TRP channels and a dextran sulfate activated rise in intracellular Ca2+ and Na+ in a subpopulation of double positive thymocytes

Abstract

This thesis presents the characteristics of the sustained rise of intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i), observed in pre-selection CD4+CD8+ (double-positive, DP) thymocytes when stimulated with dextran sulfate (DxS) and explores several candidate channels that may be involved in this rise. . This [Ca2+]i rise is proposed to mimic a physiological mechanism that influences early DP thymocyte selection and hence the T cell repertoire. During thymopoiesis, the patterning of [Ca2+]i plays a pivotal role in selection of thymocytes. In 1987, Tellam & Parish described a sustained Ca2+ rise in DP thymocytes which depended on a transmembrane influx of Ca2+. The channel through which this Ca2+ entered remained elusive.Understanding the characterisation of this rise and its underlying signalling and conductance is pivotal. I have used flow cytometry to concomitantly image [Ca2+]i and [Na+]i after DP thymocytes were exposed to 1 ug/ml DxS (500 kDa) using AM-loading of the indicators indo-1 and ANG-2 or SBFI (5 uM for all). In some instances, Mg2+ was monitored with magfluo-4. The rises were evaluated when cells were exposed to activators and blockers of receptors, cell signalling enzymes and channels. In addition, immunocytochemistry (ICC) was employed to determine if the channel was on the cell surface. In Chapter 3, I found that the earlier described Ca2+ was also accompanied by a Na+ rise. There was a delay to half-maximal amplitude of 10.8 +/- 0.2 min and a rate of ~5 min for both, and an amplitude of 165 +/- 11 nM for Ca2+. The one for Na+ could not be quantified but is likely in the range of 10 - 15 mM suggesting that a non-selective cation channel is involved that predominantly passes Na+ over Ca2+. Notably, the DxS rises are prevented by a maintained elevation of [Ca2+]i >~60 nM, but not by a transient rise. Additionally, the rises were absent if the recording temperature was <30C and if [Mg2+]o was >25 mM. Activated by DxS, critical elements in the signalling cascade leading to channel opening include CD8b and LFA-1 as the rises were absent in thymocytes from CD8b and LFA-1KO mice. Furthermore, activation of the Src tyrosine kinase Lck also appears essential as PP2 abolished the rises. Downstream of Lck, both PI3K and PLC activation is necessary as their inhibition by LY294002 and edelfosine, respectively, abolished the rises. The rises do not involve store release via IP3 receptors and SOCE, suggesting that either PIP2 hydrolysis and/or DAG generation lead to channel activation. Both, disruption of F-actin polymerization and the tarantula toxin GsMTx4 abolished the rises, suggestive that mechanosensitive stimulation is involved. All these observations indicate that a non-specific cation channel of the TRP family could be involved. In Chapter 4, commencing with a list of 14 TRP channels that share several of the above properties, ICC and flow cytometry experiments are presented that indicate that the DAG-sensitive channels TRPC6 and TRPA1 are expressed and activatable in the membrane of these cells. However, none of them underlies the rises, as they were neither blocked by SKF96365, HC030031 or AMG9810, respectively. Rather, I observed that 2-APB potentiated the rises and ruthenium red abolished them, suggestive of involvement of either TRPV2, TRPV3 or TRPM6. Since the rises were neither reduced by tranilast (TRPV2 blocker) nor NS8593 (TRPM6 blocker) it strongly suggests an involvement of TRPV3 in the cation rise in DP thymocytes. In support of this notion is the sensitization by 2-APB and temperature (at 30C) which when used independently do not cause a rise. But I cannot rule out the possibility that, upon DxS addition, the signalling cascade leading to TRPV3 activation also transiently activates a mechanosensitive channel such as piezo1. Such a Ca2+ rise, activated by a signalling cascade triggered by heparan sulfate binding to CD8b receptors, could synergistically enhance TCR signalling and fine tune selection.