receptor is made of three parts: an extracellular domain containing the ATP binding site and the extracellular vestibule of the ion channel, a transmembrane pore containing the channel gate and selectivity filter, and an intracellular vestibule in contact with the cytoplasm (7). The possibility that the permeation pathway extends outward from the extracellular vestibule was not considered until recently, when the newly solved crystal structure of a closed state of the zebrafish P2X4.1 receptor (zfP2X4.1R) revealed, but failed to discriminate between, two possible paths in the extracellular domain for ion passage to the pore (8). The first is the central pathway that spans the entire length of the extracellular domain (Fi...
We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2–6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by ∼3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking.
P2X receptors are a family of trimeric ion channels that are gated by extracellular adenosine 5′-triphosphate (ATP). These receptors have long been a subject of intense research interest by virtue of their vital role in mediating the rapid and direct effects of extracellular ATP on membrane potential and cytosolic Ca2+ concentration, which in turn underpin the ability of ATP to regulate a diverse range of clinically significant physiological functions, including those associated with the cardiovascular, sensory, and immune systems. An important aspect of an ion channel's function is, of course, the means by which it transports ions across the biological membrane. A concerted effort by investigators over the last two decades has culminated in significant advances in our understanding of how P2X receptors conduct the inward flux of Na+ and Ca2+ in response to binding by ATP. However, this work has relied heavily on results from current recordings of P2X receptors altered by site-directed mutagenesis. In the absence of a 3-dimensional channel structure, this prior work provided only a vague and indirect appreciation of the relationship between structure, ion selectivity and flux. The recent publication of the crystal structures for both the closed and open channel conformations of the zebrafish P2X4 receptor has thus proved a significant boon, and has provided an important opportunity to overview the amassed functional data in the context of a working 3-dimensional model of a P2X receptor. In this paper, we will attempt to reconcile the existing functional data regarding ion permeation through P2X receptors with the available crystal structure data, highlighting areas of concordance and discordance as appropriate.
P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO− side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors.
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