Impaired TRPM3-dependent calcium influx and restoration using Naltrexone in natural killer cells of myalgic encephalomyelitis/chronic fatigue syndrome patients

Transient receptor potential (TRP) channels are a superfamily of polymodal sensors present in many tissue and cell types, and their involvement in sensory reception is well known [1]. The TRPM3 (melastatin) channel acts as a non-selective cation channel that possesses high permeability for calcium (Ca²⁺) [2, 3]. The activation of TRPM3 channels leads to a cascade of intracellular pathways to facilitate cell function in both excitable and non-excitable cells [4]. TRPM3 shares the typical structure of all TRP channels with six transmembrane domains with both the N-terminal and C-terminal domains located within the cytosol. The endogenous neurosteroid, pregnenolone sulfate (PregS) (EC₅₀ = 12–32 µM) is commonly used in research to activate TRPM3 ion channel activity to induce an increase in intracellular Ca²⁺ concentration [5] while ononetin (IC₅₀ = 0.2–2 µM) rapidly and reversibly inhibits PregS-evoked ionic currents [6]. Changes in the function or activation of TRPM3 would result in changes to Ca²⁺ and as a consequence impacts cell function.

Ca²⁺ is a versatile, universal secondary messenger that facilitates vital biological processes in all cell types. Ca²⁺ ions are in part responsible for promoting intracellular signalling pathways, cell differentiation and proliferation, programmed cell death and transcriptional events in many cell types [7]. The intracellular concentration of Ca²⁺ ([Ca²⁺]_I) is tightly controlled in homeostatic conditions. Growing evidence suggests that the expression of TRPM3 on natural killer (NK) cells indicates a role of TRPM3 in the regulation of Ca²⁺ in immune cells [8‐11]. In addition to the processes described above, the influx of Ca²⁺ in NK cells is important for microtubule rearrangement resulting in granule polarisation, formation of the immune synapse, release of cytolytic granules and granzyme dependent target cell death [12, 13]. Therefore, disturbances in Ca²⁺ homeostasis in lymphocytes can adversely impact immune cell functions and consequentially lead to immune diseases and immunodeficiencies [14].

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe multisystemic illness hallmarked by post-exertional neuroimmune exhaustion accompanied by a range of symptoms that are broadly categorised as neurological, immunological, cardiovascular, gastrointestinal and endocrinological [15, 16]. The pathomechanism underlying ME/CFS is unknown, however immunological disturbances have been well reported with emphasis on NK cell dysfunction [17]. In 2016, five single nucleotide polymorphisms (SNPs) were identified in the TRPM3 gene (rs6560200, rs1106948, rs12350232, rs11142822, rs1891301) in NK cells from ME/CFS patient [18]. Subsequently, flow cytometry revealed a significant reduction in surface expression of TRPM3 and preliminary data reported significantly reduced Ca²⁺ mobilisation in NK cells of ME/CFS patients compared with healthy controls (HC) [8, 9]. Electrophysiological experiments have demonstrated a loss of TRPM3 ion channel function in NK cells of ME/CFS patients compared with HC [10, 11]. Thus, there is a growing body of evidence suggesting the pathomechanism of ME/CFS may be a channelopathy [8, 10, 19]. Therefore, a working hypothesis for the pathophysiology of ME/CFS is that impaired TRPM3 ion channel function limits the influx of Ca²⁺ and downstream signalling pathways, and consequentially impedes cell function.

Currently, no universal treatment exists for ME/CFS, instead treatment is aimed at alleviating targeted symptoms by administering central nervous system (CNS) stimulants, sleeping medications and analgesics [20]. Importantly, TRPM3 has been identified as a thermosensitive and nociceptive channel implicated in detecting pain and heat produced during inflammation with emphasis on heat hyperalgesia and general pain transmission in the CNS [21]. Moreover, the wide spread expression of TRPM3 in areas of the CNS including the cerebrum, brain stem, hypothalamus and hippocampus suggests a role in muscle coordination, cognitive behaviour and memory [8, 22]. These functional properties overlap considerably with symptoms of ME/CFS as epidemiological data has shown ME/CFS patients experience sensitivity for pain with up to 94% of patients reporting muscle aches and pains while 84% report multi-joint pain [23]. Therefore, TRPM3 dysfunction provides a potential therapeutic target for the treatment of ME/CFS.

Recent research has found that naltrexone (NTX) at low doses (LDN) improves the symptoms of ME/CFS [24]. Electrophysiology experiments in ME/CFS patients reported a potential benefit in restoring impaired TRPM3 ion channel activity following treatment of isolated NK cells with NTX [25]. Interestingly, NTX is a long-lasting antagonist to a subfamily of receptors known as opioid receptors (OR) used to treat opioid and alcohol dependence [26]. Mu-OR (µOR) belong to a large and diverse group of membrane receptors known as G protein coupled receptors (GPCRs). GPCRs are widely distributed in the human body where activated G proteins can interact and regulate many effectors or molecules such as Ca²⁺ sensors, ion channels and protein kinases [27]. The activity of TRPM3 can be inhibited by the activation of G protein subunits and direct binding of these subunits to the channel [28]. NTX specifically inhibits the µOR, thus negating these inhibitory effects on TRPM3 [28‐30]. Whole-cell patch clamp electrophysiology has been used to investigate the function of TRPM3 ion channels in ME/CFS patients who regularly administered LDN. TRPM3 function yielded similar results between ME/CFS patients taking LDN and HC [31]. As NTX restored TRPM3 ion channel function, it may in turn re-establish Ca²⁺ influx in NK cells leading to normalised downstream signalling pathways and immune functions.

While the function of TRPM3 ion channels have been determined in NK cells of ME/CFS patients using the whole-cell patch clamp technique, this current investigation was designed as a complementary study to evaluate Ca²⁺ influx in order to validate the loss of TRPM3 channel function using an additional method to ensure full potential of research. We aimed, for the first time, to investigate the speed and maximum response of TRPM3-dependent Ca²⁺ influx in ME/CFS patients compared with HC using an immunofluorescent technique. This current investigation provides further insights of the potential therapeutic role of NTX by examining the effect of overnight in vitro treatment on TRPM3-dependent Ca²⁺ influx.

We report, for the first time the significant reduction in Ca²⁺ influx via TRPM3 in NK cells in ME/CFS patients compared with HC using Ca²⁺ imaging technique. This current investigation provides novel findings for the rate, or speed, and insight into the maximum TRPM3-dependent Ca²⁺ influx response in ME/CFS patients. Previous investigations have demonstrated TRPM3 channel dysfunction in isolated NK cells of ME/CFS patients compared with HC using the whole-cell patch clamp technique [10, 11]. While patch-clamp is regarded as a gold standard technique for ion channel research, TRPM3-dependent Ca²⁺ influx was not clear in our previous studies due to the small inward currents activated by PregS. Therefore, our data supports the characterisation of Ca²⁺ influx and TRPM3 activity for the pathomechanism of ME/CFS and the role for NTX as a potential intervention. The combination of electrophysiology and Ca²⁺ imaging protocols provides a comprehensive and complete analysis of TRPM3-dependent Ca²⁺ changes in NK cells of ME/CFS patients.

This current investigation reports a significant reduction in the T1/2 and amplitude of maximum TRPM3-dependent Ca²⁺ responses in ME/CFS patients compared with HC at baseline. The reduction in TRPM3-dependent Ca²⁺ influx in ME/CFS patients compared with HC validates previous research which demonstrate significant loss in TRPM3 ion channel function [10, 11, 25]. Alterations in the profile of Ca²⁺ influx are strongly associated with significant physiological consequences. Changes in Ca²⁺ influx profiles play a critical role in determining the magnitude of Ca²⁺-dependent responses and are associated with physiological consequences [37]. It is hypothesised that TRPM3 ion channel dysfunction results in reduced Ca²⁺ influx in NK cells which has negative consequences on cytotoxic function in ME/CFS patients. Therefore, simultaneous reductions in amplitude and rate may augment effects of reduced Ca²⁺ concentration on cellular function and further research into Ca²⁺ mobilisation may elucidate the pathomechanism of ME/CFS.

Subsequently, the effect of IL-2 alone on Ca²⁺ influx was determined. The culturing of NK cells with the addition of IL-2 supports viability, preactivation, proliferation and development of cells; however, as IL-2 primes NK cells for activation this condition was used as a control for NTX treated cells [38]. The amplitude and T1/2 response were significantly reduced in IL-2 stimulated NK cells from ME/CFS patients compared with HC. Additionally, a significant reduction was reported for slope of Ca²⁺ influx in ME/CFS patients compared with HC. It is interesting that significance was reported for slope of Ca²⁺ influx following IL-2 stimulation, but not at baseline. Ca²⁺ measurements differed between baseline and IL-2 stimulation conditions. Stimulation of NK cells with IL-2 is known to enhance NK cell cytotoxic function through Ca²⁺-dependent pathways [39, 40]. In a recent publication, the authors suggested a crosstalk between TRPM3- and IL-2-dependent cellular pathways leading to enhanced NK cell function in vitro [41]. It is therefore likely that changes in slope reported between baseline and IL-2 stimulation that are reflected in HC indicate that pathways involved in TRPM3 and IL-2 signalling may promote Ca²⁺ mobilisation in NK cells. The consistent reduction in amplitude and T1/2 of response reported in this current investigation suggests that TRPM3 dysfunction reported in ME/CFS patients impairs sufficient Ca²⁺ entry in NK cells. Changes in ion channel function may be a consequence of an unstable open channel [42] and channel stability may be a component to consider in future research.

Ca²⁺ has a critical and beneficial role in promoting the activation of NK cell function as previous investigations using Ca²⁺ blockers have led to a significant reduction in NK cell cytotoxicity [43, 44]. The coupling between receptor and ligand initiates a Ca²⁺-dependent cascade that relies on sustained and long-term influx of the cation. Ca²⁺ facilitates the phosphorylation and activation of protein kinases such as Ras, P38, phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K) and mitogen activation protein kinases (MAPK) by enabling protein–protein and protein-phosphatase interactions [45‐47]. The significant reduction in phosphorylation of protein kinases has been reported in activated NK cells of ME/CFS patients [48]. Therefore, changes in Ca²⁺ can either enhance, or in the case of ME/CFS, impair protein kinase phosphorylation, thus impacting functional outcomes such as cytokine production and cytotoxicity. Moreover, phosphatidylinositol 4,5-bisphosphate (PIP₂) is a critical component of numerous signalling pathways including PI3K signalling and Ca²⁺ mobilisation. The function of TRPM3 also relies on the presence and binding of PIP₂ [49]. A recent publication reported a potential association between TRPM3 dysfunction and the involvement of PIP₂ in the pathomechanism of ME/CFS [41]. Therefore, TRPM3 dysfunction may contribute to ME/CFS pathomechanism due to consequences of impaired Ca²⁺ signalling, including impeding Ca²⁺-dependent cellular pathways that result in impaired NK cells function that is consistently reported in literature.

Under homeostatic conditions, the activation of TRPM3 leads to an increase in cytosolic Ca²⁺ that is important for regulating numerous biological processes including but not limited to temperature and pain sensation, insulin secretion and vascular smooth muscle contraction [50]. Furthermore, ion channels are located on different organelles throughout cells, and mitochondrial dysfunction reported in ME/CFS patients may be attributable to ion channel dysfunction, namely TRPM3 [51]. In NK cells, store-operated Ca²⁺ entry (SOCE) is a primary form of Ca²⁺ regulation [52]. However, it is possible that TRPM3 monitors intracellular Ca²⁺ levels in lymphocytes in addition to SOCE, as observed in oligodendrocytes [53, 54]. This current investigation did not aim to assess the activity of SOCE and therefore further investigation on the relationship between TRPM3 and SOCE in NK cells is proposed. It is important to outline that within the N-terminal of TRPM3 there are two calmodulin (CaM) binding sites. CaM senses changes in [Ca²⁺]_I to either up- or down-regulate TRPM3 activity [55, 56]. The interaction of CaM and TRPM3 suggests strong activity in Ca²⁺ sensing that may be augmented by Ca²⁺ store depletion or by stimulation of GPCRs [57]. The activity of CaM in TRPM3 function has not been investigated in ME/CFS patients but provides an interesting target for future research to determine any problems in TRPM3-dependent Ca²⁺ homeostasis.

Currently, there is no universal treatment to improve symptoms of ME/CFS. However, new technologies may provide future avenues to further assist in characterising TRPM3 dysfunction and impaired Ca²⁺ mobilisation in ME/CFS research. Recent electrophysiology investigations have reported the restoration of TRPM3 function following in vitro treatment of isolated NK cells with NTX [25]. In this current investigation we report the restoration of TRPM3-dependent Ca²⁺ influx following in vitro treatment of isolated NK cells overnight with NTX. The cellular mechanism involves the inhibition of µOR which would otherwise inhibit the activation of TRPM3 channels [28, 58]. Therefore, by negating the inhibitory action of µOR, it is hypothesised that this restores TRPM3 function, thus reinstating TRPM3-dependent Ca²⁺ influx in NK cells from ME/CFS patients.

The expression of µOR has been reported in lymphocytes where they are important in the inflammatory pain response [59]. The activation of µOR by certain endogenous and synthetic opioids leads to immunosuppression [60, 61, 61, 62]. Administration of the synthetic opioid, morphine, has resulted in a significant reduction in NK cell cytotoxic activity in rats, mice and humans [63‐65]. Interestingly, NTX inhibits the suppressive activity of morphine on NK cells through the µOR [66]. Research has reported that ME/CFS patients secrete insufficient opioid peptides that regulate pain and release of cytokines [31]. Beta-endorphins (β-EP) are an endogenous opioid neuropeptide produced by many cell types including lymphocytes. The release of β-EP from lymphocytes are regulated by the activity of µOR resulting in changes to cell proliferation and immune function [67]. Interestingly, within a specific dosage window of 1–5 mg/day, it is reported that LDN increases β-EP release in NK cells resulting in increased cytotoxic activity [68, 69]. An investigation by Conti and colleagues found significantly reduced β-EP in ME/CFS patients reflecting chronic immune activation [69]. Therefore, targeting OR, with NTX, on immune cells may resolve NK cell dysfunction through ion channel regulation such as the indirect effects seen with TRPM3. Future research may aim to investigate NK cell cytotoxicity in ME/CFS patients following in vitro treatment with NTX.

TRPM3 function has previously been assessed in a group of ME/CFS patients who routinely administer LDN daily [31]. ME/CFS patients who take LDN yielded similar TRPM3 function to HC and no significant differences between groups were observed [31]. Medical data from a cohort of 218 ME/CFS patients administering LDN reported a positive treatment response in 73.9% of responders [24]. In a separate investigation, self-reported retrospective comparison of ME/CFS symptoms before and after LDN commencement suggested an improvement in cognitive symptoms and immune disturbances [31]. Moreover, studies with fibromyalgia patients have found that LDN significantly reduced pain, fatigue, sleep disturbances, headaches and gastrointestinal issues [70]. Collectively, these results suggest there is potential therapeutic benefit of LDN to treat TRPM3 dysfunction in ME/CFS patients.

ME/CFS is a serious and chronic condition that affects multiple organ systems of the human body. TRPM3 dysfunction has been consistently found in NK cells of ME/CFS, with consequential implications for Ca²⁺ signalling and cell function. TRPM3-dependent Ca²⁺ influx was significantly reduced in NK cells of ME/CFS patients compared with HC. In this current investigation the treatment of isolated NK cells with NTX restored TRPM3-dependent Ca²⁺ influx in ME/CFS patients to the extent that Ca²⁺ influx measurements were either similar to HC or significantly increased. This investigation provides additional evidence for the potential therapeutic benefit of NTX for ME/CFS.