Deficient TRPM3-linked mitochondrial Ca2+ influx in natural killer cells associated with [ME/CFS], 2026, Magawa et al

[forestglip]

Deficient TRPM3-linked mitochondrial Ca2+ influx in natural killer cells associated with myalgic encephalomyelitis/chronic fatigue syndrome

Magawa, Chandi Tabeth; Eaton-Fitch, Natalie; Muraki, Katsuhiko; Marshall-Gradisnik, Sonya

Introduction
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystemic illness, commonly associated with dysregulation of the immune system including reduced cytotoxicity of natural killer (NK) cells and post-exertional neuroimmune exhaustion. Previously, transient receptor potential melastatin 3 (TRPM3) ion channel impairment associated with reduced Ca2+ mobilisation in NK cells from ME/CFS patients was reported.

To further explore the pathomechanisms involved in ME/CFS, we investigated the downstream impact of TRPM3 ion channel dysfunction on mitochondrial Ca2+ mobilisation in NK cells.

Method
Fluorescence live-cell imaging was used to investigate Ca2+ mobilisation in NK cells of (N = 10) ME/CFS, classified using Canadian Consensus Criteria, and (N = 10) healthy control (HC) participants. Cytoplasmic and mitochondrial Ca2+ entry was determined using Fluo-8 AM and Rhod-2 AM Ca2+ indicators, respectively.

The effect of TRPM3 modulation on Ca2+ mobilisation ex vivo, was examined using pregnenolone sulfate and ononetin to activate and inhibit the channel, respectively.

Results
Cytosolic Ca2+ influx amplitude and slope were significantly reduced (p < 0.001), with a significantly shorter T1/2 response (p = 0.001) in ME/CFS compared to HC. Ca2+ influx amplitude (p < 0.001) and slope (p < 0.041) into the mitochondria were significantly higher in ME/CFS compared to HC.

TRPM3 activation triggered pronounced cytosolic response (P < 0.001) accompanied by mitochondrial Ca2+ increase in HC. TRPM3-dependent cytosolic and mitochondrial Ca2+ mobilisation (P < 0.015) were significantly reduced with a shorter T1/2 response (p < 0.02) in ME/CFS compared to HC.

Conclusion
The results demonstrate that altered TRPM3-mediated cytosolic Ca2+ influx may significantly impact Ca2+ mobilisation into the mitochondria of people with ME/CFS. Alterations that interfere with the optimal function of Ca2+ permeable channels may cumulatively impact downstream signalling, leading to detrimental cellular consequences.

Collectively these findings provide an avenue for further studies on the physiological functions of TRPM3 ion channel and its role in ME/CFS.

Web | DOI | PDF | BMC Immunology | Open Access

 

[forestglip]

There was discussion about pseudoreplication potentially being an issue in previous papers from this group. See the thread for Sasso 2025 and a PubPeer comment about the paper.

Based on a quick Google Scholar search, I think this is the first paper authored by Marshall-Gradisnik that mentions pseudoreplication. It says, in the methods and discussion, respectively:

GraphPad formed statistical comparison by participant, not by individual cell, eliminating risk of pseudoreplication (40). Cell recordings were excluded if they returned an outlier value for one of the parameters (slope, T1/2, and amplitude) to minimise variability. The Shapiro-Wilk test was used for normality test, with the Mann Whitney U tests performed on nonparametric data. A p-value < 0.05 was considered statistically significant.

Due to the strict selection criteria applied for participant inclusion in this study, the sample size for this study was small. Therefore, many cells were analysed per participant, strategically increasing the population of cells analysed per group. Thus, n = 554 NK cells from N = 10 HC were compared with n = 415 NK cells from N=10 ME/CFS patients for cytosolic Ca2+ influx examination only, with a total of six different conditions performed per group. However, GraphPad formed statistical comparison by participant, not by individual cell, eliminating risk of pseudoreplication (40).

I've never used GraphPad, so I don't know what exactly "GraphPad formed statistical comparison by participant, not by individual cell" means. If it means the values from each participant were averaged, that would be sufficient to avoid pseudoreplication, but it would be preferable to see more details about what exactly was done.

There are still many very low p-values (p<.001)

Whether or not pseudoreplication is responsible for the very low p-values, all of their many studies on the topic show the same direction of effect for these TRPM3-related findings. This only seems likely to me if one of the following is true: 1) there is a real effect, 2) there are many unpublished studies with opposite direction of effect, or 3) there is some issue with the lab procedures.

Maybe it is worth a separate lab trying to replicate their findings. Maybe it could be just one of the findings, like decreased Ca2+ entry into NK cells:

Confirming previous studies, [...] average Ca2+ influx amplitude (Fig.1B, p < 0.001) into the cytosol was significantly reduced in ME/CFS patients compared to HC, following the addition of 1.8mM Ca2+ buffer.