ORIGINAL RESEARCH

published: 31 October 2019

doi: 10.3389/fimmu.2019.02545

Naltrexone Restores Impaired

Transient Receptor Potential
Melastatin 3 Ion Channel Function in
Natural Killer Cells From Myalgic
Encephalomyelitis/Chronic Fatigue
Syndrome Patients
Helene Cabanas 1,2,3*, Katsuhiko Muraki 3,4 , Donald Staines 1,2,3 and
Sonya Marshall-Gradisnik 1,2,3
1
School of Medical Science, Griffith University, Gold Coast, QLD, Australia, 2 The National Centre for Neuroimmunology and
Emerging Diseases, Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia, 3 Consortium Health
International for Myalgic Encephalomyelitis, National Centre for Neuroimmunology and Emerging Diseases, Griffith University,
Edited by: Gold Coast, QLD, Australia, 4 Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi-Gakuin University, Nagoya,
Jorge Matias-Guiu, Japan
Complutense University of
Madrid, Spain
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a seriously long-term
Reviewed by:
José Miguel Urra, and debilitating illness of unknown cause hallmarked by chronic pain and fatigue,
Hospital General Universitario de memory and concentration impairment, and inflammation. ME/CFS hypothesis involves
Ciudad Real, Spain
Carmen Scheibenbogen,
impaired Transient receptor potential melastatin 3 (TRPM3) ion channel function,
Charité Medical University of affecting calcium signaling and Natural killer (NK) cell functions. Currently, substances
Berlin, Germany called opioids, agonists of mu (µ)-opioid receptors (µOR), are the strongest painkillers
*Correspondence: clinically available for people suffering from strong or long-lasting pain characteristic of
Helene Cabanas
[email protected] ME/CFS. µOR have been reported to specifically inhibit TRPM3 and to be expressed
in immune cells where they play an immunomodulatory and immunosuppressive role.
Specialty section: Naltrexone hydrochloride (NTX) acts as an antagonist to the µOR thus negating
This article was submitted to
Multiple Sclerosis and the inhibitory function of this opioid receptor on TRPM3. Therefore, understanding
Neuroimmunology, the mechanism of action for NTX in regulating and modulating TRPM3 channel
a section of the journal
function in NK cells will provide important information for the development of effective
Frontiers in Immunology
therapeutic interventions for ME/CFS. Whole-cell patch-clamp technique was used to
Received: 07 August 2019
Accepted: 14 October 2019 measure TRPM3 activity in Interleukin-2 (IL-2) stimulated and NTX-treated NK cells
Published: 31 October 2019 for 24 h on eight ME/CFS patients and 8 age- and sex-matched healthy controls,
Citation: after modulation with a TRPM3-agonist, pregnenolone sulfate (PregS), NTX and a
Cabanas H, Muraki K, Staines D and
Marshall-Gradisnik S (2019)
TRPM3-antagonist, ononetin. We confirmed impaired TRPM3 function in ME/CFS
Naltrexone Restores Impaired patients through electrophysiological investigations in IL-2 stimulated NK cells after
Transient Receptor Potential
modulation with PregS and ononetin. Importantly, TRPM3 channel activity was restored
Melastatin 3 Ion Channel Function in
Natural Killer Cells From Myalgic in IL-2 stimulated NK cells isolated from ME/CFS patients after incubation for 24 h with
Encephalomyelitis/Chronic Fatigue NTX. Moreover, we demonstrated that NTX does not act as an agonist by directly
Syndrome Patients.
Front. Immunol. 10:2545.
coupling on the TRPM3 ion channel gating. The opioid antagonist NTX has the potential
doi: 10.3389/fimmu.2019.02545 to negate the inhibitory function of opioid receptors on TRPM3 in NK cells from

Frontiers in Immunology | www.frontiersin.org 1 October 2019 | Volume 10 | Article 2545

Cabanas et al. Naltrexone and TRPM3 in ME/CFS

ME/CFS patients, resulting in calcium signals remodeling, which will in turn affect cell
functions, supporting the hypothesis that NTX may have potential for use as a treatment
for ME/CFS. Our results demonstrate, for the first time, and based on novel patch clamp
electrophysiology, potential pharmaco-therapeutic interventions in ME/CFS.

Keywords: transient receptor potential melastatin 3, naltrexone, calcium, opioid receptor, myalgic
encephalomyelitis/chronic fatigue syndrome, natural killer cells, whole-cell patch clamp electrophysiology

INTRODUCTION pumps, and exchangers located on the plasma membrane and/or

on organelles. Disturbance of this tightly regulated homeostatic
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome system leads to disorders of Ca2+ metabolism and immune cell
(ME/CFS) is a severe systemic, acquired condition of unknown functions playing a pivotal role in the pathophysiology of several
cause characterized by persistent or recurrent incapacitating diseases and immunodeficiencies (24).
fatigue accompanied by a range of multi-system manifestations One major contributor to Ca2+ signaling is the large
(1). Currently, there are no confirmatory diagnostic tests or and diverse family of Transient Receptor Potential (TRP)
accepted specific treatments. Diagnosis is currently based non-selective cation channels, which function as polymodal
on the Canadian Consensus Criteria (CCC, 2003) and more cellular sensors involved in the fine-tuning of many biological
recently on the International Case Criteria (ICC, 2011), processes in both excitable and non-excitable cells (25).
which identifies symptoms of post-exertional malaise, fatigue The mammalian TRP melastatin (TRPM) subfamily is the
unrelieved by rest, headache, joint and muscle pain, memory and largest, consisting of eight members broadly expressed in
concentration impairment, sore throat, and lymph gland swelling a variety of cells and tissues, such as sensory ganglia, the
as components of the illness (2). Additionally, ME/CFS exhibits central nervous system (CNS), pancreatic beta cells, immune
neuro-cognitive, autonomic, cardiovascular, neuro-endocrine, cells, cardiovascular system and renal system, and are critical
and immune manifestations. for sensory and motor physiology (26). Specifically, TRPM
Although the etiology of ME/CFS remains elusive, immune member 3 (TRPM3) is a Ca2+ -permeable nonselective cation
dysfunction and abnormalities in natural killer (NK) cell channel activated by a vast array of stimuli in the environment,
functions are the most consistent laboratory immunological ranging from temperature, natural chemicals, and toxins or
features (3–18). NK cells are lymphocytes of the innate immune synthetic compounds, including the endogenous neurosteroid
system that control pathogens and tumor cells by limiting their pregnenolone sulfate (PregS), and the L-type voltage-gated
spread and subsequent tissue damage under the stimulation of Ca2+ channel inhibitor, nifedipine, to mechanical stimuli.
the endogenous interleukin-2 (IL-2) and interleukin-12 (IL-12), TRPM3 channels also respond to endogenous agents and
in addition to immune cell activation and cytokine production messengers produced during tissue injury and inflammation
(19). While there is an inconsistency in some immunological (27). Importantly, TRPM3 has been previously identified
reports, differences in NK cell phenotypes and significantly as a thermosensitive and nociceptor channel implicated in
reduced peripheral NK cell numbers resulting in significant the detection of acute heat sensing and inflammatory heat
reduction in NK cell cytotoxicity, have been reported in ME/CFS hyperalgesia as well as in pain transmission in the CNS
and implicated in disease severity (3–18). (27). TRPM3 is therefore involved in several pain-related
Importantly, NK cells require calcium (Ca2+ ) for efficient pathological conditions/modalities, including inflammatory
stimulation and functions including NK cell cytotoxicity (20–23). or neuropathic pain, and thus is identified as a potential
Intracellular Ca2+ concentration ([Ca2+ ]i ) is tightly regulated by target for analgesic treatments. Moreover, dysregulation of
numerous actors including selective and non-selective channels, thermoregulatory responses has been reported in ME/CFS
patients and generalized pain in the absence of overt tissue
damage is a characteristic of ME/CFS suggesting potential CNS
Abbreviations: 7-AAD, 7-amino-actinomycin; βEP, Beta endorphins; δOR, impairments (1).
Delta opioid receptor; µOR, Mu opioid receptor; BMI, Body Mass Index;
Ca2+ , Calcium; [Ca2+ ]i , Intracellular Ca2+ concentration; CCC, Canadian
Our previous studies have reported a loss of TRPM3 ion
Consensus Criteria; CDC, Centers for Disease Control and Prevention; CNS, channel to be associated with ME/CFS (28, 29). A previous
Central nervous system; EDTA, Ethylendiaminetetraacetic acid; FBS, Fetal bovine genomic study suggested the importance of TRPM3 in the
serum; HC, Healthy controls; ICC, International Consensus Criteria; IL-2, pathophysiology of ME/CFS and identified five single nucleotide
Interleukin-2; IL-12, Interleukin-12; I-V, Current–voltage relationship; ME/CFS,
polymorphisms (SNPs) (rs6560200, rs1106948, rs12350232,
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome; NCNED, National Centre
for Neuroimmunology and Emerging Diseases; NK, Natural killer; NTX, rs11142822, rs1891301) in TRPM3 genes in ME/CFS patients
Naltrexone hydrochloride; PBMCs, Peripheral blood mononuclear cells; PIP2 , (30). Significant reduction in TRPM3 surface expression and
Phosphatidylinositol 4,5-bisphosphate; PregS, Pregnenolone sulfate; RPMI, Ca2+ mobilization in immune cells were subsequently reported
Roswell Park Memorial Institute medium; SF-36, 36-Item Short Form Survey; in ME/CFS patients (31, 32). Recently, novel electrophysiological
SNPs, Single nucleotide polymorphisms; TRP, Transient receptor potential; TRPM,
investigations used whole-cell patch clamp techniques to report a
Transient receptor potential melastatin; TRPM3, Transient receptor potential
melastatin 3; WHODAS, World Health Organization Disability Assessment significant reduction in TRPM3 ion channel activity after PregS
Schedule. and nifedipine stimulation in NK cells from ME/CFS patients

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

(28, 29). Moreover, ionic currents in ME/CFS patients were development of effective therapeutic interventions. In addition,
resistant to ononetin in the presence of PregS and nifedipine. doses of 3–5 mg per day of LDN have been used to treat a
Consequently, dysregulation of TRPM3 function in ME/CFS variety of diseases, including ME/CFS (42, 43). However, there
patients, affecting [Ca2+ ]i and Ca2+ signaling has significant is no literature confirming the efficacy of NTX for ME/CFS
implications for NK cell regulatory machinery and functions, patients, hence the usefulness of this drug in this condition
and represents a novel and attractive therapeutic target of is yet to be determined. Additionally, the mechanisms of
ME/CFS pathology. action for NTX in immune cells and in ME/CFS pathology
There are few treatments available for people suffering from require further investigation. As TRPM3 ion channel function
severe or long-lasting pain characteristic of ME/CFS. Currently, is impaired in NK cells from ME/CFS patients (28, 29), in the
substances called opioids, agonists of mu (µ)-opioid receptors present study we sought to investigate the ability of NTX to
(µOR), are the strongest painkillers clinically available (33). restore TRPM3 ion channel function in ME/CFS using whole
Opioids mediate their effects by interacting with molecules cell patch-clamp techniques. Restoration of TRPM3 function
that belong to a group of receptor proteins called G-protein is required to promote Ca2+ signal remodeling in NK cells.
coupled receptors (GPCRs). These opioid receptors are widely Our results indicate that NTX restores TRPM3-like ionic
distributed in the CNS with the role of detecting and transmitting currents in IL-2 stimulated NK cells from ME/CFS patients
pain signals (33). It was poorly understood how activation of following 24 h incubation. Our findings also indicate that NTX
opioid receptors reduces the activity of pain-sensing nerve cells, does not affect TRPM3-dependent Ca2+ signals when directly
however recent literature suggests that activation of GPCRs applied on both IL-2 stimulated NK cells from non-fatigued
can affect TRPM3 channels and in turn decrease the flow of controls and ME/CFS patients, suggesting that NTX does not
Ca2+ ions through the pore (33–35). GPCRs interact with G- act as an agonist by directly coupling on the TRPM3 ion
proteins that, when activated by the receptor, release the Gβγ channel gating mechanism. In conclusion, our novel findings
dimers from Gα subunits of the Gi/o subfamily. Inhibition of indicate that NTX has the potential to restore the TRPM3
TRPM3 activity by stimulation of GPCRs (in particular µORs) channel activity in ME/CFS patients resulting in Ca2+ signal
is mediated through a direct binding of the Gβγ subunit to the remodeling, which will in turn affect cell functions, supporting
ion channel (34). These recent findings show that drugs already the hypothesis that NTX may have potential for use as a treatment
used in the treatment of pain can indirectly alter TRPM3 function for ME/CFS.
significantly (33).
Naltrexone hydrochloride (NTX) is a long-lasting opioid MATERIALS AND METHODS
antagonist used commonly in the treatment of opioid and
alcohol dependence (36). NTX specifically inhibits µORs and, Participant Recruitment
to a lesser extent, the delta (δ)-opioid receptors (δOR), thus Eight ME/CFS patients and 8 age- and sex-matched healthy
negating the inhibiting effects of opioid receptors agonists controls (HC) were recruited using the National Center for
(37, 38). A recent investigation demonstrated that naloxone, Neuroimmunology and Emerging Diseases (NCNED) research
a rapid response alternative to naltrexone, did not have a database between April and June 2019. Participants were aged
direct effect on TRPM3-dependent Ca2+ signals in mouse dorsal between 18 and 60 years. All ME/CFS patients had previously
root ganglion neurons (33). However, when co-applied with received a confirmed medical diagnosis and were screened
DAMGO, a highly selective µOR agonist, naloxone prevented using a comprehensive questionnaire corresponding with the
the action of DAMGO completely, indicating a possible role Centers for Disease Control and Prevention (CDC), CCC,
for naloxone in influencing TRPM3 signaling. Interestingly, and ICC case definitions. All eight ME/CFS patients were
TRPM3 activation by nifedipine and PregS was also inhibited defined by the CCC. HC reported no incidence of fatigue and
by µOR activation confirming that TRPM3 inhibition is an were in good health without evidence of illness. Participants
important consequence of peripheral µOR activation (33, 35). were excluded from this study if they were pregnant or
Moreover, it has been suggested that treatment with low- breastfeeding, or reported a previous history of smoking, alcohol
dose naltrexone (LDN) can act as an immunomodulator and abuse or chronic illness (for example, autoimmune diseases,
may be beneficial for a range of inflammatory conditions, cardiac diseases, and primary psychological disorders) or were
including Crohn’s disease, multiple sclerosis, and fibromyalgia morbidly obese (BMI ≥ 30). No participants reported use of
(39–41). Previous studies also report therapeutic effects of opioids or any other pain killers in the preceding 3 months
LDN in treatment for cancers including B cell lymphoma as well as pharmacological agents that directly or indirectly
and pancreatic cancer, as well as chronic pain syndromes, influence TRPM3 or Ca2+ signaling. This investigation was
malignancies and mental health disorders by reducing pain, approved by the Griffith University Human Research Ethics
fatigue, sleep disturbances, headaches and gastrointestinal Committee (HREC/15/QGC/63).
conditions (42).
As ME/CFS is potentially a TRP ion channel disorder Peripheral Blood Mononuclear Cell
resulting from impaired TRPM3 ion channel function (28– Isolation and Natural Killer Cell Isolation
32), understanding the mechanism(s) involved in regulating A total of 85 ml of whole blood was collected in
and modulating ion channel function will provide potentially ethylendiaminetetraacetic acid (EDTA) tubes between 8:30
important information and lead to novel targets for the and 10:30 a.m. on the Gold Coast, QLD, Australia. Routine

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

full blood analysis was performed within 4 h of collection for +110 mV, before returning to +10 mV (repeated every 10 s).
red blood cell count, white blood cell count, and granulocyte The liquid junction potential between the pipette and bath
cell count. solutions (+10 mV) was corrected when data were analyzed. A
Samples were provided to the laboratory de-identified using leak current component was not subtracted from the recorded
a unique code by an independent blood collector. Peripheral currents. Electrode was filled with the intracellular pipette
blood mononuclear cells (PBMCs) were isolated from 80 ml of solution containing 30 mM CsCl, 2 mM MgCl2 , 110 mM L-
whole blood by centrifugation over a density gradient medium Aspartic acid, 1 mM EGTA, 10 mM HEPES, 4 mM ATP, 0.1 mM
(Ficoll-Paque Premium; GE Healthcare, Uppsala, Sweden) as GTP, adjusted pH to 7.2 with CsOH and osmolality of 290
previously described (3, 44). PBMCs were stained with trypan mOsm/L with D-mannitol. The pipette solution was filtered
blue (Invitrogen, Carlsband, CA, USA) to determine cell count using a 0.22 µm membrane filter (Sigma-Aldrich, St. Louise, MO,
and cell viability. PBMCs were adjusted to a final concentration USA), divided into aliquots and stored at −20◦ C. Bath solution
of 5 × 107 cells/ml for NK cell isolation. contained: 130 mM NaCl, 10 mM CsCl, 1 mM MgCl2 , 1.5 mM
NK cells were isolated by immunomagnetic selection using CaCl2 2H2 O, 10 mM HEPES, adjusted pH to 7.4 with NaOH
an EasySep Negative Human NK Cell Isolation Kit (Stem Cell and osmolality 300 mOsm/L with D-glucose. All reagents were
Technologies, Vancouver, BC, Canada). NK Cell purification was purchased from Sigma-Aldrich, except for ATP and GTP that
determined using flow cytometry. NK cells were incubated for were purchased from Sapphire Bioscience. TRPM3 currents on
20 min at room temperature in the presence of CD56 FITC (0.25 non-treated or NTX-treated NK cells were stimulated by adding
µg/5 µl) and CD3 PE Cy7 (0.25 µg/20 µl) monoclonal antibodies 100 µM PregS (Tocris Bioscience, Bristol, UK) or 200 µM NTX
(BD Bioscience, San Jose, CA, USA) as previously described (32). hydrochloride (Sigma-Aldrich, St. Louise, MO, USA) to the bath
7-amino-actinomycin (7-AAD) (2.5 µl/test) (BD Bioscience, San solution, whereas PregS- and NTX-induced TRPM3 currents
Jose, CA, USA) was used to determine cell viability. Cells were were blocked by adding 10 µM ononetin (Tocris Bioscience,
washed and resuspended in 200 µl of stain buffer (BD Bioscience, Bristol, UK). Therefore, three different protocols have been
New Jersey, NJ, USA) and acquired at 10,000 events using the performed for each of the 8 ME/CFS patients and 8 age-
LSRFortessa X-20 (BD Biosciences, San Diego, CA, USA). Using and sex-matched HC. For each protocol, 4 different recordings
forward and side scatter, the lymphocyte population was gated were made. Hence, N refers to number of participants in each
while acquiring the sample. The NK cell population was then group (ME/CFS patients or HC) and n, to number of readings
identified as CD3− CD56+ cells. or sample size for each different protocol. All measurements
were performed at room temperature. The authors removed the
Interleukin-2 Stimulation and Drug possibility of chloride current involvement in TRPM3 assessment
Treatment by using L-Aspartic acid in the intracellular pipette solution.
Freshly isolated NK cells were stimulated with 20 IU/ml of Cells which have unstable currents were also excluded from
recombinant human IL-2 (specific activity 5 × 106 IU/mg) the analysis.
(Miltenyi Biotech, BG, Germany) in combination or not
with 200 µM NTX (Sigma-Aldrich, St. Louise, MO, USA) as Statistical Analysis
previously described (36), at a concentration of 2 × 105 cells/ml Lymphocyte populations were identified using forward and
and incubated for 24 h at 37◦ C with 5% CO2 in Roswell side scatter dot plots. Exclusions were CD3+ cells and only
Park Memorial Institute medium (RPMI)-1640 (Invitrogen Life CD3− lymphocytes were further used to characterize NK
Technologies, Carlsbad, CA, USA) supplemented with 10% fetal cell subset populations using CD56 and CD16. NK cell
bovine serum (FBS) (Invitrogen Life Technologies, Carlsbad, CA, subsets were characterized using the surface expression of
USA). NTX was freshly resuspended in endotoxin-free water CD56Bright CD16Dim/− NK cells and CD56Dim CD16Bright/+ NK
before each experiment. cells. Cytometry data was exported from FacsDiva v8.1 and
analyzed using SPSS v24 (IBM Corp, Version 24, Armonk,
Whole Cell Electrophysiology Recording NY, USA) and GraphPad Prism v7 (GraphPad Software Inc.,
Electrophysiological recordings were performed with borosilicate Version 7, La Jolla, CA, USA). Electrophysiological data were
glass capillary electrodes with an outside diameter of 1.5 mm and analyzed using pCLAMP 10.7 software (Molecular Devices,
inside diameter of 0.86 mm (Harvard Apparatus, Holliston, MA, Sunnyvale, CA, USA). Origin 2018 (OriginLab Corporation,
USA). Pipette resistance when filled with pipette solution was Northampton, MA, USA) and GraphPad Prism v7 (GraphPad
8–12 MΩ. The pipettes were mounted on a CV203BU head- Software Inc., Version 7, La Jolla, CA, USA) were used for
stage (Molecular Devices, Sunnyvale, CA, USA) connected to a statistical analysis and data presentation. Shapiro-Wilk normality
3-way coarse manipulator and a micro-manipulator (Narishige, tests were conducted to determine the distribution of data,
Tokyo, Japan). Electrical signals were amplified and recorded in addition to skewness and kurtosis tests to determine data
using an Axopatch 200B amplifier and PClamp 10.7 software normality. Statistical comparison was performed using the
(Molecular Devices, Sunnyvale, CA, USA). Data were filtered independent non-parametric Mann-Whitney U test (Table 1,
at 5 kHz and sampled digitally at 10 kHz via a Digidata 1440A Figures 1, 2, 4, 6), and Fishers exact test (Figures 3, 5, 7), to
analog to digital converter (Molecular Devices, Sunnyvale, CA, determine any significant differences. Significance was set at
USA). The voltage-ramp protocol was a step from a holding p < 0.05 and the data are presented as mean ± SEM unless
potential of +10 mV to −90 mV, followed by a 0.1 s ramp to otherwise stated.

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

TABLE 1 | Blood parameters and patient demographic. Effects of Successive Applications of

HC ME/CFS P-value
PregS on TRPM3 Activity
Electrophysiology recordings were obtained from isolated
Age (years) 34.75 ± 3.94 40.25 ± 4.66 0.442 human NK cells incubated for 24 h with IL-2 using whole-cell
Gender (%) F (100%) F (100%) 1.000 patch-clamp technique. Endogenous TRPM3 channel activity
BMI (kg/m2 ) 23.39 ± 1.17 23.78 ± 1.42 1.000 was stimulated by two successive applications of 100 µM
WHODAS 2.96 ± 1.05 24.88 ± 4.31 0.000 PregS enabling measurement of a small outwardly rectifying
SF-36 current under voltage-clamp conditions and observation of a
General health (%) 78.64 ± 6.07 24.16 ± 5.31 0.000 typical shape of the TRPM3 current–voltage relationship (I–
Physical functioning (%) 92.50 ± 5.43 34.38 ± 5.63 0.000 V) (Figure 2). Indeed, we measured a small ionic current
Role physical (%) 79.38 ± 13.58 28.13 ± 11.21 0.038 with a typical TRPM3-like outward rectification in NK cells
Role emotional (%) 96.88 ± 3.13 79.16 ± 8.33 0.083 isolated from HC after both successive additions of PregS
Social functioning (%) 92.19 ± 6.22 26.69 ± 4.05 0.003 (Figures 2B,C). In contrast and as previously reported (28,
Body pain (%) 87.81 ± 5.42 50.94 ± 7.90 0.000 29), the outward ionic current amplitudes were significantly
Pathology decreased after successive PregS stimulations in NK cells from
White cell count (×109 /L) 5.51 ± 0.31 6.24 ± 0.30 0.130
ME/CFS patients in comparison to HC (Figures 2E–G) (p <
Lymphocytes (×109 /L) 1.69 ± 0.13 2.03 ± 0.15 0.130
0.0001 and p = 0.0001). These results validate the previous
Neutrophils (×109 /L) 3.19 ± 3.15 3.60 ± 0.27 0.195
findings and report that TRPM3 channel activity is impaired after
Monocytes (×109 /L) 0.4 ± 0.03 0.38 ± 0.03 0.645
successive PregS applications on IL-2 stimulated NK cells from
Eosinophils (×109 /L) 0.16 ± 0.04 0.19 ± 0.05 0.645
ME/CFS patients.
Basophils (×109 /L) 0.05 ± 0.01 0.05 ± 0.01 0.645
Platelets (×109 /L) 239.25 ± 13.54 252.00 ± 16.27 0.505
Modulation of PregS- Evoked Currents
12
Red cell count (×10 /L) 4.57 ± 0.06 4.36 ± 0.12 0.161 With Ononetin
Haematocrit 0.41 ± 0.01 0.40 ± 0.01 0.505 Ononetin effectively inhibits PregS-induced Ca2+ -influx and
Hemoglobin (g/L) 135.75 ± 2.45 133.50 ± 3.35 0.721 ionic currents through TRPM3 ion channels (47). Therefore, to
confirm that TRPM3 activity is involved in ionic currents evoked
SF-36 scores were analyzed using participant questionnaire responses. Results from by PregS in NK cells, we next used 10 µM ononetin to modulate
routine full blood were analyzed in ME/CFS patients and HC. Data presented as
mean ± SEM. ME/CFS, myalgic encephalomyelitis/chronic fatigue syndrome; HC,
the TRPM3 ion channels (Figure 3). As previously shown (28,
healthy controls; BMI, body mass index; WHODAS, World Health Organization Disability 29), the ionic currents evoked by both successive applications
Assessment Schedule. of PregS were effectively inhibited by simultaneous application
of ononetin in isolated NK cells from HC (Figures 3G,I,K,L).
Moreover, the I–V of ononetin sensitive currents was outwardly-
RESULTS rectified and typical for TRPM3 (Figures 3B,C). In contrast,
ionic currents evoked by both successive applications of PregS
Participant Characteristics and Blood
were mostly resistant to ononetin in isolated NK cells from
Parameters ME/CFS patients (Figures 3E,F,H,J) in comparison with HC
Sixteen age- and sex-matched participants were included for (Figures 3K,L) (p = 0.0030 and p = 0.0035). Collectively, these
this investigation. Demographic and clinical data for patients results confirm the significant loss of the TRPM3 channel activity
are summarized in Table 1. There was no significant difference in NK cells from ME/CFS patients after stimulation with IL-2
in age or gender between patients and HC. The 36-Item Short for 24 h.
Form Survey (SF-36) and World Health Organization Disability
Assessment Schedule (WHODAS) were used to determine Effects of Successive Applications of
participant health-related-quality of life (45, 46). As expected,
there was a significant difference in SF-36 and WHODAS scores PregS on TRPM3 Activity After Treatment
between ME/CFS patients and HC. Moreover, full blood count With NTX
parameters were measured for each healthy participant. All Stimulated IL-2 NK cells were co-treated with 200 µM NTX for
participant results were within the specified reference ranges for 24 h as described previously (36). Indeed, at this concentration
each parameter. There were no significant differences between and incubation time, NTX has been reported to not affect
healthy participants and ME/CFS patients for these reporting cell viability or induce apoptosis of immune cells. As NTX
parameters as provided by the Gold Coast University Hospital acts as an antagonist to the µOR thus negating the inhibitory
Pathology Unit, NATA accredited laboratory. function of this opioid receptor on TRPM3, we tested whether
the amplitudes of the PregS-induced currents were modified
Natural Killer Cell Purity in NK cells treated with NTX from both HC and ME/CFS
NK cell purity (CD3− /CD56+ ) was 90.34% ± 0.6782 for HC patients. Successive PregS applications (100 µM) evoked small
and 90.9% ± 1.695 for ME/CFS patients as determined by flow ionic currents (Figures 4B,C) with a typical shape of the TRPM3
cytometry (Figure 1A). There was no significant difference in NK current–voltage relationship (I–V) in NK cells isolated from HC;
cell purity in ME/CFS patients compared with HC (Figure 1B). however, no significant difference was reported within the HC

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

FIGURE 1 | Natural Killer cell purity. (A) Gating strategy used to identify NK cells. Representative flow cytometry plots from the PBMCs of one of the study participants.
The lymphocytes were live gated during acquisition using the side and forward scatter dot plot display and then single and dead cells were excluded. Furthermore, by
using the negative and positive gating strategies, CD3− as well as CD56+ lymphocyte populations were identified. (B) Bar graphs representing isolated NK cell purity
for HC and ME/CFS patients. Data presented as mean ± SEM. HC = 90.34% ± 0.6782 and ME/CFS = 90.9% ± 1.695. 7-AAD, 7-amino-actinomycin; ME/CFS,
myalgic encephalomyelitis/chronic fatigue syndrome; FSC, forward scatter; HC, healthy controls; NK cell, natural killer cell; SSC, side scatter.

group between NK cells treated or not with NTX (Figure 4H). typical for TRPM3 (Figures 5E,F). Collectively, these results
In contrast, PregS applications in NTX-treated NK cells from confirm the restoration of TRPM3 channel activity in NK cells
ME/CFS patients mimicked the PregS-induced increase in from ME/CFS patients after treatment with NTX for 24 h.
NK cells from HC (Figures 4E–G,I). Indeed, successive PregS
stimulations induced a significant increase of the outwardly
rectified current amplitudes in NTX-treated NK cells from Effects of Successive Applications of NTX
ME/CFS patients in comparison with non-treated NK cells from and PregS on TRPM3 Activity
ME/CFS patients and to the same level as treated and non-treated Opioid antagonists, such as NTX, have been previously reported
NK cells from HC (Figures 4G–I) (p = 0.0001 and p = 0.0035). to not affect TRPM3-dependent Ca2+ signals when directly
Moreover, the PregS-evoked currents present a typical shape of applied in perfusion (33). Therefore, to verify whether NTX can
the TRPM3 current–voltage relationship (I–V) (Figures 4E,F). directly regulate TRPM3 channel activity, we stimulated TRPM3
The data suggest that NTX restores TRPM3 channel activity in in NK cells using only the opioid antagonist NTX (Figure 6).
ME/CFS patients. No TRPM3-like ionic currents were induced by NTX (200 µM).
Indeed, we did not measure any outwardly rectifying currents
Modulation of PregS- Evoked Currents under voltage-clamp conditions with a typical shape of the
With Ononetin After Treatment With NTX TRPM3 current–voltage relationship (I–V) in IL-2 stimulated
To confirm that TRPM3 activity is involved in ionic currents NK cells from either HC (Figures 6B,G) or ME/CFS patients
evoked by PregS in NTX-treated NK cells from ME/CFS (Figures 6E,G) after addition of NTX. However, as PregS is
patients, we next used 10 µM ononetin to modulate the currently the most potent TRPM3 agonist described in the
TRPM3 ion channels (Figure 5). As NTX did not modify literature (48) and to confirm that TRPM3 ion channel activity
the outwardly rectified current amplitudes in NTX-treated could be stimulated, we then applied 100 µM PregS to the
NK cells from HC, no difference was shown in HC and NK cells from both HC and ME/CFS patients (Figures 6A,B).
the ionic currents evoked by both successive applications of Application of PregS induced TRPM3-like currents in both
PregS were effectively inhibited by simultaneous application HC (Figures 6C,G) and ME/CFS patients (Figures 6F,G). The
of ononetin (Figures 5G,I,K,L). In contrast, the ionic currents I-V of the PregS-evoked currents was outwardly rectified
evoked by successive PregS applications were effectively inhibited (Figures 6C,F), which is standard for TRPM3. However, the
by simultaneous application of 10 µM ononetin in isolated NTX- outward current amplitudes decreased significantly after PregS
treated NK cells from ME/CFS patients (Figures 5E,F,H,J–L). stimulation in NK cells from ME/CFS patients (Figure 6G)
Indeed, the ionic current initially resistant to ononetin in isolated (p = 0.0006) as previously shown (Figure 4G). The data suggest
NK cells from ME/CFS patients became sensitive to ononetin that TRPM3 channel activity is not directly stimulated by NTX
after incubation with NTX for 24 h (Figure 5L). Moreover, the in either HC or ME/CFS patients. Therefore, NTX is not a direct
I–V of ononetin sensitive currents was outwardly-rectified and agonist of TRPM3 ion channel.

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

FIGURE 2 | TRPM3 activity after successive applications of PregS. Data were obtained under whole-cell patch clamp conditions. (A) A representative time-series of
current amplitude at +100 mV and −100 mV showing the effect of successive applications of 100 µM PregS (gray) on ionic currents in IL-2 stimulated NK cells from
HC. (B) I–V before and after the first PregS stimulation in a cell corresponding with (A). (C) I–V before and after the second PregS stimulation in a cell corresponding
with (A). (D) A representative time-series of current amplitude at +100 mV and −100 mV showing the effect of successive applications of 100 µM PregS (gray) on
ionic currents in IL-2 stimulated NK cells from ME/CFS patients. (E) I–V before and after the first PregS stimulation in a cell as shown in (D). (F) I–V before and after
the second PregS stimulation in a cell as shown in (D). (G) Bar graphs representing TRPM3 current amplitude at +100 mV after successive applications of 100 µM
PregS in ME/CFS patients (N = 8; n = 27 and n = 25) compared with HC (N = 8; n = 31 and n = 29). Data are represented as mean ± SEM. ME/CFS, myalgic
encephalomyelitis/chronic fatigue syndrome; HC, healthy controls; NK, natural killer; PregS, Pregnenolone sulfate; TRPM3, Transient Receptor Potential Melastatin 3.
***p = 0.0001, ****p < 0.0001.

Modulation of NTX- and PregS- Evoked mostly resistant to ononetin in isolated NK cells from ME/CFS
Currents With Ononetin patients (Figures 7F,J,L), in comparison with HC, confirming
As no TRPM3-like ionic currents were induced after stimulation that TRPM3 channel activity is not directly modulated by NTX
with NTX, no difference was reported with simultaneous compared with PregS.
application of 10 µM ononetin in either IL-2 stimulated
NK cells from HC (Figures 7B,G,K) or ME/CFS patients DISCUSSION
(Figures 7E,H,K). In contrast, the ionic currents evoked by
PregS were effectively inhibited by simultaneous application of Our previous studies have described the loss of TRPM3 ion
10 µM ononetin in isolated NK cells from HC (Figures 7C,I,L). channel function to be associated with ME/CFS (28, 29). In
In addition, ionic currents in the presence of PregS were the present study, we used whole-cell patch-clamp technique

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

FIGURE 3 | Modulation of PregS- evoked currents with Ononetin. Data were obtained under whole-cell patch clamp conditions. (A) A representative time-series of
current amplitude at +100 mV and −100 mV showing the effect of 10 µM ononetin (gray) on ionic currents in the presence of 100 µM PregS in IL-2 stimulated NK
cells from HC. (B) I–V before and after the first application of ononetin in the presence of PregS in a cell as shown in (A). (C) I–V before and after the second
application of ononetin in the presence of PregS in a cell as shown in (A). (D) A representative time-series of current amplitude at +100 mV and −100 mV showing the
effect of 10 µM ononetin (gray) on ionic currents in the presence of 100 µM PregS in IL-2 stimulated NK cells ME/CFS patients. (E) I–V before and after the first
application of ononetin in the presence of PregS in a cell as shown in (D). (F) I–V before and after the second application of ononetin in the presence of PregS in a cell
as shown in (D). (G,H) Scatter plots representing change of each current amplitude before and after the first application of ononetin in presence of PregS in all NK
cells from HC and ME/CFS patients. Each cell represented as red lines had reduction in currents by ononetin. (I,J) Scatter plots representing change of each current
amplitude before and after the second application of ononetin in presence of PregS in all NK cells from HC and ME/CFS patients. Each cell represented as red lines
had reduction in currents by ononetin. (K) Table summarizing data for sensitive and insensitive cells to the first application of 10 µM ononetin in presence of PregS in
HC (N = 8; n = 31) compared to ME/CFS patients (N = 8; n = 27). (L) Table summarizing data for sensitive and insensitive cells to the second application of 10 µM
ononetin in presence of PregS in HC (N = 8; n = 29) compared to ME/CFS patients (N = 8; n = 24). Data are analyzed with Fisher’s exact test. ME/CFS, myalgic
encephalomyelitis/chronic fatigue syndrome; HC, healthy controls; NK, natural killer; PregS, Pregnenolone sulfate; TRPM3, Transient Receptor Potential Melastatin 3.

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

FIGURE 4 | Effects of treatment with NTX on TRPM3 activity after successive applications of PregS. Data were obtained under whole-cell patch clamp conditions. (A)
A representative time-series of current amplitude at +100 mV and −100 mV showing the effect of successive applications of 100 µM PregS (gray) on ionic currents in
IL-2 stimulated NK cells treated with 200 µM NTX for 24 h from HC. (B) I–V before and after the first PregS stimulation in a cell corresponding with (A). (C) I–V before
and after the second PregS stimulation in a cell corresponding with (A). (D) A representative time-series of current amplitude at +100 mV and −100 mV showing
(Continued)

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

FIGURE 4 | the effect of successive applications of 100 µM PregS (gray) on ionic currents in IL-2 stimulated NK cells treated with 200 µM NTX for 24 h from ME/CFS
patients. (E) I–V before and after the first PregS stimulation in a cell as shown in (D). (F) I–V before and after the second PregS stimulation in a cell as shown in (D).
(G) Bar graphs representing the effect of NTX treatment on TRPM3 current amplitude at +100 mV after successive applications of 100 µM PregS in ME/CFS patients
(N = 8; n = 22 and n = 16) compared with HC (N = 8; n = 20 and n = 19). (H) Bar graphs representing TRPM3 current amplitude at +100 mV after successive
applications of 100 µM PregS in NK cells treated with 200 µM NTX (N = 8; n = 20 and n = 19) or NK cells non-treated (N = 8; n = 31 and n = 29) from HC. The
same control has been used as Figure 2G. (I) Bar graphs representing TRPM3 current amplitude at +100 mV after successive applications of 100 µM PregS in NK
cells treated with 200 µM NTX (N = 8; n = 22 and n = 16) or NK cells non-treated (N = 8; n = 27 and n = 25) from ME/CFS patients. The same control has been
used as Figure 4G. Data are represented as mean ± SEM. ME/CFS, myalgic encephalomyelitis/chronic fatigue syndrome; HC, healthy controls; NK, natural killer;
NTX, naltrexone hydrochloride; PregS, Pregnenolone sulfate; TRPM3, Transient Receptor Potential Melastatin 3. **p = 0.0035, ***p = 0.0001.

as a method to measure endogenous TRPM3 activity in IL-2 are characteristics of ME/CFS (49). Physiologically, endogenous
stimulated NK cells from HC and ME/CFS patients, enabling the opioids are synthesized in vivo to modulate pain mechanisms
ion channel current recordings under voltage-clamp conditions and inflammatory pathways. Endogenous and exogenous opioids
and observation of the shape of the TRPM3 current–voltage mediate analgesia in response to painful stimuli binding to
relationship (I–V). This novel study confirms the significant loss opioid receptors, members of the family of GPCRs, on neuronal
of the TRPM3 channel activity after modulation with PregS and cells (38). Interestingly, molecules that bind to and activate
ononetin on a new cohort of ME/CFS participants (Figures 2, 3). opioid receptors significantly reduce TRPM3-dependent pain.
Additionally, we report a similar effect after a second modulation Recent reports suggest that the activation of TRPM3 channels is
with PregS and ononetin on IL-2 stimulated NK cells from greatly reduced if the channels are also stimulated by any of a
ME/CFS patients. Indeed, successive application of PregS enabled variety of GPCRs, and particularly by the µOR responsible for
the measurement of small ionic currents with a typical TRPM3- analgesic, rewarding and unwanted effects of opioids (33, 34).
like outward rectification in IL-2 stimulated NK cells isolated Under activation by opioids—whether produced by the body
from HC. In contrast, the amplitude of outward ionic currents (endogenously) or taken as a drug—µOR can mediate a spectrum
decreased significantly after successive PregS-stimulation in IL-2 of acute signaling and longer-term regulatory behaviors (50).
stimulated NK cells from ME/CFS patients, confirming impaired Such functional versatility cannot be explained by a simple “on-
TRPM3 channel activity in ME/CFS patients. In addition, off ” switch model of receptor activation and is more compatible
PregS-evoked ionic currents through TRPM3 ion channels were with dynamic and flexible receptor structures. Indeed, µOR
significantly modulated by ononetin in IL-2 stimulated NK cells interact with, and activate heterotrimeric G proteins to amplify
from HC compared with ME/CFS patients. Indeed, ionic currents the signal and regulate downstream effectors (51).
in the presence of PregS were mostly resistant to ononetin in IL-2 Recent studies have reported that TRPM3 inhibition requires
stimulated NK cells from ME/CFS patients. the heterotrimeric G protein to dissociate in order to prevent
We now report, for the first time, that TRPM3 channel the inhibitory Gαi subunit from interacting with the activated
activity was restored in IL-2 stimulated NK cells isolated µOR, locking the complex in the resting trimeric state (33–
from ME/CFS patients after incubation with 200 µM NTX for 35). On the other hand, it has been demonstrated that TRPM3
24 h (Figures 4, 5). We demonstrated that the treatment with activity is strongly inhibited by the direct interaction with Gβγ
NTX for 24 h had no effect on the PregS-evoked TRPM3- as the flow of ions through TRPM3 channels is strongly and
like currents in HC, whereas the outward current amplitudes reversibly inhibited when the cell membrane is flushed with
increased significantly after successive PregS stimulations in purified Gβγ (33, 35). Interestingly, TRPM3 channels require
ME/CFS patients. In addition, treatment with NTX also restored Phosphatidylinositol 4,5-bisphosphate (PIP2 ) in the membrane
the sensitivity of NK cells from ME/CFS patients to ononetin, in order to open, and some other Gα subunits enhance the
confirming that TRPM3 activity was involved in ionic currents inhibition of TRPM3, by acting on the localized depletion of PIP2
evoked by PregS in NTX-treated NK cells from ME/CFS (35). Therefore, specific assembly of µOR—G proteins complexes
patients. Finally, we report that direct application of NTX regulate the opening and closing of the TRPM3 ion channel
does not affect TRPM3-dependent Ca2+ signals when tested pore and in turn impact on the Ca2+ signaling cascade and
before PregS stimulation on both IL-2 stimulated NK cells biological responses.
from HC and ME/CFS patients (Figures 6, 7), suggesting that Importantly, opioid receptors are widely distributed on
NTX does not act as an agonist by directly coupling on the tissues and organ systems outside the CNS, such as the
TRPM3 ion channel gating but may involve an upstream lasting cells of the immune system, indicating that opioids are
regulatory mechanism. capable of exerting additional effects in the periphery, such
TRPM3 ion channels act as integrators of several stimuli as immunomodulatory and immunosuppressive effects (38).
and signaling pathways. TRPM3, expressed abundantly in Evidence now exists showing that the analgesic effects of
sensory neurons, are defined as thermosensitive and nociceptor opioids are not exclusively mediated by opioid receptors in
channels that help organisms to sense heat and make them the brain but can also be mediated via the activation of
more sensitive to pain during inflammation (27). Dysregulation receptors in immune cells. In turn this mechanism results in
of thermoregulatory responses has been reported in ME/CFS different side effects including immune suppressive functions
patients and generalized pain as well as brain inflammation and exacerbation of certain disease states (38). Although opioids

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

FIGURE 5 | Modulation of PregS- evoked currents with Ononetin after treatment with NTX. Data were obtained under whole-cell patch clamp conditions. (A) A
representative time-series of current amplitude at +100 mV and −100 mV showing the effect of 10 µM ononetin (gray) on ionic currents in the presence of 100 µM
PregS in IL-2 stimulated NK cells treated with 200 µM NTX from HC. (B) I–V before and after the first application of ononetin in the presence of PregS in a cell as
shown in (A). (C) I–V before and after the second application of ononetin in the presence of PregS in a cell as shown in (A). (D) A representative time-series of current
amplitude at +100 mV and −100 mV showing the effect of 10 µM ononetin (gray) on ionic currents in the presence of 100 µM PregS in IL-2 stimulated NK cells
treated with 200 µM NTX ME/CFS patients. (E) I–V before and after the first application of ononetin in the presence of PregS in a cell as shown in (D). (F) I–V before
and after the second application of ononetin in the presence of PregS in a cell as shown in (D). (G,H) Scatter plots representing change of each current amplitude
before and after the first application of ononetin in presence of PregS in all NK cells treated with 200 µM NTX from HC and ME/CFS patients. Each cell represented as
red lines had reduction in currents by ononetin. (I,J) Scatter plots representing change of each current amplitude before and after the second application of ononetin
in presence of PregS in all NK cells treated with 200 µM NTX from HC and ME/CFS patients. Each cell represented as red lines had reduction in currents by ononetin.
(K) Table summarizing data for sensitive and insensitive cells treated with 200 µM NTX to the first application of 10 µM ononetin in presence of PregS in HC (N = 8; n
= 20) compared to ME/CFS patients (N = 8; n = 22). (L) Table summarizing data for sensitive and insensitive cells treated with 200 µM NTX to the second application
of 10 µM ononetin in presence of PregS in HC (N = 8; n = 19) compared to ME/CFS patients (N = 8; n = 16). Data are analyzed with Fisher’s exact test. ME/CFS,
myalgic encephalomyelitis/chronic fatigue syndrome; HC, healthy controls; NK, natural killer; NTX, naltrexone hydrochloride; PregS, Pregnenolone sulfate; TRPM3,
Transient Receptor Potential Melastatin 3.

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

FIGURE 6 | TRPM3 activity after successive applications of NTX and PregS. Data were obtained under whole-cell patch clamp conditions. (A) A representative
time-series of current amplitude at +100 mV and −100 mV showing the effect of 100 µM PregS and 200 µM NTX (gray) on ionic currents in IL-2 stimulated NK cells
from HC. (B) I–V before and after NTX stimulation in a cell corresponding with (A). (C) I–V before and after PregS stimulation in a cell corresponding with (A). (D) A
representative time-series of current amplitude at +100 mV and −100 mV showing the effect of 100 µM PregS and 200 µM NTX (gray) on ionic currents in IL-2
stimulated NK cells from ME/CFS patients. (E) I–V before and after NTX stimulation in a cell as shown in (D). (F) I–V before and after PregS stimulation in a cell as
shown in (D). (G) Bar graphs representing TRPM3 current amplitude at +100 mV after successive applications of 100 µM PregS in ME/CFS patients (N = 8; n = 19
and n = 19) compared with HC (N = 8; n = 23 and n = 21). Data are represented as mean ± SEM. ME/CFS, myalgic encephalomyelitis/chronic fatigue syndrome;
HC, healthy controls; NK, natural killer; PregS, Pregnenolone sulfate; TRPM3, Transient Receptor Potential Melastatin 3. ***p = 0.0006.

modulate both innate and adaptive immune systems, defects in inflamed rat paw (52). βEP acts primarily on µOR and
in innate immunity appear to have broader consequences. For δOR and modulates the functions of lymphocytes and other
example, opioids act as immunosuppressors on migration and cells involved in host defense and immunity (53). Recently,
functional activity of innate immune responders by modulating NTX has been shown to promote δOR activity and enhance
leukocyte recruitment, cytokine secretion and bacterial clearance NK cell cytolytic activity in response to βEP in vitro on
leading to impairment of the hosts’ ability to eradicate pathogens rat splenocytes, suggesting the potential use of NTX in the
(38). Interestingly, immune-derived opioid peptides also play treatment of immune deficiency (54). The δOR agonistic
a substantial role in the modulation of inflammatory pain. activity of NTX on splenocytes appears to be due to its
For example, (β)-endorphin (βEP), an endogenous opioid potent µOR antagonistic function as δOR expression is tightly
neuropeptide and peptide hormone, has been reported to be controlled by a negative feedback regulation of µOR. NTX
produced by T- and B-lymphocytes as well as in monocytes, disrupts this feedback control by reducing µOR function,

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

FIGURE 7 | Modulation of NTX- and PregS- evoked currents with Ononetin. Data were obtained under whole-cell patch clamp conditions. (A) A representative
time-series of current amplitude at +100 mV and −100 mV showing the effect of 10 µM ononetin (gray) on ionic currents in the presence of 200 µM NTX or 100 µM
PregS in IL-2 stimulated NK cells from HC. (B) I–V before and after application of ononetin in the presence of NTX in a cell as shown in (A). (C) I–V before and after
application of ononetin in the presence of PregS in a cell as shown in (A). (D) A representative time-series of current amplitude at +100 mV and −100 mV showing the
effect of 10 µM ononetin (gray) on ionic currents in the presence of 200 µM NTX or 100 µM PregS in IL-2 stimulated NK cells ME/CFS patients. (E) I–V before and
after application of ononetin in the presence of NTX in a cell as shown in (D). (F) I–V before and after application of ononetin in the presence of PregS in a cell as
shown in (D). (G,H) Scatter plots representing change of each current amplitude before and after application of ononetin in presence of NTX in all NK cells from HC
and ME/CFS patients. Each cell represented as red lines had reduction in currents by ononetin. (I,J) Scatter plots representing change of each current amplitude
before and after application of ononetin in presence of PregS in all NK cells from HC and ME/CFS patients. Each cell represented as red lines had reduction in
currents by ononetin. (K) Table summarizing data for sensitive and insensitive cells to 10 µM ononetin in presence of NTX in HC (N = 8; n = 23) compared to ME/CFS
patients (N = 8; n = 19). (L) Table summarizing data for sensitive and insensitive cells to 10 µM ononetin in presence of PregS in HC (N = 8; n = 19) compared to
ME/CFS patients (N = 8; n = 19). Data are analyzed with Fisher’s exact test. ME/CFS, myalgic encephalomyelitis/chronic fatigue syndrome; HC, healthy controls; NK,
natural killer; NTX, naltrexone hydrochloride; PregS, Pregnenolone sulfate; TRPM3, Transient Receptor Potential Melastatin 3.

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Cabanas et al. Naltrexone and TRPM3 in ME/CFS

thereby upregulating δOR binding that results in enhanced NK has been reported to contribute to the pathological state of
cell cytolytic response to the ligands (54). Interestingly, an ME/CFS (28–32). In the present study, we confirmed impaired
investigation by Conti et al. reported significantly reduced βEP TRPM3 activity in ME/CFS patients through electrophysiological
in ME/CFS patients reflecting the condition of chronic immune investigations in IL-2 stimulated NK cells after both successive
activation (55). activation with PregS and inhibition with ononetin. Importantly,
Importantly, NTX acts as an antagonist to the µOR thus we demonstrated that TRPM3 channel activity was restored
negating the inhibitory function of this opioid receptor on in IL-2 stimulated NK cells isolated from ME/CFS patients
TRPM3 (37, 38). Indeed, opioids binding to µOR block Ca2+ after incubation with NTX for 24 h. Indeed, the opioid
influx through inhibition of TRPM3 channels. The disinhibiting antagonist NTX negates the inhibitory function of opioid
effect of NTX restores the TRPM3 ion channel activity in receptors on TRPM3 in NK cells from ME/CFS patients.
ME/CFS patients and in turn re-establishes the appropriate This study not only helps to provide an understanding of
Ca2+ signaling. Each cell type, including NK cells, has a the etiology and pathomechanism of ME/CFS, but confirms
unique signaling phenotype capable of delivering Ca2+ signals that opioid receptors in immune cells have an important
with the spatial and temporal properties necessary to regulate role in analgesic effects. Finally, the restoration of TRPM3
its particular function (24). Phenotypic stability seems to be channel activity suggests an immune-modulating action of
maintained by Ca2+ - dependent feedback mechanisms that NTX by remodeling the TRPM3-dependent Ca2+ signals,
adjust the expression levels of individual toolkit components which will in turn affect NK cell functions, and support the
that contribute to these cell-specific signaling systems. These hypothesis that NTX may have potential for use as a treatment
homoeostatic systems are highly plastic and can undergo a for ME/CFS.
process of phenotypic remodeling, resulting in the Ca2+ signals
being set either too high or too low. Such subtle dysregulation DATA AVAILABILITY STATEMENT
of Ca2+ signals may highly impact cell functions and result
in diseases (24, 56). Release of Ca2+ by TRPM3 seems to All datasets generated for this study are included in the
play a central role in NK cells’ activation and function as manuscript/supplementary files.
dysregulation of TRPM3 function in ME/CFS patients has been
shown to affect Ca2+ signaling, leading to impaired NK cell ETHICS STATEMENT
regulatory machinery and functions. Restoration of TRPM3
channel activity by NTX leads to Ca2+ signal remodeling All participants gave written consent to participate and to
that will in turn contribute to cells’ activation, effector publish. Ethics approval was under Human Research Ethics
functions, gene expression or differentiation. Thus, restoring Committee Griffith University (HREC/15/QGC/63).
Ca2+ homeostasis in NK cells will help to reactivate and
rebalance the different Ca2+ dependent mechanisms including AUTHOR CONTRIBUTIONS
ongoing transcriptional processes, modulation of TRPM3 surface
expression, or constitutive and regulated vesicular trafficking of HC, KM, SM-G, and DS designed the study and wrote the
the ion channel itself or of accessory and regulating proteins. manuscript. HC performed experiments. HC and KM performed
Hence restoration of Ca2+ homeostasis results also in the data analysis.
restoration of the integrity and stability of these NK cell-specific
signaling systems. FUNDING
In conclusion, ME/CFS is a long-term illness with a wide
range of symptoms, including chronic pain, impaired memory This study was supported by the Mason Foundation, McCusker
and concentration, and inflammation (1). The widely expressed Charitable Foundation, Stafford Fox Medical Research
Ca2+ permeable nonselective cation channel, TRPM3, functions Foundation, Mr. Douglas Stutt, Alison Hunter Memorial
as a chemo- and thermo-sensor enabling the detection of Foundation, Buxton Foundation, Blake Beckett Trust, Henty
painful and innocuous thermal stimuli (27). TRPM3 dysfunction Donation, and the Change for ME Charity.

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