Ruth D. Murrell-Lagnado
University of Cambridge
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Ruth D. Murrell-Lagnado.
Molecular Pharmacology | 2007
Chang Guo; Omar S. Qureshi; Ruth D. Murrell-Lagnado
The cytolytic ionotropic ATP receptor P2X7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X4, they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X4 and P2X7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X4 and P2X7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X7 although there was no change in overall expression. Coexpression of a dominant-negative P2X4 mutant (C353W) with P2X7, inhibited P2X7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non–dominant-negative mutant (S341W) did not. Coexpression of P2X4S341W with P2X7 produced a current that was potentiated by ivermectin and inhibited by 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X4 and P2X7, which suggests that they associate to form heteromeric receptors.
Journal of Biological Chemistry | 2005
Nelson P. Barrera; Susan J. Ormond; Robert M. Henderson; Ruth D. Murrell-Lagnado; J. Michael Edwardson
P2X receptors are cation-selective channels activated by extracellular ATP. The architecture of these receptors is still not completely clear. Here we have addressed this issue by both chemical cross-linking and direct imaging of individual receptors by atomic force microscopy (AFM). Cross-linking of the P2X2 receptor produced higher order adducts, consistent with the presence of trimers. The mean molecular volume of the receptor determined by AFM (409 nm3) also points to a trimeric structure. P2X2 receptors bearing His6 epitope tags were incubated with anti-His6 antibodies, and the resultant complexes were imaged by AFM. For receptors with two bound antibodies, the mean angle between the antibodies was 123°, again indicating that the receptor is a trimer. In contrast, cross-linking of the P2X6 receptor did not produce higher order adducts, and the mean molecular volume of the receptor was 145 nm3. We conclude that P2X2 receptors are trimers, whereas the P2X6 receptor subunits do not form stable oligomers.
Journal of Cell Science | 2007
Omar S. Qureshi; Anbalakan Paramasivam; Jowie C. H. Yu; Ruth D. Murrell-Lagnado
The P2X4 receptor has a widespread distribution in the central nervous system and the periphery, and plays an important role in the function of immune cells and the vascular system. Its upregulation in microglia contributes to neuropathic pain following nerve injury. The mechanisms involved in its regulation are not well understood, although we have previously shown that it is constitutively retrieved from the plasma membrane and resides predominantly within intracellular compartments. Here, we show that the endogenous P2X4 receptors in cultured rat microglia, vascular endothelial cells and freshly isolated peritoneal macrophages are localized predominantly to lysosomes. Lysosomal targeting was mediated through a dileucine-type motif within the N-terminus, together with a previously characterized tyrosine-based endocytic motif within the C-terminus. P2X4 receptors remained stable within the proteolytic environment of the lysosome and resisted degradation by virtue of their N-linked glycans. Stimulation of phagocytosis triggered the accumulation of P2X4 receptors at the phagosome membrane. Stimulating lysosome exocytosis, either by incubating with the Ca2+ ionophore ionomycin, for normal rat kidney (NRK) cells and cultured rat microglia, or the weak base methylamine, for peritoneal macrophages, caused an upregulation of both P2X4 receptors and the lysosomal protein LAMP-1 at the cell surface. Lysosome exocytosis in macrophages potentiated ATP-evoked P2X4 receptor currents across the plasma membrane. Taken together, our data suggest that the P2X4 receptor retains its function within the degradative environment of the lysosome and can subsequently traffic out of lysosomes to upregulate its exposure at the cell surface and phagosome.
Journal of Biological Chemistry | 2009
Annette Nicke; Yung-Hui Kuan; Jürgen Rettinger; Benjamin Marquez-Klaka; Olaf Bender; Dariusz C. Górecki; Ruth D. Murrell-Lagnado; Florentina Soto
The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-d-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7−/− mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7−/− strains has important implications for our understanding of the role of this receptor in health and disease.
The Journal of Physiology | 1999
Ivan H M Ho; Ruth D. Murrell-Lagnado
1 G protein‐gated inwardly rectifying K+ (GIRK) channels are activated independently by Gβγ and internal Na+ via mechanisms requiring phosphatidylinositol phosphates. An aspartate (Asp) at position 226 in GIRK2 is crucial for Na+‐dependent activation of GIRK1‐GIRK2 heteromeric channels. We expressed wild‐type and mutant GIRK1‐GIRK2 channels in Xenopus oocytes and tested the effects of Na+ and neutralizing Asp226 on the functional interactions of the channels with phosphatidylinositol 4,5‐bisphosphate (PIP2). 2 The rate of inhibition of GIRK1‐GIRK2 currents by application of anti‐PIP2 antibody to inside‐out membrane patches was slowed > 2‐fold by the D226N mutation in GIRK2 and by increasing internal [Na+]. The reverse mutation in GIRK1 (N217D) increased the rate of inhibition. 3 The dose‐response relationship for activation by purified PIP2 was shifted to lower concentrations in the presence of 20 mM Na+. 4 Three synthetic isoforms of PIP2, PI(4,5)P2, PI(3,4)P2 and PI(3,5)P2, activated GIRK channels with similar potencies. 5 We conclude that Na+ directly interacts with Asp226 of GIRK2 to reduce the negative electrostatic potential and promote the functional interaction of the channels with PIP2.
Journal of Biological Chemistry | 2009
Miyyada Boumechache; J. Michael Edwardson; Dariusz C. Górecki; Ruth D. Murrell-Lagnado
P2X4 and P2X7 are the predominant P2X receptor subtypes expressed in immune cells. Having previously shown a structural and functional interaction between the two recombinant receptors, our aims here were to identify the preferred assembly pathway of the endogenous receptors in macrophage-like cells and to investigate the trafficking of these receptors between the plasma membrane and intracellular sites. We exploited the difference in size between the two subunits, and we used a combination of cross-linkers and blue native-PAGE analysis to investigate the subunit composition of complexes present in primary cultures of rat microglia and macrophages from wild type and P2X7–/– mice. Our results indicate that the preferred assembly pathway for both receptors is the formation of homotrimers. Homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. In both macrophages and microglia, P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular. There were clear cell type-dependent differences in the extent to which P2X4 receptors trafficked to and from the surface; trafficking was much more dynamic in microglia than in the macrophages, and further activation of cultured microglia with relatively short (3-h) incubations with lipopolysaccharide caused an ∼4-fold increase in the fraction of receptors at the surface with only a 1.2-fold increase in total expression. The redistribution of intracellular receptors is thus an efficient means of enhancing the functional expression of P2X4 at the plasma membrane of microglia.
Journal of Biological Chemistry | 1999
Ivan H M Ho; Ruth D. Murrell-Lagnado
G protein-gated inwardly rectifying K+ channels (GIRKs) are activated by a direct interaction with Gβγ subunits and also by raised internal [Na+]. Both processes require the presence of phosphatidylinositol bisphosphate (PIP2). Here we show that the proximal C-terminal region of GIRK2 mediates the Na+-dependent activation of both the GIRK2 homomeric channels and the GIRK1/GIRK2 heteromeric channels. Within this region, GIRK2 has an aspartate at position 226, whereas GIRK1 has an asparagine at the equivalent position (217). A single point mutation, D226N, in GIRK2, abolished the Na+-dependent activation of both the homomeric and heteromeric channels. Neutralizing a nearby negative charge, E234S had no effect. The reverse mutation in GIRK1, N217D, was sufficient to restore Na+-dependent activation to the GIRK1N217D/GIRK2D226N heteromeric channels. The D226N mutation did not alter either the single channel properties or the ability of these channels to be activated via the m2-muscarinic receptor. PIP2 dramatically increased the open probability of GIRK1/GIRK2 channels in the absence of Na+ or Gβγ but did not preclude further activation by Na+, suggesting that Na+ is not acting simply to promote PIP2binding to GIRKs. We conclude that aspartate 226 in GIRK2 plays a crucial role in Na+-dependent gating of GIRK1/GIRK2 channels.
Journal of Biological Chemistry | 2002
Stephen J. Royle; Laura K. Bobanovic; Ruth D. Murrell-Lagnado
Rapid modulation of the surface number of certain ionotropic receptors is achieved by altering the relative rates of insertion and internalization. These receptors are internalized by a clathrin-mediated pathway; however, a motif that is necessary for endocytosis of ionotropic receptors has not yet been identified. Here, we identified a motif that is required for constitutive and agonist-regulated internalization of the ionotropic P2X4 receptor. Three amino acids in the C terminus of P2X4 (Tyr378, Gly381, and Leu382) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X4 protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathioneS-transferase fusion of the P2X4 C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the μ2 subunit of adaptor protein-2 or the YXXGL motif in the receptor C terminus caused a decrease in receptor internalization and a dramatic increase in the surface expression of P2X4 receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X4 receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.
Journal of Cell Science | 2005
Stephen J. Royle; Omar S. Qureshi; Laura K. Bobanovic; Philip R. Evans; David J. Owen; Ruth D. Murrell-Lagnado
During clathrin-mediated endocytosis, proteins on the cell surface are selected for inclusion in clathrin-coated vesicles by clathrin adaptors, mainly the adaptor complex AP2. The P2X4 subtype of ATP-gated ion channel has in its C-terminus two putative endocytic motifs: a canonical YXXΦ motif and a non-canonical YXXGΦ motif (YEQGL). We demonstrate that endocytosis of P2X4 receptors is mediated preferentially by the YXXGΦ motif because the YXXΦ motif is inaccessible to AP2 owing to the structure of the channel. The crystal structure of a complex between residues 160-435 of the μ2 subunit of AP2 and a P2X4 C-terminal peptide showed that the YEQGL motif binds to μ2 at the same site as YXXΦ motifs. Y and Φ residues are accommodated in the same hydrophobic pockets in μ2 with the extra residue between them being accommodated by changes in the peptides backbone configuration, when compared to YXXΦ motifs. These data demonstrate that the family of potential tyrosine-based endocytic signals must be expanded to include motifs with an additional glycine at Y+3 (YXXGΦ).
British Journal of Pharmacology | 2012
Christopher N. J. Young; KoiNi Lim; Sara J Barnes; Xing Jian Xu; Viola Marschall; Wojciech Brutkowski; Elizabeth R. Mooney; Dariusz C. Górecki; Ruth D. Murrell-Lagnado
BACKGROUND AND PURPOSE Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor‐mediated responses. Here, we investigated expression and function of C‐terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7−/− mice (Pfizer KO).