Structural and functional interaction between P2X(4), P2X(7) and pannexin-1

Boumechache, Miyyada, Ali, Saira, Shahid, Sumayya, Masin, Marianela and Murrell-Lagnado, Ruth (2010) Structural and functional interaction between P2X(4), P2X(7) and pannexin-1. Purinergic Signalling, 6 (1). p. 73. ISSN 1573-9538

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Abstract

Activation of P2X7 receptors triggers an increase in membrane permeability to large molecules and, in immune cells, the processing and release of inflammatory cytokines. The hemi-channel Pannexin-1 (Px1) was recently shown to interact with P2X7 and to be involved in both processes [1]. There is also evidence that the P2X4 receptor interacts with P2X7 [2]. In this study, we set out to further investigate the trafficking and functional interaction between P2X7, Px1 and P2X4. Proteins were overexpressed in HEK293 cells, and we used siRNA and carbenoxolone (CBX) to inhibit Px1. In HEK293 cells transiently overexpressing P2X7, incubation with CBX produced a dose-dependent inhibition of ethidium uptake evoked by ATP or BzATP. Surprisingly, in HEK293 cells expressing recombinant Px1 as well as P2X7, there was a reduction in the rate of ethidium uptake compared to cells transfected with P2X7 alone, and also incubation with CBX enhanced rather than inhibited BzATP-evoked ethidium uptake. The expression levels of P2X7 receptors in wild-type and Px1 stable cells were very similar, yet the fraction of P2X7 at the cell surface was slightly increased in Px1-cells as determined by biotinylation and selective cross-linking of surface receptors. The role of the cytoskeleton in large pore formation and in P2X7 coupling to Px1 was also examined. As shown previously, disruption of actin filaments with latrunculin had no effect on ethidium uptake; in contrast, depolymerisation of microtubules with colchicine resulted in a significant increase in both the rate and maximal dye uptake in response to ATP. The effects of co-expressing P2X4 on the interaction between P2X7 and Px1 are being investigated.

Item Type: Article
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QH Natural history > QH0301 Biology
Depositing User: Tom Gittoes
Date Deposited: 25 Mar 2015 13:36
Last Modified: 25 Mar 2015 13:36
URI: http://sro.sussex.ac.uk/id/eprint/52598
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