calcium ion channels – American ME and CFS Society

Investigation into the restoration of TRPM3 ion channel activity in post-COVID-19 condition: a potential pharmacotherapeutic target

Abstract:

Introduction: Recently, we reported that post COVID-19 condition patients also have Transient Receptor Potential Melastatin 3 (TRPM3) ion channel dysfunction, a potential biomarker reported in natural killer (NK) cells from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) patients. As there is no universal treatment for post COVID-19 condition, knowledge of ME/CFS may provide advances to investigate therapeutic targets. Naltrexone hydrochloride (NTX) has been demonstrated to be beneficial as a pharmacological intervention for ME/CFS patients and experimental investigations have shown NTX restored TRPM3 function in NK cells. This research aimed to: i) validate impaired TRPM3 ion channel function in post COVID-19 condition patients compared with ME/CFS; and ii) investigate NTX effects on TRPM3 ion channel activity in post COVID-19 condition patients.

Methods: Whole-cell patch-clamp was performed to characterize TRPM3 ion channel activity in freshly isolated NK cells of post COVID-19 condition (N = 9; 40.56 ± 11.26 years), ME/CFS (N = 9; 39.33 ± 9.80 years) and healthy controls (HC) (N = 9; 45.22 ± 9.67 years). NTX effects were assessed on post COVID-19 condition (N = 9; 40.56 ± 11.26 years) and HC (N = 7; 45.43 ± 10.50 years) where NK cells were incubated for 24 hours in two protocols: treated with 200 µM NTX, or non-treated; TRPM3 channel function was assessed with patch-clamp protocol.

Results: This investigation confirmed impaired TRPM3 ion channel function in NK cells from post COVID-19 condition and ME/CFS patients. Importantly, PregS-induced TRPM3 currents were significantly restored in NTX-treated NK cells from post COVID-19 condition compared with HC. Furthermore, the sensitivity of NK cells to ononetin was not significantly different between post COVID-19 condition and HC after treatment with NTX.

Discussion: Our findings provide further evidence identifying similarities of TRPM3 ion channel dysfunction between ME/CFS and post COVID-19 condition patients. This study also reports, for the first time, TRPM3 ion channel activity was restored in NK cells isolated from post COVID-19 condition patients after in vitro treatment with NTX. The TRPM3 restoration consequently may re-establish TRPM3-dependent calcium (Ca²⁺) influx. This investigation proposes NTX as a potential therapeutic intervention and TRPM3 as a treatment biomarker for post COVID-19 condition.

Source: Etianne Martini Sasso, Katsuhiko Muraki, Natalie Eaton-Fitch, Peter Smith, Andrew Jeremijenko, Paul Griffin, Sonya Marshall-Gradisnik. Investigation into the restoration of TRPM3 ion channel activity in post-COVID-19 condition: a potential pharmacotherapeutic target. Front. Immunol., 02 May 2024; Sec. Multiple Sclerosis and Neuroimmunology; Volume 15 – 2024 | https://doi.org/10.3389/fimmu.2024.1264702. https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1264702/full (Full text)

Altered TRPM7-Dependent Calcium Influx in Natural Killer Cells of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling multisystemic condition. The pathomechanism of ME/CFS remains unestablished; however, impaired natural killer (NK) cell cytotoxicity is a consistent feature of this condition. Calcium (Ca²⁺) is crucial for NK cell effector functions.

Growing research recognises Ca²⁺ signalling dysregulation in ME/CFS patients and implicates transient receptor potential ion channel dysfunction. TRPM7 (melastatin) was recently considered in the pathoaetiology of ME/CFS as it participates in several Ca²⁺-dependent processes that are central to NK cell cytotoxicity which may be compromised in ME/CFS. TRPM7-dependent Ca²⁺ influx was assessed in NK cells isolated from n = 9 ME/CFS patients and n = 9 age- and sex-matched healthy controls (HCs) using live cell fluorescent imaging techniques.

Slope (p < 0.05) was significantly reduced in ME/CFS patients compared with HCs following TRPM7 activation. Half-time of maximal response (p < 0.05) and amplitude (p < 0.001) were significantly reduced in the HCs compared with the ME/CFS patients following TRPM7 desensitisation.

Findings from this investigation suggest that TRPM7-dependent Ca²⁺ influx is reduced with agonism and increased with antagonism in ME/CFS patients relative to the age- and sex-matched HCs. The outcomes reported here potentially reflect TRPM3 dysfunction identified in this condition suggesting that ME/CFS is a TRP ion channelopathy.

Source: Du Preez S, Eaton-Fitch N, Smith PK, Marshall-Gradisnik S. Altered TRPM7-Dependent Calcium Influx in Natural Killer Cells of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. Biomolecules. 2023; 13(7):1039. https://doi.org/10.3390/biom13071039 https://www.mdpi.com/2218-273X/13/7/1039 (Full text)

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

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious disorder of unknown aetiology. While the pathomechanism of ME/CFS remains elusive, reduced natural killer (NK) cell cytotoxic function is a consistent immunological feature. NK cell effector functions rely on long-term sustained calcium (Ca²⁺) influx. In recent years evidence of transient receptor potential melastatin 3 (TRPM3) dysfunction supports the hypothesis that ME/CFS is potentially an ion channel disorder. Specifically, reports of single nucleotide polymorphisms, low surface expression and impaired function of TRPM3 have been reported in NK cells of ME/CFS patients. It has been reported that mu (µ)-opioid receptor (µOR) agonists, known collectively as opioids, inhibit TRPM3. Naltrexone hydrochloride (NTX), a µOR antagonist, negates the inhibitory action of µOR on TRPM3 function. Importantly, it has recently been reported that NTX restores impaired TRPM3 function in NK cells of ME/CFS patients.

Methods: Live cell immunofluorescent imaging was used to measure TRPM3-dependent Ca²⁺ influx in NK cells isolated from n = 10 ME/CFS patients and n = 10 age- and sex-matched healthy controls (HC) following modulation with TRPM3-agonist, pregnenolone sulfate (PregS) and TRPM3-antaognist, ononetin. The effect of overnight (24 h) NTX in vitro treatment on TRPM3-dependent Ca²⁺ influx was determined.

Results: The amplitude (p < 0.0001) and half-time of Ca²⁺ response (p < 0.0001) was significantly reduced at baseline in NK cells of ME/CFS patients compared with HC. Overnight treatment of NK cells with NTX significantly improved TRPM3-dependent Ca²⁺ influx in ME/CFS patients. Specifically, there was no significance between HC and ME/CFS patients for half-time response, and the amplitude of Ca²⁺ influx was significantly increased in ME/CFS patients (p < 0.0001).

Conclusion: TRPM3-dependent Ca²⁺ influx was restored in ME/CFS patients following overnight treatment of isolated NK cells with NTX in vitro. Collectively, these findings validate that TRPM3 loss of function results in altered Ca²⁺ influx supporting the growing evidence that ME/CFS is a TRP ion channel disorder and that NTX provides a potential therapeutic intervention for ME/CFS.

Source: Eaton-Fitch N, Du Preez S, Cabanas H, Muraki K, Staines D, Marshall-Gradisnik S. Impaired TRPM3-dependent calcium influx and restoration using Naltrexone in natural killer cells of myalgic encephalomyelitis/chronic fatigue syndrome patients. J Transl Med. 2022 Feb 16;20(1):94. doi: 10.1186/s12967-022-03297-8. PMID: 35172836. https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-022-03297-8 (Full text)

Loss of Transient Receptor Potential Melastatin 3 ion channel function in natural killer cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients

Abstract:

BACKGROUND: Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME) is a debilitating disorder that is accompanied by reduced cytotoxic activity in natural killer (NK) cells. NK cells are an essential innate immune cell, responsible for recognising and inducing apoptosis of tumour and virus infected cells. Calcium is an essential component in mediating this cellular function. Transient Receptor Potential Melastatin 3 (TRPM3) cation channels have an important regulatory role in mediating calcium influx to help maintain cellular homeostasis. Several single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in patients with CFS/ME and have been proposed to correlate with illness presentation. Moreover, a significant reduction in both TRPM3 surface expression and intracellular calcium mobilisation in NK cells has been found in CFS/ME patients compared with healthy controls. Despite the functional importance of TRPM3, little is known about the ion channel function in NK cells and the epiphenomenon of CFS/ME. The objective of the present study was to characterise the TRPM3 ion channel function in NK cells from CFS/ME patients in comparison with healthy controls using whole cell patch-clamp techniques.

METHODS: NK cells were isolated from 12 age- and sex-matched healthy controls and CFS patients. Whole cell electrophysiology recording has been used to assess TRPM3 ion channel activity after modulation with pregnenolone sulfate and ononetin.

RESULTS: We report a significant reduction in amplitude of TRPM3 current after pregnenolone sulfate stimulation in isolated NK cells from CFS/ME patients compared with healthy controls. In addition, we found pregnenolone sulfate-evoked ionic currents through TRPM3 channels were significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients.

CONCLUSIONS: TRPM3 activity is impaired in CFS/ME patients suggesting changes in intracellular Ca2+ concentration, which may impact NK cellular functions. This investigation further helps to understand the intracellular-mediated roles in NK cells and confirm the potential role of TRPM3 ion channels in the aetiology and pathomechanism of CFS/ME.

Source: Cabanas H, Muraki K, Eaton N, Balinas C, Staines D, Marshall-Gradisnik S. Loss of Transient Receptor Potential Melastatin 3 ion channel function in natural killer cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients. Mol Med. 2018 Aug 14;24(1):44. doi: 10.1186/s10020-018-0046-1.

New light shed on cause of chronic fatigue syndrome

New research findings may shed new light on the potential cause of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME).

Researchers from Griffith University’s National Centre for Neuroimmunology and Emerging Diseases (NCNED) — part of the new Menzies Health Institute Queensland — have uncovered significant factors contributing to the pathology of this illness.

The results reveal genetic changes in important receptors associated with immunological and cellular function and contribute to the development of this complex illness.

“These findings have been achieved through a team effort involving researchers, patients, funding bodies, clinicians and the support of Griffith University and the Queensland Government,” say chief investigators Professor Sonya Marshall-Gradisnik and Professor Donald Staines.

Co-researcher and consultant immunologist Professor Pete Smith said that important signalling mechanisms are disrupted as a result of these genetic changes involving the detection and response to threats.

“These are primitive genes that are involved in many cellular signals in the brain, gut, cardiovascular and immune systems, as well as in the mediation of pain.”

These research findings coincide with International Neuroimmune Awareness week commencing Monday 11 May.

The Griffith Health Centre on the university’s Gold Coast campus is being lit up each evening from 10 -12 May to raise awareness of neurological conditions such as CFS/ME as well as other conditions such as Fibromyalgia and Gulf War Syndrome.

“The lighting up of the Griffith Health Centre signifies Griffith’s commitment to the CFS patient community and our team approach to this research,” says Pro-Vice Chancellor (Health) Professor Allan Cripps.

CFS/ME is a highly debilitating disorder characterized by profound fatigue, muscle and joint pain, cerebral symptoms of impaired memory and concentration, impaired cardiovascular function, gut disorder and sensory dysfunction such as noise intolerance and balance disturbance. Many cases can continue for months or years. It is believed to affect around 250,000 Australians.

The research findings are to be presented at an international conference in London later this month.

Journal Reference: Sonya Marshall-Gradisnik, Donald Staines, Pete Smith, Bernd Nilius, Ekua Brenu, Sandra Ramos. Examination of Single Nucleotide Polymorphisms (SNPs) in Transient Receptor Potential (TRP) Ion Channels in Chronic Fatigue Syndrome Patients. Immunology and Immunogenetics Insights, 2015; 1 DOI: 10.4137/III.S25147

Source: Griffith University. “New light shed on cause of chronic fatigue syndrome.” ScienceDaily. ScienceDaily, 11 May 2015. https://www.sciencedaily.com/releases/2015/05/150511172755.htm

Single nucleotide polymorphisms and genotypes of transient receptor potential ion channel and acetylcholine receptor genes from isolated B lymphocytes in myalgic encephalomyelitis/chronic fatigue syndrome patients

Abstract:

OBJECTIVE: The pathomechanism of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is unknown; however, a small subgroup of patients has shown muscarinic antibody positivity and reduced symptom presentation following anti-CD20 intervention. Given the important roles of calcium (Ca2+) and acetylcholine (ACh) signalling in B cell activation and potential antibody development, we aimed to identify relevant single nucleotide polymorphisms (SNPs) and genotypes in isolated B cells from CFS/ME patients.

METHODS: A total of 11 CFS/ME patients (aged 31.82 ± 5.50 years) and 11 non-fatigued controls (aged 33.91 ± 5.06 years) were included. Flow cytometric protocols were used to determine B cell purity, followed by SNP and genotype analysis for 21 mammalian TRP ion channel genes and nine mammalian ACh receptor genes. SNP association and genotyping analysis were performed using ANOVA and PLINK analysis software.

RESULTS: Seventy-eight SNPs were identified in nicotinic and muscarinic acetylcholine receptor genes in the CFS/ME group, of which 35 were in mAChM3. The remaining SNPs were identified in nAChR delta (n = 12), nAChR alpha 9 (n = 5), TRPV2 (n = 7), TRPM3 (n = 4), TRPM4 (n = 1) mAChRM3 2 (n = 2), and mAChRM5 (n = 3) genes. Nine genotypes were identified from SNPs in TRPM3 (n = 1), TRPC6 (n = 1), mAChRM3 (n = 2), nAChR alpha 4 (n = 1), and nAChR beta 1 (n = 4) genes, and were located in introns and 3′ untranslated regions. Odds ratios for these specific genotypes ranged between 7.11 and 26.67 for CFS/ME compared with the non-fatigued control group.

CONCLUSION: This preliminary investigation identified a number of SNPs and genotypes in genes encoding TRP ion channels and AChRs from B cells in patients with CFS/ME. These may be involved in B cell functional changes, and suggest a role for Ca2+ dysregulation in AChR and TRP ion channel signalling in the pathomechanism of CFS/ME.

Source: Marshall-Gradisnik S, Johnston S, Chacko A, Nguyen T, Smith P, Staines D. Single nucleotide polymorphisms and genotypes of transient receptor potential ion channel and acetylcholine receptor genes from isolated B lymphocytes in myalgic encephalomyelitis/chronic fatigue syndrome patients. J Int Med Res. 2016 Nov 10. pii: 0300060516671622. [Epub ahead of print] https://www.ncbi.nlm.nih.gov/pubmed/27834303

Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels

Abstract:

Transient receptor potential melastatin subfamily 3 (TRPM3) ion channels play a role in calcium (Ca2+ ) cell signalling. Reduced TRPM3 protein expression has been identified in chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients. However, the significance of TRPM3 and association with intracellular Ca2+ mobilization has yet to be determined.

Fifteen CFS/ME patients (mean age 48·82 ± 9·83 years) and 25 healthy controls (mean age 39·2 ± 12·12 years) were examined. Isolated natural killer (NK) cells were labelled with fluorescent antibodies to determine TRPM3, CD107a and CD69 receptors on CD56dim CD16+ NK cells and CD56bright CD16dim/- NK cells. Ca2+ flux and NK cytotoxicity activity was measured under various stimulants, including pregnenolone sulphate (PregS), thapsigargin (TG), 2-aminoethoxydiphenyl borate (2APB) and ionomycin.

Unstimulated CD56bright CD16dim/- NK cells showed significantly reduced TRPM3 receptors in CFS/ME compared with healthy controls (HC). Ca2+ flux showed no significant difference between groups. Moreover, PregS-stimulated CD56bright CD16dim/- NK cells showed a significant increase in Ca2+ flux in CFS/ME patients compared with HC. By comparison, unstimulated CD56dim CD16+ NK cells showed no significant difference in both Ca2+ flux and TRPM3 expression. PregS-stimulated CD56dim CD16+ NK cells increased TRPM3 expression significantly in CFS/ME, but this was not associated with a significant increase in Ca2+ flux.

Furthermore, TG-stimulated CD56dim CD16+ NK cells increased K562 cell lysis prior to PregS stimulation in CFS/ME patients compared with HC. Differential expression of TRPM3 and Ca2+ flux between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in CFS/ME.

Source: Nguyen T, Johnston S, Clarke L, Smith P, Staines D, Marshall-Gradisnik S. Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels. Clin Exp Immunol. 2017 Feb;187(2):284-293. doi: 10.1111/cei.12882. Epub 2016 Nov 23. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217865/ (Full article)

Natural killer cells and single nucleotide polymorphisms of specific ion channels and receptor genes in myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

AIM: The aim of this paper was to determine natural killer (NK) cytotoxic activity and if single nucleotide polymorphisms (SNPs) and genotypes in transient receptor potential (TRP) ion channels and acetylcholine receptors (AChRs) were present in isolated NK cells from previously identified myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) patients.

SUBJECTS AND METHODS: A total of 39 ME/CFS patients (51.69±2 years old) and 30 unfatigued controls (47.60±2.39 years old) were included in this study. Patients were defined according to the 1994 Centers for Disease Control and Prevention criteria. Flow cytometry protocols were used to examine NK cytotoxic activity. A total of 678 SNPs from isolated NK cells were examined for 21 mammalian TRP ion channel genes and for nine mammalian AChR genes via the Agena Bioscience iPlex Gold assay. SNP association and genotype was determined using analysis of variance and Plink software.

RESULTS: ME/CFS patients had a significant reduction in NK percentage lysis of target cells (17%±4.68%) compared with the unfatigued control group (31%±6.78%). Of the 678 SNPs examined, eleven SNPs for TRP ion channel genes (TRPC4, TRPC2, TRPM3, and TRPM8) were identified in the ME/CFS group. Five of these SNPs were associated with TRPM3, while the remainder were associated with TRPM8, TRPC2, and TRPC4 (P<0.05). Fourteen SNPs were associated with nicotinic and muscarinic AChR genes: six with CHRNA3, while the remainder were associated with CHRNA2, CHRNB4, CHRNA5, and CHRNE (P<0.05). There were sixteen genotypes identified from SNPs in TRP ion channels and AChRs for TRPM3 (n=5), TRPM8 (n=2), TRPC4 (n=3), TRPC2 (n=1), CHRNE (n=1), CHRNA2 (n=2), CHRNA3 (n=1), and CHRNB4 (n=1) (P<0.05).

CONCLUSION: We identified a number of SNPs and genotypes for TRP ion channels and AChRs from isolated NK cells in patients with ME/CFS, suggesting these SNPs and genotypes may be involved in changes in NK cell function and the development of ME/CFS pathology. These anomalies suggest a role for dysregulation of Ca(2+) in AChR and TRP ion channel signaling in the pathomechanism of ME/CFS.

Source: Marshall-Gradisnik S, Huth T, Chacko A, Johnston S, Smith P, Staines D. Natural killer cells and single nucleotide polymorphisms of specific ion channels and receptor genes in myalgic encephalomyelitis/chronic fatigue syndrome. Appl Clin Genet. 2016 Mar 31;9:39-47. doi: 10.2147/TACG.S99405. ECollection 2016. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821384/ (Full article)