Contribution of the juxta-transmembrane intracellular regions to the time-course and permeation of ATP-gated P2X7 receptor ion channels.
journal contributionposted on 2015-05-12, 14:27 authored by Rebecca C. Allsopp, Richard J. Evans
P2X7 receptors are ATP gated ion channels that contribute to inflammation and cell death. They have the novel property of showing marked facilitation to repeated applications of agonist and the intrinsic channel pore dilates to allow passage of fluorescent dyes. A 60s application of ATP to hP2X7 receptors expressed in Xenopus oocytes gave rise to a current that had a biphasic time course with initial and secondary slowly developing components. A second application of ATP evoked a response with a more rapid time to peak. This facilitation was reversed to initial levels following a 10 minute agonist free interval. A chimeric approach showed replacement of the pre-TM1 amino terminal region with the corresponding P2X2 receptor section (P2X7-2Nβ) gave responses that quickly reached steady state and did not show facilitation. Subsequent point mutations of variant residues identified N16 and S23 as important contributors to the time-course/facilitation. The P2X7 receptor is unique in having an intracellular carboxyl terminal cysteine rich region (Ccys). Deletion of this region removed the secondary slowly developing current and when expressed in HEK293 cells ethidium bromide uptake was only ~5% of WT levels indicating reduced large pore formation. Dye uptake was also reduced for the P2X7-2Nβ chimera. Surprisingly, combination of the chimera and the Ccys deletion (P2X7-2NβdelCcys) restored current rise time and ethidium uptake to WT levels. These findings suggest there is a co-evolved interaction between the juxta-transmembrane amino and carboxyl termini in regulation of P2X7 receptor gating.
This work was supported by the Wellcome Trust (080487/Z/06/Z) and the Medical Research Council (MR/K027018/1).
CitationThe Journal of Biological Chemistry, 2015, 290 (23), 14556-14566
Author affiliation/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/School of Biological Sciences/Department of Cell Physiology and Pharmacology
- VoR (Version of Record)