Our previous investigations have proposed that NK cells from CFS/ME patients have significantly reduced expression of TRPM3 and subsequent reduction in intracellular Ca2+ mobilisation compared with HC (Nguyen et al., 2017; Nguyen et al., 2016). This present study used electrophysiological methods to characterise endogenous TRPM3 activity in peripheral NK cells from HC and CFS/ME patients. We provide evidence suggesting PregS-dependent channel activity for TRPM3 is significantly lower in CFS/ME patients compared with HC. Moreover, ionic currents in CFS/ME patients were resistant to ononetin in the presence of PregS.

The patch clamp technique is regarded as a gold standard for ion channel research and offers direct insight into ion channel properties through the characterization of ion channel activity. In this study, we characterised, for the first time, the TRPM3 ion channel current in isolated human NK cells. We report a significant reduction amplitude of TRPM3 current after PregS stimulation in isolated NK cells from CFS/ME patients compared with HC. This is consistent with our previous findings showing significantly reduced TRPM3 expression as well as significantly reduced intracellular Ca2+ mobilisation in isolated NK cells from CFS/ME patients compared with HC (Nguyen et al., 2017; Nguyen et al., 2016). In addition, we found PregS-evoked ionic currents through TRPM3 channels were significantly modulated by ononetin in isolated NK cells from HC compared with CFS/ME patients. Indeed, isolated NK cells from CFS/ME were resistant to ononetin suggesting that PregS may activate non-TRPM3 cationic currents in CFS/ME patients. Alternatively, CFS/ME patients may express different spliced isoforms of TRPM3 that are non-sensitive to ononetin. Although we demonstrate TRPM3 channel activity dysfunction in CFS/ME patients, further investigations are required to elucidate the mechanisms involved in the impaired TRPM3 channel activity as well as the different TRPM3 isoform types that are expressed in NK cells.

Previous electrophysiological investigations have shown that TRPM3 forms an ion channel permeable to Ca2+, sodium (Na+), magnesium (Mg2+), and manganese (Mn2+) (Grimm et al., 2005; Oberwinkler et al., 2005). Ca2+ plays an important role in intracellular signalling pathways, cell differentiation and division, apoptosis, and transcriptional events. In non-excitable cells, such as immune cells, a main Ca2+ entry pathway is known as store-operated Ca2+ entry (SOCE) and some TRP ion channels are associated with this pathway. While the sub-family TRPC have been traditionally associated with this important cellular mechanism (Cheng et al., 2013; Ong et al., 2016; Salido et al., 2009), recent research has also identified TRPM3 as a component for SOCE in white matter of the central nervous system (CNS) (Papanikolaou et al., 2017). Upon TRPM3 channel activation, changes in [Ca2+] i occur, resulting in the regulation of many biological processes that correspond to an array of cells expressing this channel. TRPM3 is located and linked to vascular smooth muscle contraction, modulation of glucose-induced insulin release from pancreatic islets, detection of noxious heat in dorsal root ganglia and development of epithelial cells of the choroid plexus, as well as function of oligodendrocytes and neurons (Hoffmann et al., 2010; Oberwinkler et al., 2005; Vriens et al., 2011; Wagner et al., 2008). Therefore, dysregulation of TRPM3 family, affecting SOCE and more generally, Ca2+ signalling has significant implications for cell regulatory machinery.

Significant reduction in NK cell cytotoxicity is a consistent feature reported in CFS/ME patients (Brenu et al., 2012; Brenu et al., 2011; Curriu et al., 2013; Hardcastle et al., 2015; Huth et al., 2016a; Klimas et al., 1990; Maher et al., 2005; Natelson et al., 2002; Nijs & Frémont, 2008; Sharpe et al., 1991; Siegel et al., 2006; Stanietsky & Mandelboim, 2010). NK cell cytotoxic activity is a Ca2+ dependent process, which drives the intracellular microtubule reorganisation, polarisation of cytoplasmic granules, release of lytic proteins and the creation of the immune synapse (Anasetti et al., 1987; Henkart, 1985). Moreover, Ca2+-dependent cytotoxic processes allow for the production and recruitment of lytic proteins (Voskoboinik et al., 2015). Following cytotoxic granule delivery to the immune synapse, the formation of perforin pores and granzyme-induced cell apoptosis are highly dependent on Ca2+ (Orrenius et al., 2003; Voskoboinik et al., 2005). Previous studies have reported impaired Ca2+ signalling in NK cells from CFS/ME patients demonstrated through changes to ERK1/2 and mitogen-activated protein kinase (MAPK) pathways (Chacko et al., 2016; Huth et al., 2016b). CFS/ME patients have significantly decreased ERK1/2 following incubation with K562 cells (Huth et al., 2016b). ERK1/2 is activated in a Phosphatidylinositol-4,5-bisphosphate 3-kinase (P1 3 K)-dependent manner that may also be associated with cytoplasmic Ca2+ ion levels through activation of TRPM3 (Lee et al., 2003). In the absence of phosphatidylinositol 4,5-biphosphate (PIP 2 ), TRPM3 is not activated, resulting in reduced cytosolic Ca2+ (Tóth et al., 2015). Previous research completed by Nguyen and colleagues reported that the expression of TRPM3 ion channel and [Ca2+] i were significantly reduced in NK cells from CFS/ME patients (Nguyen et al., 2017). In addition, ERK1/2 requires Ca2+ as the final activator to initiate NK cell lysis (Huth et al., 2016b). Changes in Ca2+ signalling may impair cytokine production, including Interferon (IFN)-γ and Tumor Necrosis Factor (TNF), therefore interfering with systemic inflammation and anti-tumour responses (Romee et al., 2013). Previous investigations have reported both an increased and decreased inflammatory profile of CFS/ME patients along with reduced IFN-γ (Klimas et al., 1990; Lorusso et al., 2009). TRPM3-related Ca2+ dysfunction may then result in a reduction of [Ca2+] i , which may lower the function and cytotoxic capacity of the NK cells in CFS/ME patients.

Importantly, TRPM3 ion channels have a role in the detection of heat and in pain transmission in the CNS (Held et al., 2015). TRPM3 has been previously identified as a nociceptor channel involved in acute heat sensing and inflammatory heat hyperalgesia, and thus as a potential target for analgesic treatments (Vriens et al., 2011). Dysregulation of thermoregulatory responses has been reported in CFS/ME patients (Wyller et al., 2007). Generalised pain is a characteristic of CFS/ME and occurs in the absence of overt tissue damage, and this is suggestive of potential CNS impairments (Barnden et al., 2015; Barnden et al., 2016; Shan et al., 2016; Shan et al., 2017). Our present findings suggest TRPM3 ion channels may be involved in the pathomechansim of CFS/ME and hence have a possible role in nociception and thermoregulation.

While this study provides evidence of TRPM3 channel activity dysfunction in CFS/ME patients, this study is not without limitations with the low sample numbers. Indeed, these findings need to be validated in a larger cohort of patients. Additionally, the use of high and single doses of PregS and Ononetin may reduce the drugs specificity and selectivity and consequently modulate other non-TRPM3 cationic currents. Further investigations are also required to assess the impact of PregS and Ononetin on the different TRPM3 isoforms. This could explain the differences observed between HC and CFS/ME patients as well as within groups. Finally, TRPM4, TRPM5, TRPM2 and TRPM7 surface expression has been reported on B cells, bone marrow cells, splenic cells, lymph node B cells and T and mast cells (Zierler et al., 2017), suggesting our recent findings may also be pertinent to other TRPM channel functions and Ca2+ − mediated roles, such as SOCE.