The data presented within this Chapter provides information as to how the PH domain, C2 domain and EF-hand regulate the enzyme activity of PLCη2. The first step in the initial characterisation was the identification of a Ca2+-mobilising agent as a specific trigger for PLCη2 activity in a cellular environment (COS-7 cells). Unexpectedly, only monensin induced a significant elevation in PLCη2 activity of all Ca2+-mobilising agents tested (ionomycin, A23187, thapsigargin and bafilomycin). Kim et al. also demonstrated that ionomycin stimulation of the endogenous PLC- activity was retained after PLCη2 knock-down in Neuro-2a cells (Kim et al. 2011). Ca2+ ionophores ionomycin and A23187 might have been incapable of inducing the special spatial and/or temporal distribution of Ca2+ which was developed by monensin. It was demonstrated earlier that monensin is able to increase intracellular Ca2+ level by 2.3-fold in FRTL-5 thyroid cells by reversing the NCXs and this was coupled to increased PLC activity. This seems to confirm that the effect of monensin on PLCη2 is due to the elevated Ca2+ levels. Although, in this case the Ca2+- ionophores (ionomycin and A23187) should have had at least some effect on PLCη2 activity. To better understand the nature of Ca2+ dynamics following monensin stimulation Ca2+-chelation and non-specific inhibition of Ca2+ channels were used. In the presence of EGTA, PLCη2 activity was reduced to the control level. The compound SKF-96365, which is a non-specific inhibitor of the voltage- and receptor- operated Ca2+-entry (Merritt et al. 1990; Singh et al. 2010) also greatly reduced PLCη2 activity in transfected cells. These agents further demonstrated that the action of monensin on PLCη2 is clearly Ca2+-dependent. Depending on the localisation of PLCη2, its activation might be controlled by Ca2+release from a specific store. A specific inhibitor of the mitochondrial Na+/Ca2+-exchanger (CPG-3757) greatly
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interfered with the monensin-induced activity of PLCη2. This suggests that the monensin-induced Ca2+ release from mitochondria – either directly or indirectly- influenced PLCη2 activity. At least some of the inhibition of SKF-96365 on monensin-induced Ca2+-release was likely derived from its action on the Na+/Ca2+- exchanger. This was shown by the co-administration of SKF-96365 and CPG-3757 which resulted in only a slight additional inhibition. Collectively these results show that monensin is an activator of PLCη2 and in this activation mitochondrial Ca2+ release is likely to play an important role. However, it is plausible that monensin has no direct effect and other factors might transmit its effect on PLCη2 activity via an unknown mechanism.
The cellular distribution of the GFP-tagged PLCη2 protein highlighted that this enzyme may be present on both plasma- and organelle membranes. However this construct did not provide sufficient evidence that PLCη2 would primarily localise on mitochondrial membranes as was suggested by previous results. Interestingly, localisation of the same construct was found to be predominant on the plasma membrane in HeLa S3 cells (Nakahara et al. 2005). Immunostaining of the overexpressed PLCη2 yielded in a somewhat contradictory result placing the majority of PLCη2 onto the mitochondria. Moreover, mitochondrial labelling highlighted a morphological change in the intracellular structure of transfected COS- 7 cells. One possibility is that PLCη2 may act as a link between mitochondrial membranes directed by the homodimerisation of the molecule. PLCβ was previously found to compose dimers via its C-terminal extension (Singer et al. 2002). If PLCη2 is also able to form such structures, the dimer-partners may attack different membranes with their PH domains and the overexpression of PLCη2 may lead to the development of extended PLC-membrane complexes. In PLCη2-transfected COS-7 cells a considerably higher level of mitochondrial-staining was observed compared to untransfected cells. This suggests that the rate of mitochondrial fissure was elevated in transfected cells that might be caused by the high levels of PLCη2. Similar structural changes are detected when hFis1, a protein involved in mitochondrial
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fissure is overexpressed in HeLa cells (Frieden et al. 2004). The greater number of mitochondria and the high localisation of the overexpressed untagged PLCη2 on mitochondria-related membranes might partially explain why mitochondrial Ca2+ release possessed such a great effect on PLCη2 activity. ηηIn addition, the future assessment of the effect of PLCη2 on mitochondrial fissure might be of great importance and should be followed up in the future. Elevated mitochondrial fissure may be a hallmark of degenerative diseases such as Alzheimer`s Disease (Wang et al. 2009) so it is plausible that imbalanced PLCη2 activity also contribute to the development of age-related cell abnormalities.Nakahara et al. identified the first time that PLCη2 possesses a unique sensitivity towards Ca2+ in vitro. According to this study, PLCη2 is activated by a 10-fold lower Ca2+ concentration (1 µM) than PLCδ1. Unfortunately, results of this assay were not convincing due to the low level of activation and the high variance in values (Nakahara et al. 2005). My aim was to study Ca2+-sensitivity of PLCη2 in a cellular model providing the physiological environment necessary for the activation of the enzyme. Permeabilised COS-7 cells offered the desired tool whereby the Ca2+-sensitivity of wild-type and mutant PLCη2 proteins were quantitatively determined. PLCη2 enzyme activity displayed the greatest increase between 100 nM and 1 µM [Ca2+]. This renders the enzyme 10-fold less sensitive to Ca2+ than the sperm-specific PLCζ, although 10-fold more than PLCδ1 (Kouchi et al. 2005; Nakahara et al. 2005).