N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake.

Purinergic Signalling
Karin DreisigBirgitte Rahbek Kornum

Abstract

The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a...Continue Reading

References

Sep 26, 2000·American Journal of Physiology. Cell Physiology·B J GuJ S Wiley
Jun 11, 2002·American Journal of Physiology. Cell Physiology·Megan L SmartSteven Petrou
May 16, 2006·Progress in Neurobiology·Beáta SperlághPeter Illes
Oct 13, 2006·British Journal of Pharmacology·A D MichelP P A Humphrey
Oct 15, 2008·The Journal of General Physiology·Zonghe YanStanko S Stojilkovic
Sep 12, 2012·CNS & Neurological Disorders Drug Targets·C VolontéG Burnstock
Apr 25, 2013·PloS One·Chengqun SunRussell D Salter
Aug 15, 2014·Biomedical Journal·Ana Carolina MorandiniRobson Coutinho-Silva
Dec 18, 2014·Current Medicinal Chemistry·N R JørgensenM Ellegaard
Mar 18, 2015·Reproduction, Fertility, and Development·Jing WangChunping Zhang

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Methods Mentioned

BETA
flow cytometry
transfection
confocal microscopy

Software Mentioned

FACSuite
GraphPad Prism

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