Chapter Eight - Regulation of P2X Purinergic Receptor Signaling by Cholesterol

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Abstract

P2X receptors are cation-selective channels that are activated by the binding of extracellular ATP. They have a high permeability to Ca2 +, Na+, and K+ and are expressed widely throughout the nervous, immune, cardiovascular, skeletal, gastrointestinal, respiratory, and endocrine systems. Seven mammalian subtypes of P2X receptor subunits have been identified, P2X1–7, and those that function as homotrimeric receptors (P2X1, 2, 3, 4, and 7) are targeted to lipid rafts, although they show limited resistance to solubilization by Triton X-100. Recent crystal structures of P2X3 and P2X4 receptors have provided considerable high-resolution information about the architecture of this family of receptors and yet the molecular details of how they are regulated by cholesterol are unknown. Currents mediated by the P2X1–4 receptors are either inhibited or relatively insensitive to cholesterol depletion, but there is no clear evidence to support the direct binding of cholesterol to these receptors. In contrast, the activity of the low-affinity, proinflammatory P2X7 receptor is potentiated by cholesterol depletion and regions within the proximal C-terminus play an important role in coupling changes in cholesterol to the gating of the pore. Based upon our understanding of the lipid signaling events that are triggered downstream of P2X7 receptor activation, a change in the levels of cholesterol may contribute to the sensitization of receptor currents and the dilation of the pore that occurs following prolonged, high-level stimulation. This chapter focuses on the regulation of P2X7 receptor signaling by cholesterol and our current understanding of the mechanisms that underlie this.

Introduction

P2X receptors have a very widespread distribution throughout the human body and purinergic signaling mediated by these receptors takes place in all tissues (Khakh & North, 2006). All cells release ATP by a combination of mechanisms including vesicular release, ion channel-mediated release, and cell lysis (Burnstock & Verkhratsky, 2012). Under normal physiological conditions, the levels of extracellular ATP are very low because of the highly active, membrane-bound ectonucleotidases that rapidly break down ATP to ADP, AMP, and adenosine. However, levels of extracellular ATP are much higher following injury or in diseases that are associated with inflammation. These include cancer, neurodegenerative, and metabolic diseases, in addition to the well-characterized chronic inflammatory diseases such as arthritis and atherosclerosis. High ATP concentrations are recognized as a danger signal, the effects of which are mediated by the uniquely low-affinity P2X7 receptor (Khakh & North, 2006). Conditions that lead to enhanced P2X7 receptor signaling are also conditions that result in perturbations of membrane lipids and remodeling of rafts (Fessler & Parks, 2011).

Lipid rafts are specialized, liquid-ordered microdomains in the plasma membrane enriched in cholesterol, sphingolipids, and saturated phospholipids. They can be identified biochemically by their low buoyant density on a sucrose density gradient, their resistance to solubilization in detergent, and the presence of protein markers such as caveolin (Lingwood and Simons, 2010, Pike, 2004). The precise nature of rafts can differ with respect to their size and lipid/protein composition. For example, caveolae are a special type of lipid raft that form small invaginations of the plasma membrane and contain caveolin, an integral membrane protein. Some other proteins, such as glycosylphosphatidylinositol-anchored proteins, are targeted to rafts, but others are excluded, resulting in compartmentalization and organization of signaling events. Dynamic remodeling of rafts is important in signaling and the control of cholesterol trafficking through rafts by environmental factors and signaling cascades can be an important determinant of the cellular response (Fessler & Parks, 2011).

Section snippets

P2X Receptors Form a Structurally Distinct Class of Ligand-Gated Ion Channels

P2X receptors assemble as homo- or heterotrimeric complexes. The seven identified subtypes share the same general structure, possessing two transmembrane regions connected via a large extracellular loop and intracellular N- and C-termini. The seven P2X subunits are 40%–50% identical in their amino acid sequence, but they range in size because of a very divergent C-terminal domain which is short in P2X1, P2X3, P2X4, and P2X6 (~ 30–50 residues) longer in P2X2 and P2X5 and considerably longer in

P2X Receptors Are Localized to Lipid Rafts

Several members of the P2X family are detected in the buoyant lipid raft fractions including P2X1, P2X2, P2X3, P2X4, and P2X7 (Allsopp et al., 2010, Barth et al., 2008, Barth et al., 2007, Gnanasekaran et al., 2011, Gonnord et al., 2009, Vacca et al., 2004, Vial and Evans, 2005, Vial et al., 2006). The other two subtypes, P2X5 and P2X6, have very limited or no function as homomeric receptors, and their association with rafts has not been reported. The presence of P2X receptors within lipid

P2X1 Receptors

The P2X1 receptor is expressed in smooth muscle where it mediates contraction of arteries, resistant arterioles, vas deferens, and bladder (Vial & Evans, 2000). Similar to the recombinant receptor expressed in HEK293 cells, the native receptor in smooth muscle is predominantly within lipid rafts (Vial and Evans, 2005, Vial et al., 2006). P2X1 receptor currents are profoundly sensitive to disruption of lipid rafts, with the current mediated by the recombinant receptor expressed in HEK293 cells

P2X7 Receptors and the Mechanisms by Which They Associate With Rafts

P2X7 receptors are highly expressed in immune cells, including mast cells, monocytes, macrophages, microglia, and T-lymphocytes, but are also found in other cells including epithelia, vascular endothelial cells, fibroblasts, osteoclasts, and osteoblasts (Surprenant & North, 2009). The nature of lipid rafts differs in these cells, with caveolae being well described in fibroblasts and epithelial cells, but less well characterized in macrophages (Fessler & Parks, 2011). Thus the regulation of P2X7

The Effects of Cholesterol on P2X7 Receptor Function and Signaling

There are several ways in which the recruitment of the P2X7 receptor to lipid rafts and the cholesterol content of the rafts could affect receptor signaling: first, by affecting the plasma membrane expression of the receptor, second, by affecting receptor function such as the gating of the channel pore, and third, by concentrating signaling molecules that act either as downstream effectors of the receptor or as regulators of the receptor.

Little is known about what controls the trafficking of

The Determinants of P2X7 Receptor Cholesterol Sensitivity

It is still unclear whether cholesterol exerts its effect on P2X receptor activity directly, by binding to a cholesterol-sensing motif (CSM) within the receptor, or indirectly, via an associated protein and/or by changing the biophysical properties of the membrane. Unlike the cholesterol regulation of P2X1 receptors, the potentiation of P2X7 receptor activity that occurs in response to cholesterol depletion is not affected by the cytoskeletal-stabilizing agent jasplakinolide, which argues

Future Work

In this review, we have focused on the inhibitory regulation of P2X7 receptors by cholesterol but have also briefly touched on other lipids, such as the products of the PLA2 pathway, which positively regulate the receptor. The underlying molecular mechanisms of this regulation are not yet established, but with the publishing of P2X receptor structures in the close, open, and desensitized states, the field is well placed to elucidate the detailed nature of how lipids directly or indirectly

Acknowledgments

I thank Dr. Andrew Thompson for assistance with Fig. 1 and for discussions. This work have been supported by the BM1406 COST Action.

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