Novel characterization of endogenous transient receptor potential melastatin 3 ion channels from Gulf War Illness participants 2024 Marshall-Gradisnik

Andy · Jun 26, 2024 at 10:06 AM

Abstract

Gulf War Illness (GWI) is a chronic condition characterized by multisystem symptoms that still affect up to one-third of veterans who engaged in combat in the Gulf War three decades ago. The aetiology of GWI is mainly explained by exposure to multiple toxic agents, vaccines, and medications. As there is a significant overlap in symptoms between GWI and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), the objective of this study was to investigate a biomarker widely reported in Natural Killer (NK) cells from ME/CFS patients, the Transient Receptor Potential Melastatin 3 (TRPM3) ion channel.

NK cells from 6 healthy controls (HC) and 6 GWI participants were isolated, and TRPM3 function was assessed through whole-cell patch-clamp. As demonstrated by prior studies, NK cells from HC expressed typical TRPM3 function after pharmacomodulation. In contrast, this pilot investigation demonstrates a dysfunctional TRPM3 in NK cells from GWI participants through application of a TRPM3 agonist and confirmed by a TRPM3 antagonist. There was a significant reduction in TRPM3 function from GWI than results measured in HC. This study provides an unprecedented research field to investigate the involvement of TRP ion channels in the pathomechanism and potential medical interventions to improve GWI quality of life.

Open access, https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0305704

Andy · Jun 26, 2024 at 10:10 AM

For my own 'amusement', I discovered that the phrase "gold standard" was used 3 times in this paper in reference to the method of testing.

John Mac · Jun 26, 2024 at 10:33 AM

Andy said: ↑

the objective of this study was to investigate a biomarker widely reported in Natural Killer (NK) cells from ME/CFS patients, the Transient Receptor Potential Melastatin 3 (TRPM3) ion channel.
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Widely reported mostly by themselves. Have they reached a dead with this finding in ME and are now just moving on to new diseases? What next TRMP3 in Lyme disease, TRMP3 in Long Covid?

Kitty · Jun 26, 2024 at 10:39 AM

John Mac said: ↑

What next TRMP3 in Lyme disease, TRMP3 in Long Covid?
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They've got the whole FND field to go at too.

Hutan · Jun 26, 2024 at 10:53 AM

Does anyone know, has any other group ever tried to replicate this finding?

NCNED got a fair chunk of the NHMRC AUD3 million funds, back in, what was it, 2021? to get on with things. But, they have never scaled things up. And now they are doing the same small study in a different disease.

I haven't read this paper yet and I forget if we have discussed the TRPM3 finding in ME/CFS and dismissed it. But why doesn't NCNED cooperate with some other lab to replicate their finding? They've been claiming that they have solved ME/CFS for years, you'd think they would want someone else to say 'yep, it really looks like they have'.

Hutan · Jun 26, 2024 at 10:56 AM

Oops, I take some of that back.
Authors:

Sonya Marshall-Gradisnik ,

Etianne Martini Sasso ,

Natalie Eaton-Fitch,

Peter Smith,

James N. Baraniuk,

Katsuhiko Muraki

Looks as though the Baraniuk lab has got on board. Perhaps they had some Department of Defence GWI funding sloshing around. The paper could be worth checking out to see if it holds up.

Hutan · Jun 26, 2024 at 11:09 AM

Introduction

A remarkable feature of ME/CFS is the post-exertional neuroimmune exhaustion caused by an inappropriate response to stressors and involves symptoms exacerbated following mild physical or mental activity [20–22], a char- acteristic also reported in GWI patients [12,23].
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Our previous studies have demonstrated the role of Transient Receptor Potential (TRP) ion channels in the pathophysiology of ME/CFS which also promoted research into novel thera- pies [24–29]. The Mammalian TRP ion channels family is compounded by six subfamilies totaling 28 members: ankyrin (TRPA), canonical (TRPC), melastatin (TRPM), mucolipin (TRPML), polycystin (TRPP), and vanilloid (TRPV) [30–32]. Furthermore, a large number of the TRP superfamily members are highly sensitive to diverse chemical, physical and biological stimuli, therefore these ion channels act as molecular sensors to perceive modifications in the internal and external environment for the purpose of maintaining homeostasis [33–38]. In addition, many TRP channels participate in the regulation of calcium (Ca2+) signalling to pre- serve cellular homeostasis. Overall, TRP channels also contribute to crucial processes, includ- ing neurogenesis, plasticity, immune functions, inflammation control, cell proliferation and survival, and pain perception [32,37,39–45].
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Although GWI symptoms are the result of the long-term effects of exposure during military service, and the potential for TRP channels to be modulated by various environmental and toxic stimuli, to our knowledge this is the first study to analyse a TRP channel function in vet- erans with GWI. We specifically selected TRPM3 as the first TRP channel to be investigated in the GWI pathomechanism due to TRPM3’s association with ME/CFS, even though other TRP channels may also be associated with the symptomatology presented by patients with GWI. Hence, in this pilot study, the aim was to evaluate TRPM3 ion channel activity in NK cells from GWI participants in comparison with the same cells from healthy controls (HC).
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Hutan · Jun 26, 2024 at 11:37 AM

Methods

The GWI group was composed of six Australian males who engaged in combat at the GW, were diagnosed with GWI previously, and met CDC Case Definition [14] and Kansas criteria [15] for GWI. Six HC male participants were selected for this study, all those reported to be in good health, absence of illness and no fatigue episodes.
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This investigation was approved by the Griffith University Human Research Ethics Committee (GU HREC 2022/666)
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So, it's looking as though it was done in the NCNED lab.

Data about their symptoms was collected.

To assess disability data the ques- tionnaire includes the World Health Organization Disability Assessment Schedule (WHODAS)....
WHODAS is subdivided into seven domains of life: (1) Communication and understanding; (2) Mobility; (3) Self-care; (4) Interpersonal connections; (5) Life activities; (6) Work or School participation; and (7) Participation in society.
Scores converted from 0% to 100%, disability are inversely proportional to the scale, whereby lower scores indicate less disability and correspond 100% to full disability [47].
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Whole blood sample
(We get told a lot of detail in some places, but I didn't see anything about how quickly the blood was processed)
Peripheral blood mononuclear cells centrifuged out
NK cells isolated by immunomagnetic selection

Flow cytometry was performed to identify the NK cell purification from each NK cell isola- tion. Immediately after NK cell isolation, cells were incubated with CD56 APC (0.25g/20l) and CD3 PE Cy7 (0.25g/5l) monoclonal antibodies (Becton Dickinson (BD) Bioscience, San Jose, CA, USA) for 20 minutes in the dark at room temperature. NK cells were washed and resus- pended in 350 ml of stain buffer (BD Bioscience, New Jersey, USA) and acquired at 10,000 events using the BD LSR- FortessaTM X-20 flow cytometer (BD Biosciences, San Diego, CA, USA). The NK cell population was then identified using phenotypic surface expression as CD3-CD56+.
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Samples of NK cells didn't differ in purity.

The gold standard patch-clamp technique was conducted to determine TRPM3 ion channel activity in NK cells freshly isolated from HC and people with GWI.
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google said:

The patch clamp technique is the generally recognized approach to study ion channel function. During a patch clamp recording, a micropipette containing a recording electrode connected to an amplifier, and filled with electrolyte solution, is brought into contact with the membrane of an isolated cell.
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There were a lot of salts and other things in the solutions.

As previously validated by NCNED, pharmacological agents were included in the extracel- lular solution to assess TRPM3 ionic currents [26]. Briefly, a gravity perfusion system was used to apply extracellular solution for 50 seconds to establish a baseline current. Subsequently, extracellular solution containing 100 μM of PregS was added to stimulate TRPM3 ion channels for 2.5 minutes. Following on from this addition extracellular solution with 10 μM Ononetin and 100 μM of PregS was applied for another 2.5 minutes to block TRPM3 ion channels. After the conclusion of the drugs application, another cycle of 100 seconds of only extracellular solu- tion was applied to remove the drugs.
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All recordings were analysed individually by a blinded researcher and posteriorly data was reviewed one by one by another blinded researcher, as detailed in S2 Fig.
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data are presented as mean ± standard error of the mean (SEM) unless otherwise stated.
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Hutan · Jun 26, 2024 at 12:13 PM

Results

GWI and control groups matched on age and BMI.
No differences in full blood counts between the groups

WHODAS

there were signifi- cant differences between HC and GWI results in communication and under-standing
(p = 0.029), mobility (p = 0.007), self-care (p = 0.022), interpersonal connections (p = 0.024), life activities (p = 0.022), work participation (p = 0.007) and participation in society
(p = 0.004). GWI participants had higher scores in all WHODAS domains, which indicates a significant increase in disability levels.
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Interest- ingly, GWI participants from this study met ME/CFS clinical criteria, specifically all GWI met Canadian Consensus Criteria (CCC) for ME/CFS [49] and one GWI met the International Consensus Criteria (ICC) for ME/CFS [20].
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Electrophysiology experiments

TRPM3 agonist pregnenolone sulfate (PregS) activates these ion channels inducing an increase in intracellular Ca2+ concentration in HC cells, as represented in Fig 1A and 1B. Under voltage-clamp conditions, 100 μM PregS induced small outward rectifying currents in most NK cells isolated from HC which showed characteristic TRPM3 current–volt- age relationship (I–V) (Fig 1B). However, in NK cells from the GWI group, the application of 100 μM of PregS stimulated only a few NK cells. To statistically compare TRPM3 ion channel function between both groups, amplitudes were determined for each recording as a change in amplitude from baseline to PregS induced peak, as represented in time-series graphs (Fig 1A and 1D). In this investigation a significantly smaller amplitude of PregS-evoked currents was found in NK cells isolated from GWI participants in comparison to cells from HC (Fig 1G,p < 0.0001), a result that indicates people diagnosed GWI have TRPM3 impaired function. Fig 1 provides examples of recordings in a NK cell from a HC (Fig 1A–1C) and GWI participant (Fig 1D–1F).
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Figure 2

Scatter plots representing change of each current amplitude before and after application of Ononetin in presence of PregS in NK cells from HC and GWI participants respectively. Each red line represented a cell sensitive to Ononetin as a reduction in amplitude was recorded.
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It does look interesting. Hopefully someone familiar with the approach can tell us if it all holds together.

Hutan · Jun 26, 2024 at 12:22 PM

Discussion

Our data is the first to report significant impairment in TRPM3 ion channel function in NK cells from participants with GWI compared to HC. TRPM3 is a non-selective cation channel that highly exhibits permeability to Ca2+ and plays a critical role in a variety of biological pro- cesses due to its contribution in the Ca2+ signalling regulation
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abnormal intracellular Ca2+ concentration associated with dysfunctional TRPM3 may cause inadequate cell function and imbalance intracellular signal- ling pathways [26,27,61].
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TRP channels are involved in oxidative stress, pain and inflammatory responses, while TRPM3 specifically in peripheral nociceptors have pronociceptive and pro-inflammatory properties [37,64,65]. Elhaj et al recently identified an increase in interleukin 6 (IL-6) and C-reactive protein (CRP) levels in GWI patients compared with other veterans without GWI, which is consistent with previ- ous research [62,66] and corroborate the role of chronic neuroimmune and neuroinflamma- tion disturbances [1].
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Notably, our present finding of decreased TRPM3 function in NK cells from GWI is compatible with results reported in ME/ CFS and long COVID cohorts [26,61,67], suggesting a common overlap of TRPM3 dysfunc- tion, or TRP ion channels, in the pathomechanism of each of these diseases. Support for this argument is highlighted by TRP ion channels being modulated by numerous stimuli, for example, by environmental (e.g. temperature, light, chemicals), mechanical (e.g. osmolarity, pressure), natural (e.g. herbs or spices, venoms, toxins), endogenous factors, tissue damage, infection and inflammation [40,41,68–71].
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Furthermore, TRP ion channels have been recognized as potentially associated with multi- system diseases and emerging as important drug targets due to their ubiquitous expression in cells from human organs and peripheral tissues [30,40,41,44,73,74]. Fonfria et al. characterized the expression of all TRPM family members in many human tissues, TRPM3 specifically was detected in cells from the brain, pituitary, kidney, adipose tissue, pancreas, prostate, and bone [75]. Other researchers also reported TRPM3 in different parts of the central nervous and car- diovascular systems, immune cells, liver, urinary and genital systems [24,26,50,73,76,77]. Despite the present study having assessed only TRPM3 ion channels in NK cells, these findings suggest this ion channel is dysfunctional in other tissue cells in GWI participants. Hence, the ubiquitous expression of TRPM3 ion channels is compatible with the constellation of GWI symptoms.
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the novel findings of these studies provide the perspective [prospect] of developing a test focused on the characterization of ion channel function using an easily acquired biological sample.
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(There's quite a lot of typos in the paper)

Conclusion

In conclusion, the present study creates a rationale for future studies analysing other TRP channels in plasma membrane, as well as organelles. It further provides an opportunity to lead investigations of therapeutic strategies to treat and manage the GWI condition, to alleviate severe symptoms and consequently improve their quality of life. Further, ion channel studies are necessary to investigate the contribution of other TRP channels in the pathophysiology of GWI.
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So, I guess the authors are suggesting GWI is a channelopathy, as is ME/CFS and Long Covid. Unfortunately, it looks as though we still don't have replication in a lab that isn't NCNED.

But, maybe they are right? How would new NK cells be being produced with the channelopathy?

Hutan · Jun 26, 2024 at 7:30 PM

The following paper has some background on TRPM3. But the paper is from 2008, and so surely more is known. I'd be interested to know why Sonya Marshall-Gradinisk has focussed on TRPM3 - was work done on a whole range of ion channels in ME/CFS and this was the one that wasn't working properly, or was there some reason that this particular ion channel was selected for investigation?

Herbal Compounds and Toxins Modulating TRP Channels, 2008

TRPM3 transcripts are expressed in kidney (though disputed), eye and brain, in the latter specifically in regions such as the dentate gyrus, the intermediate lateralseptal nuclei, the indusium griseum, and the tenia tecta. Strongest Trpm3 expression was found in the epithelialcells of choroid plexus [86]. Studies on the functional role of TRPM3 are fairly complicated by the fact that the TRPM3 gene is alternatively spliced into multiple functional variants. To date it was reported that at least TRPM3α1 and TRPM3α2 form cation-selective channels, though only TRPM3α2 is a Ca2+ permeable channel. Both exhibit constitutively active, outwardly rectifying currents that are blocked by intracellular Mg2+ [86].

Interestingly, TRPM3α2 is reported to be activated by some naturally occurring steroidal compounds, unlike TRPM3(1, but further information is lacking at the time of writing [72, 80]. Another splice variant, the 1325 amino acid variant of TRPM3 is activated by the sphingolipid D-erythro-sphingosine (SPH) and by SPH analogs, but not sphingosine-1-phosphate or ceramide. SPH is a central metabolite arising during the de novo synthesis of cellular sphingolipids [30]. Further insight in the physiological importance of these activating compounds will inevitably require elucidation of the expression of specific splice variants in specific tissues, an issue that is highly unclear at the moment.
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Nightsong · Jun 26, 2024 at 7:43 PM

May also be of interest that the authors have made at least three patent applications relating to their TRP cation channel work:

https://patentimages.storage.googleapis.com/37/24/63/a52cbb3716cf87/US20210069180A1.pdf
https://patentimages.storage.googleapis.com/7b/99/34/688d61f40e71a2/WO2016023077A1.pdf
https://patentimages.storage.googleapis.com/42/8c/0f/235d4ab44f175a/CA3021399A1.pdf

Hutan · Jun 26, 2024 at 8:40 PM

Transient receptor potential (TRP) channels: a clinical perspective, 2013
A slightly more recent paper

From the paper:

TRPM3
Endogenous agonists:
Pregnenolone sulphate Wagner et al. (2008) [as used in this Marshall-Gradinisk study, with the GWI NK cells not responding to the stimulation]
D-erythro-sphingosine Grimm et al. (2005)

Endogenous antagonist:
Progesterone Majeed et al. (2012) [interesting given female preponderance of ME/CFS, but not sure how it fits with reported improvements in pregnancy and a seeming lack of change in symptoms post-menopause]

Exogenous antagonist:
Rosiglitazone Majeed et al. (2011a)
Mefenamic acid Klose et al. (2011)
Naringenin, hesperetin, ononetin, eriodictyol Straub et al. (2013)
TM3E3 (polyclonal antibody) Naylor et al. (2008)

As a general rule, TRP channels have six transmembrane spanning domains (S1–S6) with a pore-forming loop between S5 and S6 (Figure 1; Wu et al., 2010). Both –NH2 and –COOH termini are located intracellularly. Many TRP channels are non-selective Ca2+-permeable channels with permeability ratios PCa/PNa < 10. TRPM4 and TRPM5, in particular, are only permeable to monovalent cations and they do not conduct Ca2+ and Mg2+, while TRPV5 and TRPV6 are highly Ca2+ selective with PCa/PNa > 100 (Owsianik et al., 2006). Most TRPs form functional channels as homotetramers, but heteromultimerization is frequently observed (Cheng et al., 2010). This creates a potential problem for drug discovery efforts as heteromultimers (that are not easily recreated in heterologous expression systems) may have distinct pharmacological properties.
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So, I'm imagining these molecules sitting in the cell membrane, with loops sticking out of the cell that can capture the molecules it is made to sense. Interesting about the possibility of multiple TRPs joining together and that they may have distinct pharmacological properties when they do that. This paper has a picture of a standard TRP:

I think this Marshall-Gradisnik GWI paper was suggesting that these channels might also be operating on the surface of organelles i.e. within the cell.

TRP channels are ‘cellular sensors’ (Clapham, 2003) that respond to changes in the cellular environment, including temperature, stretch/pressure, chemicals, oxidation/reduction, osmolarity and pH, both acidic and alkaline (Moran et al., 2011; Nieto-Posadas et al., 2011a).
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Activation of TRP channels allows cations pass through the membrane and depolarize cells, leading to a wide range of cellular responses. Stimulated by a broad range of stimuli and expressed probably in all the cells in the body (Nilius, 2013), TRP channels are thought to play diverse physiological roles.
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TRPM3-deficient mice showed impaired behavioural response to noxious heat and failed to develop inflammatory heat hyperalgesia (Vriens et al., 2011). Naturally occurring TRPM3 blockers include the citrus fruit flavanones, naringenin and hesperetin (Straub et al., 2013).
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Hutan · Jun 26, 2024 at 9:08 PM

So, I guess the question I have is why hasn't the report of faulty TRPM3 in ME/CFS got more attention? The answer might be in forum threads on the older papers. Certainly the small size of studies and the lack of replication have contributed.

But, it doesn't seem like a particularly difficult thing to attempt a replication of. Why haven't other labs had a look at this? Perhaps with Baraniak's involvement in this paper, there will be more attention and followup.

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