Validation of impaired Transient Receptor Potential Melastatin 3 ion channel activity in natural killer cells from ME/CFS patients

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Mol Med. 2019 Apr 23;25(1):14. doi: 10.1186/s10020-019-0083-4.
Validation of impaired Transient Receptor Potential Melastatin 3 ion channel activity in natural killer cells from Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis patients.
Cabanas H1,2,3, Muraki K4,5, Balinas C6,7,5, Eaton-Fitch N6,7,5, Staines D6,7,5, Marshall-Gradisnik S6,7,5.
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Abstract
BACKGROUND:
Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis (CFS/ME) is a complex multifactorial disorder of unknown cause having multi-system manifestations. Although the aetiology of CFS/ME remains elusive, immunological dysfunction and more particularly reduced cytotoxic activity in natural killer (NK) cells is the most consistent laboratory finding. The Transient Receptor Potential (TRP) superfamily of cation channels play a pivotal role in the pathophysiology of immune diseases and are therefore potential therapeutic targets.

We have previously identified single nucleotide polymorphisms in TRP genes in peripheral NK cells from CFS/ME patients. We have also described biochemical pathway changes and calcium signaling perturbations in NK cells from CFS/ME patients. Notably, we have previously reported a decrease of TRP cation channel subfamily melastatin member 3 (TRPM3) function in NK cells isolated from CFS/ME patients compared with healthy controls after modulation with pregnenolone sulfate and ononetin using a patch-clamp technique.

In the present study, we aim to confirm the previous results describing an impaired TRPM3 activity in a new cohort of CFS/ME patients using a whole cell patch-clamp technique after modulation with reversible TRPM3 agonists, pregnenolone sulfate and nifedipine, and an effective TRPM3 antagonist, ononetin. Indeed, no formal research has commented on using pregnenolone sulfate or nifedipine to treat CFS/ME patients while there is evidence that clinicians prescribe calcium channel blockers to improve different symptoms.
METHODS:
Whole-cell patch-clamp technique was used to measure TRPM3 activity in isolated NK cells from twelve age- and sex-matched healthy controls and CFS/ME patients, after activation with pregnenolone sulfate and nifedipine and inhibition with ononetin.
RESULTS:
We confirmed a significant reduction in amplitude of TRPM3 currents after pregnenolone sulfate stimulation in isolated NK cells from another cohort of CFS/ME patients compared with healthy controls. The pregnenolone sulfate-evoked ionic currents through TRPM3 channels were again significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients. In addition, we used nifedipine, another reversible TRPM3 agonist to support the previous findings and found similar results confirming a significant loss of the TRPM3 channel activity in CFS/ME patients.
CONCLUSIONS:
Impaired TRPM3 activity was validated in NK cells isolated from CFS/ME patients using different pharmacological tools and whole-cell patch-clamp technique as the gold standard for ion channel research. This investigation further helps to establish TRPM3 channels as a prognostic marker and/ or a potential therapeutic target for CFS/ME.
KEYWORDS:
Calcium; Chronic fatigue syndrome/Myalgic encephalomyelitis; Natural killer cells; Patch-clamp; Transient receptor potential Melastatin 3
PMID: 31014226 DOI: 10.1186/s10020-019-0083-4
 

Gemini

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Full paper is Open Access. Excerpt [my bold]:

Therefore, TRPM3-related Ca2+ dysfunction may then result in a reduction of [Ca2 +]i, which may lower the function and cytotoxic capacity of the NK cells in CFS/ME patients. Changes in Ca2+ signalling may also impair cytokine production, including Interferon (IFN)-γ and Tumor Necrosis Factor (TNF), therefore interfering with systemic inflammation and anti-tumour responses (Romee et al., 2013).
 

Gijs

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I tried calcium blockers it made me very very sick. So i don't understand this finding.
 

Wishful

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If the treatments based on this finding doesn't actually reduce overall ME symptom severity, then it may just be a secondary effect, which may cause some symptoms in some patients, but not in others.
 

hixxy

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I tried calcium blockers it made me very very sick. So i don't understand this finding.
Then the calcium blockers may still be hitting on central pathology just not in a desirable way but that's still somewhat illuminating. Also there are many many different types of calcium channels and calcium channel blockers.
 

Diwi9

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I believe @Dechi has reported improvements from calcium channel blockers. I also believe they were on Dr. Goldstein's list.
 

Dechi

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I believe @Dechi has reported improvements from calcium channel blockers. I also believe they were on Dr. Goldstein's list.
You are right. After 3 years of taking Nimodipine (now at a lower dose), I can say it helps alleviate a certain amount of cognitive difficulties. To be honest, sometimes I think it doesn’t make a difference but then when I stop it, I notice the difference,

It doesn’t drastically changes my symptoms, but it helps keeping the edge off.
 

Gingergrrl

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Also there are many many different types of calcium channels and calcium channel blockers.
There definitely are and I wish I understood all of this better. I have autoantibodies that block the N-type Calcium Channel and because of this, I have been told not to take any medications or anesthesia that are calcium channel blockers. But if this relates whatsoever to this article, I have no idea :xpem:
 

Dan_USAAZ

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I found this research update useful in describing the paper in layperson's terms.

If I understand correctly, it appears to be saying that proper transport of calcium into the cell is impaired and may be causing ME/CFS symptoms.

TRP ion channels play a crucial role in healthy cells – transferring calcium and other cations from outside the cell to the inside, resulting in regulation of many cell functions. Therefore, abnormal TRP ion channels lead to dysregulation of calcium signalling.
The NCNED team have now reported again that immune cells from a new cohort of ME/CFS* patients have faulty cell receptors compared with healthy participants. This results in reduced calcium being transported into the immune cell where cells are unable to function optimally causing immune system changes seen in ME/CFS* patients.

@Gijs, I am not familiar with the mechanism of calcium blockers, but based simply on the name, this treatment may be the opposite direction of what is needed and could exacerbate the problem?
I tried calcium blockers it made me very very sick. So i don't understand this finding.

All, The research team has stated that treatment studies (drugs?) are under way, but I was not able to find what or even a category. Has someone found that they are suggesting treatment with calcium blockers? I would think not.

As mentioned, I am not familiar with the mechanism of calcium blockers and I do not have a science background. Please feel free to let me know if I have misunderstood.

Thanks!
 

sb4

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Calcium ion has a role in regulation of PDC in muscle tissue, because it activates PDP, stimulating glycolysis on its release into the cytosol - during muscle contraction. Some products of these transcriptions release H2 into the muscles. This can cause calcium ions to decay over time.
It appears lack of calcium in the cell could lead to inactivation of PDH.
 

Gingergrrl

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If I understand correctly, it appears to be saying that proper transport of calcium into the cell is impaired and may be causing ME/CFS symptoms.
@Gijs, I am not familiar with the mechanism of calcium blockers, but based simply on the name, this treatment may be the opposite direction of what is needed and could exacerbate the problem?
@Dan_USAAZ How do these new results match with someone like me who has an autoantibody that blocks the N-type Calcium Channels? I am like @Gijs and do not tolerate calcium channel blockers and once we found I had this autoantibody (in 2016), I was told by neurologists not to take meds that are CCB's.

Does this study match with my case, or is it the opposite, or is it not related? I wish I could understand these studies! Tagging @Inara @JasonUT just in case this is of interest :)
 

JasonUT

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Interesting info. My tests were slightly positive for N-type Calcium Channel AB; however, I am not sure what to make of it yet. I am trying to see Dr. Steven Vernino who helped design the Mayo DYS1 panel. Hopefully, I can get help straight from the expert.

I have never tried a prescription CCB; however, I feel wonderful on Mg Glycinate 100 mg TID. Mg acts as a natural CCB.
 

Dan_USAAZ

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@Dan_USAAZ How do these new results match with someone like me who has an autoantibody that blocks the N-type Calcium Channels? I am like @Gijs and do not tolerate calcium channel blockers and once we found I had this autoantibody (in 2016), I was told by neurologists not to take meds that are CCB's.

Does this study match with my case, or is it the opposite, or is it not related? I wish I could understand these studies! Tagging @Inara @JasonUT just in case this is of interest :)
Hi @Gingergrrl ,
I really do not have the knowledge and understanding to compare and contrast your illness with that which Is described in this research paper, so please take my comments with a grain of salt.

It would appear to me that there may be some parallels between the two, but also a distinct difference. Both appear to result in reduced intra-cellular calcium. If I understand the research paper, they are suggesting that this calcium deficit within the cell can cause many of the ME/CFS symptoms we experience.

The primary difference would be the cause and possibly the target cells. Your illness is caused by autoantibodies effecting the N-type Calcium Channels while the research paper describes impaired function of the TRPM3 ion channel in immune cells. I do not know enough about N-type CCs and TRPM3 CCs to understand how it could impact you differently. If these are effecting two distinct sets of cells, I assume the resulting symptoms would be related to what functions those cells were responsible for.

I would think this line of research could be interesting for you to follow, especially for understanding the downstream effects of the calcium deficits.

If anyone can provide better input and clarification or correction to my speculation above, please do so.

Thanks,
Dan
 

Gingergrrl

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@Inara I'm tagging you again (and am not sure why the first tag did not work) but this is the thread that I was referring to...

Interesting info. My tests were slightly positive for N-type Calcium Channel AB; however, I am not sure what to make of it yet. I am trying to see Dr. Steven Vernino who helped design the Mayo DYS1 panel. Hopefully, I can get help straight from the expert.
I am SO hoping that you will still get to see Dr. Vernino (so you can be my proxy :nerd: ) and I can learn his interpretation of what it means to test positive for the N-type Calcium Channel Auto-AB on the Mayo Panels as I have from 2016 to the present (and possibly earlier but will never know).

I have never tried a prescription CCB; however, I feel wonderful on Mg Glycinate 100 mg TID. Mg acts as a natural CCB.
This is fascinating to me since I don't do well with too much Mg (let alone a CCB).

Hi @Gingergrrl, I really do not have the knowledge and understanding to compare and contrast your illness with that which Is described in this research paper, so please take my comments with a grain of salt.
Grain of salt taken and I really appreciate your explanation and comments.

It would appear to me that there may be some parallels between the two, but also a distinct difference. Both appear to result in reduced intra-cellular calcium.
Thx for clarifying that both mechanisms results in reduced intra-cellular calcium (and I actually was not sure).

The primary difference would be the cause and possibly the target cells. Your illness is caused by autoantibodies effecting the N-type Calcium Channels while the research paper describes impaired function of the TRPM3 ion channel in immune cells.
I hope I can ask this where it makes sense... (to Dan or anyone reading :D)

1) Is the impaired function of the TRPM3 ion channel (in the paper) also of an autoimmune mechanism or is it something different?

2) Does the mechanism that was found in the paper ONLY pertain to NK cells or to all cells?

3) Is the mechanism in the paper considered a "calcium channelopathy" (like mine)? If yes, then it seems that whatever was found in the paper is very similar to whatever my illness will ultimately be named. Did the subjects in the paper have muscle weakness or dysautonomia?

I read these abstracts but don't know how to follow them, or how put the pieces together, or to compare them to my own situation.

I do not know enough about N-type CCs and TRPM3 CCs to understand how it could impact you differently. If these are effecting two distinct sets of cells, I assume the resulting symptoms would be related to what functions those cells were responsible for.
That all makes sense and makes me assume (which might be totally incorrect) that the TRMP3 calcium channels are in the NK cells but the N-type (mine) are in other types of cells?

I would think this line of research could be interesting for you to follow, especially for understanding the downstream effects of the calcium deficits.
It is extremely interesting to follow and frustrates me to no end that I am not able to understand it better :bang-head:
 

Inara

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I tried calcium blockers it made me very very sick. So i don't understand this finding.
The researchers found lowered intracellular calcium. Lowering it further (e.g. by blockage) is maybe not the right thing to do...assuming the calcium blocker you used really blocks TRPM3 channels (I somehow don't think so).

Calcium signaling is much more than "too low/too high intracellular calcium", it's a finely tuned system where all the calcium channels interplay. I would be highly interested in knowing the quality of the calcium channel they measured, not only the quantity. And what the other channels are doing while they measure TRPM3.

Because in NK cells, TRPM3 isn't the only calcium channel. There are ORAI1 and IP3Rs, L-type voltage gated calcium channels, TRPM2, most probably also RyRs and others. Which channel did what? An example: Researchers wanted to study IP3R. For this, they looked at the calcium flux of ORAI1, assuming that, since ORAI1 only opens if endoplasmic reticulum calcium stores are empty (so-called SOCE), IP3R works normal if ORAI1 does. This is an incorrect assumption: You cannot deduce from ORAI1 to IP3R (or the other way around), you can only say, yes, IP3R spits out calcium so that ORAI1 can work properly (i.e. the quantity is normal), but what about the calcium signal quality which directs so many processes? Is sth similar happening here?

Ok, voltage-gated channels need a certain voltage to open, so quantity plays an important role here. Does quality, too? I don't know enough about voltage-gated calcium channels to answer that. I do wonder if the researchers are looking at a TRPM3 effect or a downstream effect. Can someone tell from their "Methods" chapter? I don't see any mention of other channels and if and how they silenced them.

Authors said:
TRPM3 currents were stimulated by adding 100 μM PregS (Tocris Bioscience, Bristol, UK) or 100 μM nifedipine (Sapphire Bioscience, NSW, Australia) to the bath solution, whereas PregS- and nifedipine-induced TRPM3 currents were blocked by adding 10 μM ononetin (Tocris Bioscience, Bristol, UK).
Do PregS and nifedipine only affect TRPM3? It seems nifedipine blocks L-type calcium channels, too, so no.
 

Inara

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It appears lack of calcium in the cell could lead to inactivation of PDH.
This is correct. Calcium (via IP3R3 on the endoplasmic reticulum to some voltage-gated calcium channel, whose name I forgot, on the mitochondria associated membrane into the mitochondrion) is needed to activate PDH.

There can be many, many reasons why not enough calcium (or the wrong calcium signal) reaches the mitochondria, it doesn't have to be TRPM3. It could be one reason.
 

Inara

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Does this study match with my case, or is it the opposite, or is it not related? I wish I could understand these studies!
L-type calcium channels and TRPM3 are different (how exactly, please don't ask :) ). As you know, in my case another calcium channel is affected, so that I start to think that many ways lead to Rome, i.e. different (let me call it) calcium channelopathies maybe lead to similar symptoms, maybe even to very similar pathomechanisms. Because if one part in the calcium signaling cascade is affected, the rest may be too.

1) Is the impaired function of the TRPM3 ion channel (in the paper) also of an autoimmune mechanism or is it something different?
This isn't known yet. It could be autoimmunity, it could be "mutated" channels, it could be something else (viruses?).

2) Does the mechanism that was found in the paper ONLY pertain to NK cells or to all cells?
Not many human calcium channels can be researched, human NK cells seem to be suited (at least this is how I understood a researcher who explained to me the tests they will be doing on "my" calcium channel).

3) Is the mechanism in the paper considered a "calcium channelopathy" (like mine)?
I don't know. Re. TRPM3 this is too early I think. It would make sense to call a calcium channel that produces a dysfunctional calcium signal (and which leads to symptoms/disease) a "calcium channelopathy" , wouldn't it?
 
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