ION CHANNELS: THEIR CELL BIOLOGY, STRUCTURE AND FUNCTION
We are interested in the structure, function and cell biology of ion channels. In recent years our focus has been the P2X family of membrane receptors but we also have a long-standing interest in potassium channels that regulate neuronal and cardiac function. P2X receptors are cation channels that open on binding extracellular ATP and are widely distributed throughout all major systems in the body, playing a key role in afferent signalling, in the regulation of blood flow and the generation of inflammatory responses. Extracellular ATP signals tissue injury, sites of inflammation and cancers. P2X receptors are therefore involved in many pathologies and are important targets for the development of new drug therapies.
ASSEMBLY AND TRAFFICKING OF P2X RECEPTORS
Many cellular signalling pathways are regulated through the processes that insert and remove receptors from the cell surface, and we have focused much effort in understanding the assembly and trafficking of multimeric receptor complexes. Structurally the P2X family forms a distinct group from the Cys-loop and glutamate receptor families of ligand gated ion channels. Each subunit has only two membrane spanning segments and they assemble as trimers. The existence of multiple subtypes of P2X receptor and the ability to form homo- and heteromeric complexes increases receptor diversity. In addition, there is evidence that P2X receptors interact with other membrane proteins to form part of larger signalling complexes. We use a combination of molecular biological, biochemical and electrophysiological methods combined with atomic force microscopy (AFM) to determine the stoichiometry and subunit arrangement of receptor complexes. AFM is carried out in collaboration with Prof J.M. Edwardson. Mechanisms involved in the trafficking and targeting of receptors are studied using a combination of mutagenesis analysis, immunofluorescence and live cell imaging techniques (Figs. 1 and 2). We have shown that one member of the P2X family, P2X4 is normally targeted to lysosomes, where it resists degradation and can subsequently be delivered to the cell surface to up-regulate the cellular response to ATP. This work was the first to demonstrate that an ion channel can be regulated in this way and there is now a growing body of evidence which indicates that lysosomes are not simply sites of degradation but are multifunctional structures, involved in release of chemical mediators, membrane trafficking and signal transduction. Targeting of the P2X4 receptor to lysosomes suggests that it might function within these acidic intracellular compartments as well as at the cell surface, as has recently been suggested for P2X-like channels in Dictyostelium. Identifying a role for intracellular mammalian P2X receptors is a current area of investigation within the laboratory.
|Fig 1. (A) Confocal image showing intracellular distribution of P2X4-EGFP expressed in NRK cells. Expanded images show P2X4-positive structures accumulate lysotracker (red), scale bars = 1 µm. (B) Intracellular P2X4-EGFP positive compartments adjacent to the plasma membrane were imaged by total internal reflection microscopy (TIRFM). Maximum projection of images acquired over 30 seconds at 4 frames/second. Larger compartments were docked and exhibited small random movements. Small compartments moved directionally at rate of ~1µm/second.||Fig 2. Confocal images showing the endogenous P2X4 receptors are localized to lysosomes in peritoneal macrophages. Cells were fixed, permeabilized and stained with anti-P2X4 (green) and co-labelled with anti-LAMP-1. Scale bars = 10 µm. Lower panels show high magnification images of intracellular structures, scale bars = 1 µm|
REGULATION OF P2X7 RECEPTOR SIGNALLING AND IMMUNE CELL FUNCTION
The P2X7 receptor is unique within the P2X family, both with respect to its structure and function. It has an extra long cytoplasmic C-terminal domain that interacts with cytoskeletal and signalling proteins, low affinity for ATP, is resistant to desensitization and can induce membrane permeability to large molecules. It is highly expressed in macrophages and other immune cells and its ligation by extracellular ATP is an important second stimulus for Toll-like receptor 4 dependent IL-1 and IL-18 secretion. It also induces both macrophage apoptosis and killing of intracellular mycobacteria in infected human macrophages. Polymorphic alleles with reduced function are associated with an increased susceptibility to mycobacterial infections. In other cell types including epithelial cells, it plays an important role in the regulation of cell proliferation and cell death and its dysregulation is associated with cancers. Generally the expression of the receptor in many tissues is up regulated in disease states or injury and it mediates the effects of ATP as a ‘danger signal’. Its involvement in the many diseases associated with chronic inflammation make it as attractive target for development of novel therapies.
We study mechanisms involved in the regulation of P2X7 receptor signalling. This includes regulation at the level of alternative splicing of the RNA transcript, trafficking of the receptor complex to and from the plasma membrane, its association and regulation of lipid microdomains and its interaction with other proteins involved in its targeting and signalling. We use a range of approaches including fluorescence imaging techniques, electrophysiology and biochemical assays combined with molecular biology and tissue culture.
AN INTRACELLULAR ROLE FOR P2X RECEPTORS WITHIN LYSOSOMES AND PHAGOSOMES
The up-regulation and activation of P2X receptors in microglia contributes to neuropathic and inflammatory pain; hence the receptors are potential targets for the development of novel therapies in the management of chronic pain. The predominant subtypes expressed are P2X4 and P2X7 and their respective roles in mediating the effects of ATP are not well understood. The high affinity P2X4 receptor resides predominantly within lysosomes and is up-regulated at the plasma membrane and the phagosome membrane following stimulation of lysosome exocytosis and phagocytosis, respectively (Figs. 3 and 4). We have evidence to suggest that activation of the low affinity P2X7 receptor can promote the recruitment of the high affinity receptor to the cell surface and to the phagosome. We are investigating a possible intracellular role for these highly calcium permeable receptors within lysosomes and phagosomes.
|Fig 3. Lysosome exocytosis upregulates P2X4 receptors at the plasma membrane. (LHS) Surface expression of P2X4 and LAMP-1 in peritoneal macrophages following 15 minutes incubation with 50 µM of the weak base, methylamine (MA). Surface receptors were detected by biotinylation at 12°C followed by immunoblotting. Samples from the total cell lysates show that the overall expression of P2X4 and LAMP-1 was unchanged by MA treatment. (RHS) Whole cell currents measured using the perforated patch clamp technique following application of 30 µM ATP at a holding potential of -60 mV in LPS primed mouse peritoneal macrophages. Currents were measured in control cells, and cells preincubated with MA.||Fig. 4. Trafficking of P2X4 receptors to phagosomes. (A) Primary macrophages were incubated with zymosan particles for 30 minutes, followed by a 60 minute chase, fixed, permeabilized and stained with anti- P2X4 (green) and anti-LAMP-1 (red). (B)RAW264.7 cells expressing P2X4-GFP (green) were incubated with latex beads for 30 minutes followed by a 1 hour chase, fixed, permeabilized and stained with anti-LAMP-1 (red). Scale bars = 10 µm. (C) Detection of P2X4 and LAMP-1 in latex bead phagosomes isolated by sucrose density gradient flotation from bone marrow derived macrophages by immunoblotting (Phag) and dilutions of total cell lysates (Total).|