ION CHANNELS: THEIR CELL BIOLOGY, FUNCTION AND REGULATION
We are interested in the structure and function of ion channels and understanding their regulation and misregulation in health and disease. Changes in ion channel signaling underlies many pathologies, hence they are important targets for development of novel therapeutics. We utilize a combination of molecular and cell biological techniques combined with functional analysis of channel properties to investigate ion channel regulation at the level of alternative splicing of the primary transcript, assembly of the channel complex, trafficking and targeting of the complex within the cell, and the identification of protein and non-protein regulatory molecules.
In recent years our attention has focused on the P2X family of membrane receptors. P2X receptors are cation selective channels that open upon binding extracellular ATP and are widely distributed throughout all major systems in the body, playing a key role in afferent signaling, in the regulation of blood flow and in the generation of inflammatory responses. High extracellular ATP occurs following tissue injury, at sites of inflammation and in cancers and it is known as a DAMP (Damage Associated Molecular Pattern) molecule. The effects of pathologically high levels of ATP are mediated primarily via the low affinity P2X7 receptor. This is up regulated in many disease states and contributes to inflammation and cell death. Our work in this area is currently focused on three main objectives:
- To understand the mechanisms of regulation of P2X7 receptor signaling and how this changes during inflammation and with age and its implication for cancer and traumatic brain injury.
- To understand the role of intracellular P2X4 receptors in lysosome function and dysfunction.
- To develop novel legends acting at P2X receptors for use as analgesics, cardioprotectants and antithrombotics.
We also study the mechanisms of action of the Sigma1 receptor. This protein, resident within the endoplasmic reticulum (ER) and known to act as a chaperone protein, is also a regulator of ion channels, both within the ER and at the plasma membrane. Our work in this area is currently focused on elucidating the mechanisms by which the Sigma1 receptor controls Calcium Release Activated Calcium (CRAC) channels and luminal ER and mitochondrial calcium concentrations. Our main objective is to understand the molecular mechanisms that underlie the proliferative and anti-apoptotic actions of the Sigma1 receptor in cancer cells.
MECHANISMS REGULATING P2X7 RECEPTOR SIGNALLING
The low affinity, non-desensitizing P2X7 receptor is unique within the P2X family, both with respect to its structure and function. In epithelial, endothelia and immune cells it regulates cell growth and proliferation as well as programmed cell death and the release of inflammatory mediators. It mediates the effects of extracellular ATP as a DAMP signal and is up regulated in many tissues throughout the periphery and CNS in response to damage and inflammation. This up regulation contributes to pathologies and we are particularly interested in its role in cancer and CNS damage following traumatic brain injury. We study its regulation by alternative splicing of the primary transcript (Nicke et al, 2009; Masin et al., 2012, Xu et al., 2012), by its trafficking and targeting within the cell (Boumechache et al. 2009) and by its lipid environment at the plasma membrane (Robinson et al., 2014).
P2X7 receptor signaling is dependent upon the opening of a ‘small’ cation selective pore upon binding ATP, and the rate at which this dilates to form a ‘large’ pore, permeable to molecules up to 900Da. Large pore formation is assayed by the uptake of fluorescent dye (red) in HEK293 cells that express the receptor (green).
Small to large pore transition is regulated by the following:
1) Expression of alternatively splice variants. 2) Species variation and Single Nucleotide Polymorphisms (SNPs). 3) Membrane environment, e.g. cholesterol
Opening of the ‘small’, calcium permeable pore can be measured using whole cell patch clamping. Shown on the left are inward currents evoked by multiple, 5 second applications of ATP in HEK293 cells expressing the P2X7 receptor. Current amplitude is regulated by changing plasma membrane cholesterol levels. Elevated cholesterol (chol) inhibits receptor activation (chol), whereas its depletion by MCD, strongly potentiates the response.
THE ROLE OF INTRACELLULAR P2X4 RECEPTORS IN LYSOSOME FUNCTION AND DYSFUNCTION
We previously showed that one member of the P2X family, P2X4, is targeted to lysosomes, where it resists degradation and can subsequently be delivered to the cell surface to up-regulate the cellular response to ATP (Bobanovic et al. 2002; Royle et al. 2002; Royle et al. 2005; Qureshi et al. 2007, Boumechache et al., 2009). This targeting to lysosomes suggests that the receptor might also function within these acidic intracellular compartments as well as at the cell surface, similar to P2X-like channels in Dictyostelium. Currents mediated by lysosomal P2X4 receptors were recently shown as part of a collaboration with Xianping Dong (Huang et al., 2014). P2X4 receptors are highly calcium permeable and can promote lysosome fusion. They are pH dependent and would normally be inhibited by the acidic environment of the lysosome. Thus, within the lysosome, P2X4 receptors are likely to be gated by changes in luminal pH rather than changes in luminal ATP. We are currently investigating a synergistic interaction between P2X4 and P2X7 receptors and its role in regulating lysosome fusion and function. This include fusion with the plasma membrane and fusion with late endosomes and autophagosomes and the regulation of autophagy. We use fluorescence microscopy, electrophysiology and molecular and biochemical techniques.
(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. Final image (RHS) shows a peritoneal macrophage that has phagocytosed 2 zymosan particles and then stained with anti-P2X4 antibody to show targeting of this receptor to the phagosome.
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.
DRUG DISCOVERY TARGETED AT P2X RECEPTORS
Fragment-Based Drug-Discovery (FBDD) is being utilized to identify novel P2X ligands for treatment of conditions indicated. The initial 96 well plate assay is based upon the use of a voltage-sensitive fluorescent dye and FlexStation. Hits are further characterised using two-electrode voltage clamp and radio ligand binding. P2X1 is targeted for development of antithrombotics, P2X4 for analgesics and for heart failure and P2X7 for treatment of neuropathic and inflammatory pain and cancer.
REGULATION OF CALCIUM SIGNALLING BY THE SIGMA1 RECEPTOR
The Sigma1 receptor is resident within the endoplasmic reticulum (ER) and acts as a chaperone protein and a regulator of ion channels, both within the ER and at the plasma membrane. It is widely distributed in the brain and in peripheral tissues and is also highly expressed in cancer cells. It binds a wide variety of ligands including neurosteroids, benzomorphans and psychotropic drugs and has been implicated in several diseases that range from cancers to cocaine and alcohol addiction, to neurodegenerative disorders. It also contributes to neuroprotection and synaptic plasticity. Our work in this area is currently focused on elucidating the mechanisms by which Sigma1R controls Calcium Release Activated Calcium (CRAC) channels and luminal ER and mitochondrial calcium concentrations. Our main objective is to understand how this underlies its proliferative and anti-apoptotic action in cancer cells. The techniques being utilized include [Ca2+] measurements using Fluo-4 (Flexstation and confocal microscoy), TIRF microscopy, Atomic Force Microscopy (AFM) (Balasuriya et al., 2014) and co-immunoprecipitation, either with overexpression or deletion of Sigma1R by siRNA.