# paper.pdf ## Metadata - PDFFormatVersion=1.6 - Language=en - IsLinearized=true - IsAcroFormPresent=false - IsXFAPresent=false - IsCollectionPresent=false - IsSignaturesPresent=false - CreationDate=D:20260708180446+05'30' - Creator=LaTeX with hyperref package - ModDate=D:20260708180522+05'30' - Producer=Acrobat Distiller 26.0 (Windows) - Title=Altered TRPM3‐Dependent Cytosolic and Mitochondrial Calcium Influx in Natural Killer Cells of Post‐COVID‐19 Condition Patients - Custom.WPS-ARTICLEDOI=10.1002/eji.70240 - Custom.WPS-JOURNALDOI=10.1002/(ISSN)1521-4141 - Custom.WPS-PROCLEVEL=3 - xmp:createdate=2015-08-18T08:05:36+05:30 - xmp:modifydate=2016-06-21T14:17:29-04:00 - xmp:metadatadate=2016-06-21T14:17:29-04:00 - pdf:producer=Acrobat Distiller 9.0 (Windows) - pdfx:wps-articledoi=10.1002/eji.70240 - pdfx:wps-journaldoi=10.1002/(ISSN)1521-4141 - pdfx:wps-proclevel=3 - jav:journal_article_version=VoR - prism:doi=10.1002/eji.70240 - dc:format=application/pdf - dc:title=Altered TRPM3‐Dependent Cytosolic and Mitochondrial Calcium Influx in Natural Killer Cells of Post‐COVID‐19 Condition Patients - xmpmm:documentid=uuid:1405c44d-9096-34e6-81da-a85197a96014 - xmpmm:instanceid=uuid:9495f05a-1630-39ed-ac01-77c7bc29b0d2 ## Contents ### Page 1 European Journal of Immunology European Journal of Immunology ![logo]() ![logo]() RESEARCH ARTICLE ![logo]() Immunity to Infection # Altered TRPM3-Dependent Cytosolic and MitochondrialCalcium Influx in Natural Killer Cells of Post-COVID-19Condition Patients Chandi Tabeth Magawa,, Natalie Eaton-Fitch, Katsuhiko Muraki Sonya Marshall-Gradisnik, 1 National Centre for Neuroimmunology and Emerging Diseases, Health Group, Griffith University, Gold Coast Campus, Gold Coast, Australia 2 ConsortiumHealth International For Myalgic Encephalomyelitis, Griffith University, Gold Coast Campus, Gold Coast, Australia 3 School of Pharmacy and MedicalSciences, Griffith University, Gold Coast Campus, Gold Coast, Australia 4 Laboratory of Cellular Pharmacology, School of Pharmacy, Aichi-Gakuin University,Nagoya, Japan Correspondence: Chandi Tabeth Magawa (chandi.magawa@griffithuni.edu.au; c.magawa@griffith.edu.au) Received: 9 January 2026 Revised: 1 June 2026 Accepted: 29 June 2026 Keywords: calcium signalling mitochondria natural killer cells post-COVID-19 condition TRP channels TRPM3 ABSTRACTAccording to the World Health Organization (WHO), approximately 6% of COVID-19 cases develop serious long-term sequelaereferred to as post-COVID-19 condition (PCC). Immunological disturbances such as persistent activation of immune cells andreduced cytotoxicity by natural killer (NK) cells are reported as key aspects in PCC. Recently, electrophysiological studies byour group demonstrated impairment of transient receptor potential melastatin 3 (TRPM3) ion channels in NK cells from PCCpatients. The significant reduction in TRPM3 channel function and reduced functional activity by NK cells warrants furtherinvestigation. Hence, using live cell calcium (Ca2+ ) imaging ex vivo, we examined the downstream impact of TRPM3 ion channeldysfunction on intracellular and mitochondrial Ca2+ mobilization in NK cells from N = 8 PCC patients, age and sex matchedto N = 8 PCC healthy controls (HC). Our findings provide new evidence of altered passive and TRPM3-mediated Ca2+ influx,significantly impacting cytoplasmic and mitochondrial Ca2+ mobilization in PCC. Passive cytosolic Ca2+ influx amplitude (p< 0.0001) was significantly reduced in PCC; however, passive mitochondrial Ca2+ mobilization (p < 0.0001) was significantlyincreased. Importantly, cytoplasmic and mitochondrial response rates (slope, p < 0.001) to pregnenolone sulphate stimulationwere significantly reduced in PCC. Consequently, TRPM3-dependent cytosolic (p < 0.001) and mitochondrial (p < 0.0005) Ca2+mobilization were significantly reduced in PCC compared with HC. Altered ion channel Ca2+ signalling can severely impactboth the immune system and bioenergetic processes, potentially leading to broader systemic dysregulations underpinning thepathomechanism of the PCC condition, and warrants further investigations. Abbreviations: ATP, adenosine triphosphate; BMI, body mass index; Ca2+ , calcium; CaM, calmodulin; CNS, central nervous system; COVID-19, coronavirus disease 2019;CRAC, calcium-release-activated channel; ER/SR, endoplasmic/sarcoplasmic reticulum; F0, minimum fluorescence; FBC, full blood count; FCCP, carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone; Fmax, fluorescence at maximum peak; HC, healthy control; MCC, Manders’ colocalization coefficients; ME/CFS, myalgicencephalomyelitis/chronic fatigue syndrome; N, number of participants; NCNED, National Centre for Neuroimmunology and Emerging Diseases; NK cells, natural killercells; PCC, post-COVID-19 condition; Pearson’s r, Pearson’s correlation coefficient; PregS, pregnenolone sulfate; RFI, relative fluorescence intensity; ROI, region of interest;SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SD, standard deviation; SERCA, sarco/endoplasmic reticulum calcium-ATPase; SF-36, 36-item short-formhealth survey; SOCE, store operated Ca2+ entry; SPSS, statistical package for the social sciences; Stim1, stromal interaction molecule 1; T1 , time at minimum fluorescenceintensity; T1/2 , half-time to peak Ca2+ influx; T2 , time at maximum fluorescence intensity; TRP, transient receptor potential; TRPM3, transient receptor potential melastatin3; WHO, World Health Organization. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided theoriginal work is properly cited. © 2026 The Author(s). European Journal of Immunology published by Wiley-VCH GmbH. European Journal of Immunology, 2026; 56:e70240 https://doi.org/10.1002/eji.70240 ### Page 2 ## 1 Background The repercussions of the coronavirus disease 2019 (COVID-19)caused by the severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) remain a global public health concern [].Although most people with COVID-19 recover fully within 1month after infection, approximately 6% of cases are reportedto develop serious long-term sequelae post-infection []. LongCOVID, post-COVID-19 condition (PCC), post-acute sequelae ofSARS-CoV-2 (PASC) and long-haul COVID are interchangeablyused to describe the persistent and chronic symptoms followingacute infection []. The World Health Organization (WHO)defined PCC as occurring in patients with a history of probableor confirmed SARS-CoV-2 infection, usually three months fromthe acute infection, with symptoms that last for two or moremonths and that an alternative diagnosis cannot explain [].Patients with PCC exhibit a constellation of symptoms such aschronic fatigue, shortness of breath, cognitive disturbances, sleepdisturbances, dyspnoea, myalgia and post-exertional malaise,which indicate interconnected consequences of disruptions inmultiple body systems []. Currently, there is an absenceof a universal standard laboratory-based diagnostic tests, hencediagnosis for PCC is reliant on the fulfilment of clinical casedefinitions [].Although the pathomechanism underlying the extended per-sistent symptoms exhibited in PCC is yet to be elucidated,research has demonstrated structural and functional dysregula-tion of neurological, immunological, cardiovascular, pulmonaryand metabolic systems []. A key feature is immunedysregulation, potentially induced by numerous factors suchas the presence of viral reservoirs, viral fragments, metabolicdysregulation and complex immune responses to SARS-CoV-2 infection []. Immunological dysregulations such aspersistent activation of immune cells, such as neutrophils andmonocytes, altered immune communication, exhausted T cells,altered B cell function, elevated cytokine levels and functionallyimpaired natural killer (NK) cells in PCC, have been reported invarious studies []. Furthermore, previous researchin severely ill COVID-19 patients has reported suppression ofgenes involved in cytotoxic activity in CD8 T lymphocytes and NKcells, which was associated with disease severity [].In humans, NK cells are part of the innate immune system thatform the first line of defence against viral infections by both lysingtarget cells and producing early immunoregulatory cytokines[]. They are identified by the surface expression of the neuralcell-adhesion molecule CD56 (positive), the low-affinity Fc IgGreceptor III (FcγRIII, CD16) and negative expression of the CD3T cell marker []. Functionally, the CD3− CD56dim CD16brightsubset has cytotoxic properties, and the CD3− CD56bright CD16dimsubset has immunoregulatory roles []. Persistent NK cellfunctional disturbances associated with disease severity werereported in patients with COVID-19 []. The regulation of NKcell function is reliant on a synergistic network of receptors, pro-teins, cytolytic granules and the dynamic mitochondrial function,which all together influence their target killing potential throughprecise regulation of intracellular Ca2+ ions, that modulates theireffector efficacy []. In activated NK cells, Ca2+drives signalling, making the ion essential for lytic granule polar- isation, immune synapse formation and exocytosis of cytolyticproteins [].Ca2+ ions serve as highly crucial cellular signalling messengersfor various biological processes such as cell growth, metabolism,gene transcription, muscle contraction and cell death [].Importantly, maintaining Ca2+ homeostasis is crucial. Ca2+permeable ion channels such as transient receptor potential(TRP) channels and calcium-release-activated calcium (CRAC)channels, together with a dynamic network of organelles suchas the endoplasmic/sarcoplasmic reticulum (ER/SR) and themitochondria, synergistically function to precisely mediate cyto-plasmic Ca2+ entry and intracellular signalling processes [].The mitochondria play a key role as adenosine triphosphate(ATP) producers; in addition, they are also involved in variousinterconnected processes, including cell growth, informationtransmission, immune responses, Ca2+ storage and regulation []. Intracellular Ca2+ entry is facilitated by various mechanismsincluding in response to receptor stimulation, depletion of Ca2+stores, changes in membrane potential electrical signalling andbinding of specific ligands; however, the depletion of intracellularCa2+ stores induces the store operated Ca2+ entry (SOCE), a majorCa2+ signalling pathway that leads to activation and regulationof various Ca2+ sensitive ion channels in immune cells [].During SOCE activation, the mitochondrial network regulatesCa2+ mobilization by actively taking up Ca2+ , thereby indirectlyinhibiting inactivation of Ca2+ permeable ion channels []. Theprocess prolongs cytosolic Ca2+ influx, which is required forimmune cell activation [].Importantly, since NK cells require Ca2+ to exert their targeteffector cytotoxicity [], in mylagic encephalomyelitis orchronic fatigue syndrome (ME/CFS), reduced NK cell cytotoxicitywas linked to decreased Ca2+ influx via the transient receptorpotential melastatin 3 (TRPM3) ion channel in previous studies[]. ME/CFS is a complex multisystemic illness, and researchover the past few years has reported a considerable overlap inclinical manifestations and pathomechanisms with PCC [].In addition, it is hypothesised that SARS-CoV-2 is a potentialinfectious trigger for ME/CFS [], and more recently, elec-trophysiology investigations reported impairment of the TRPM3ion channel in NK cells from patients with ME/CFS and PCC;however, further research is required [].TRPM3 belongs to a family of transient receptor potential (TRP)ion channels, a large and diverse family of nonselective cationchannels that respond to a wide range of chemical and physicalstimuli []. TRPM3 is a Ca2+ permeable polymodal ion channelthat is expressed in a wide variety of tissues and cells, but is highlyexpressed in the brain, spinal cord, eye, pituitary gland, kidney,adipose tissue and pancreatic beta cells []. TRP channels areinvolved in numerous critical physiological processes, includingtemperature sensing, taste, vision, nociception, epithelial iontransport, and mineral homeostasis []. Therefore, due totheir biological roles and wide expression in the body, dysregula-tion of expression or activity of TRP channels has been linked tosome disease onset, such as neuropathic pain, neurodegenerativediseases, and cancer, collectively referred to as TRP-relatedchannelopathies []. TRPM3 ion channel impairment hasalso been linked to various illnesses such as intellectual disability, ### Page 3 epilepsy, musculoskeletal anomalies, altered pain perception,glaucoma and ME/CFS [].The TRPM3 ion channel can be activated by both thermal andchemical stimuli, such as the endogenous neurosteroid preg-nenolone sulfate (PregS), and reversibly inhibited by ononetin[]. Furthermore, the TRPM3 ion channel was indicatedto form plasmalemmal Ca2+ channels with Orai1 and stromalinteraction molecule 1 (Stim1) in white matter (WM) glia, con-tributing to increased Ca2+ influx in the central nervous system(CNS) cells []. The TRPM3 ion channel senses the changesin Ca2+ concentration via calmodulin (CaM), a Ca2+ bindingprotein located on the N-terminus of the channel, leading toeither up- or downregulation of the channel’s activity [].Activation of the TRPM3 ion channel leads to a Ca2+ increase,engaging downstream transcriptional regulators such as nuclearfactor of activated T cells (NFAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and c-Jun N-terminalkinase (JNK) []. This coupling of membrane-proximal ion fluxto transcriptional activity underpins the role of TRPM3 in regu-lating Ca2+ -dependent immune gene expression and downstreamimmune responses [].Given previous research reporting significant dysfunction ofTRPM3 and calcium influx in NK cells of PCC patients comparedwith HCs, the aim of this current investigation was to determineTRPM3 ion channel function on Ca2+ influx into the cytoplasmand mitochondria in NK cells using fluorescence live cell imagingfrom PCC patients compared with HCs. ## 2 Materials and Methods ### 2.1 Study Participants This research was conducted in accordance with Griffith Univer-sity’s Human Research Ethics guidelines, the National Statementon Ethical Conduct in Human Research (2007, updated 2018), theAustralian Code for the Responsible Conduct of Research (2018)and in accordance with the Declaration of Helsinki. The humanresearch ethics application was approved by the Griffith Univer-sity Human Research Ethics Committee (GU HREC 2022/666),and all participants provided informed and written consentbefore blood collection. PCC patients and HC were recruited byclinical referral or the National Centre for Neuroimmunologyand Emerging Diseases (NCNED) patient database. Between May2024 and May 2025, eight PCC patients and eight age and sexmatched HC volunteered for this study. Patients with PCC wererequired to have received a clinical diagnosis and meet the clinicalcase definition by Delphi consensus published by the WHO [],while HC participants were in good health without incidence offatigue or evidence of illness. Participants were excluded from thisstudy if pregnant, breastfeeding, reported previous alcohol abuse,obese (body mass index (BMI) ≥ 30), aged under 18 years or over65 years, or suffered from chronic illness such as autoimmunediseases, cardiovascular disease, diabetes, metabolic syndrome,thyroid disease, malignancies, insomnia, chronic fatigue, or pri-mary psychological disorders. Participants were excluded if theywere routinely taking any pharmacological agents that directly orindirectly influence TRPM3 or Ca2+ signalling. Participants wereprovided with the option to cease any conflicting medications for a minimum of 14 days before blood donation after approval fromtheir physician.All participants completed an online questionnaire providingsociodemographic background, medical history, medicationstaken, and quality of life (QoL) assessed using the 36-item shortform health survey (SF-36) and the WHO Disability AssessmentSchedule (DAS). The SF-36 was scored across eight domains,including physical functioning, role limitations due to physi-cal health problems, bodily pain, general health perceptions,energy/vitality, social functioning, role limitations due to per-sonal or emotional problems, and emotional wellbeing/generalmental health. Domains were scored on a scale of 0% to 100%,whereby 0 indicated very severe, and 100 indicated no symptom[]. The WHO DAS assessed functional capacity across sixdomains of life: cognition, mobility, self-care, getting along, workand school participation, general life activities, and participationin society. Each of the 36 items of the WHO DAS 2.0 was scoredon a five-point scale: none, mild, moderate, severe, and extreme orcannot do. The subscale scores were generated by first convertingeach item score into the corresponding, predefined weightedvalues as outlined in the WHO DAS 2.0 manual (available onlineat: https://apps.who.int/iris/handle/10665/43974.) []. Low per-centage scores were associated with no disability, while a highdegree of difficulty or greater disability was represented by higherpercentages (0 = no symptom, 100 = very severe) []. Participantswith PCC reported on the presence, frequency, and severity ofsymptoms. Symptoms were categorised in 10 diverse groups,including cognitive difficulties, pain, sleep disturbances, cardio-vascular, gastrointestinal, respiratory, urinary, immunological,and thermoregulatory intolerances, and sensory disturbances. ### 2.2 Natural Killer Cells Isolation 84 mL of whole blood was collected in ethylenediaminetetraaceticacid ([EDTA], Becton Dickinson, BD Biosciences, San Diego,CA, USA) tubes from the antecubital vein of each participantvia venepuncture. All blood collections were performed between6:30 am and 10:00 am at convenient locations for participants,including the Gold Coast campus of Griffith University, RobinaHospital, patient homes, or private laboratories in South EastQueensland and North-East New South Wales. Full blood countwas performed within 4 h of collection for each participant,analysing red blood cell count, white blood cell count, andgranulocyte cell count at Gold Coast University Hospital orprivate laboratories in Australia that are NATA certified.All samples were de-identified using a unique alphanumericcode before arriving at the NCNED laboratories. PBMCs wereisolated from 80 mL of whole blood samples by density gradi-ent centrifugation (Ficoll Paque Plus (Cytiva), GE Healthcare,Uppsala, Sweden), as previously described []. Cell countsand cell viability were determined using trypan blue stain(Invitrogen, Carlsbad, CA, USA) and an automatic cell counter(TC20 Automated cell counter, Bio-Rad Laboratories, Hercules,CA, USA).NK cells were isolated from PBMCs by immunomagnetic selec-tion using the EasySep human NK cell enrichment kit (StemCell Technologies, Vancouver, BC, Canada). The purity of isolated ### Page 4 NK cells was determined by CD3− CD56+ phenotypic surfaceexpression using flow cytometry. NK cells were incubated for20 min at room temperature in the presence of CD3 PE-Cy7(5 μL/test) and CD56 APC (20 μL/test) monoclonal antibodies(BD, Biosciences). Cells were acquired at 10,000 events using theBD X20 flow cytometer (BD Biosciences). Acceptable NK cellpurity was ≥75% (Figure). ### 2.3 Cell Staining Cells were initially prepared and washed in 0 mM Ca2+ EDTA-free buffer, unless otherwise stated. The 0 mM Ca2+ buffer wasprepared in milliQ water and contained Sodium chloride (NaCl)140 mM, Potassium chloride (KCl) 5.4 mM, Magnesium chloride(MgCl) 2 1.0 mM, and HEPES 10 mM. The pH was adjusted to 7.40± 0.05 using NaOH, and the osmolality was adjusted to 300 ± 10mOsm/L using D-glucose.NK cells were adhered to 24-well glass bottom plates (Celvis,P24-1.5H-N) using Corning Cell-Tak cell and tissue adhesive (Bio-Strategy, Campbellfield, Vic, Australia) as previously described[]. For specific mitochondrial identification and Ca2+ indi-cation, NK cells were stained with 1 μM Mito Tracker GreenFM for 30 min at 37◦C, washed, then stained with 1 μMHoechst 33342 and 1 μM Rhod-2 AM in the presence of 0.02 %Pluronic F127 (Thermofisher, Waltham, MA, USA) for 15 minat room temperature (RT). To promote sequestration of Rhod-2 AM into the mitochondria, Rhod-2 AM was first reducedto a colourless nonfluorescent dihydrorhod-2 AM by adding asmall solid excess of sodium borohydride (NaBH4 ). For cytosoliccalcium determination, NK cells were stained with 1 μM Fluo-8-acetoxymethyl ester (AM) (Abcam, Cambridge, UK) in thepresence of 0.02% Pluronic F127 for 30 min at 37◦C. After awash cycle, cells were pre-depleted with 1 μM Thapsigargin(Sigma-Aldrich) for 15 min at RT, then left for a further 15 min,allowing de-esterification of Fuo-8-AM and Rhod 2-AM, ready forimaging. ### 2.4 Live-Cell Imaging To investigate TRPM3-dependent intracellular Ca2+ mobilization,pharmacomodulations were administered via a continuous grav-ity perfusion system in 1.8 mM Ca2+ buffer. The 1.8 mM Ca2+buffer was prepared in milliQ water and contained NaCl 140 mM,KCl 5.4 mM, Calcium chloride (CaCl2 ) 1.8 mM, MgCl2 1.0 mM,and HEPES 10 mM. The pH was adjusted to 7.40 ± 0.05 usingNaOH, and the osmolality was adjusted to 300 ± 10 mOsm/Lusing D-glucose. Stained NK cells adhered to 24-well glass-bottomplates were imaged using the Evident IXplore IX83 SpinSR SuperResolution microscope system (60× objective, oil immersion,spinning disk confocal configuration) with a Hamamatsu ORCA-Fusion camera and Olympus Cellsens Dimension 4.2 acquisitionsoftware (Evident Corporation, Tokyo, Japan). Fluo-8 fluores-cence was acquired at excitation/emission (Ex/Em) 489/508 nm,Mito Tracker Green FM fluorescence at Ex/Em: 490/516 nm,Rhod-2 fluorescence at 552/581 nm and Hoechst 33342 at Ex/Em:341/542 nm wavelengths. Time-lapse images were acquired at15 s intervals. All experiments were performed at RT (20–25◦C).For TRPM3 stimulation, three wells were recorded per partic- ipant (Fluo-8 alone well, Fluo-8 combined with Rhod-2 well,Mitotracker Green + Rhod-2 + Hoechst 33342), the baseline wasrecorded in 0 mM Ca2+ for 60 s, sequentially followed by addition(for 3 min each) of 1.8 mM Ca2+ , then activation with 50 μM PregS(in vitro technology), ending the experiment by addition of 1 μMCarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)to monitor and indicate mitochondrial uncoupling. The perfusionline was adequately washed with 0 mM Ca2+ solution betweensubsequent recordings. To confirm Ca2+ influx via TRPM3 ionchannel mediation, Ononetin was used for TRPM3 inhibition,and the baseline was recorded in 0 mM Ca2+ for 60 s, sequentiallyfollowed by addition (all for 3 min each) of 1.8 mM Ca2+ ,inhibition by Ononetin alone, combined modulation using PregSand Ononetin, TRPM3 activation using PregS alone and endingthe experiment with mitochondrial uncoupling using 1 μM FCCP(Figure). ### 2.5 Image Analysis Time-lapse image data were extracted using Olympus cellSensDimension Desktop 4.3 software by selecting regions of interest(ROIs) per well. The background was subtracted by drawingan ROI on an area without visible cells. Cells were selectedbased on good morphology (cells with intact membranes) andtheir presence throughout the duration of the recording. ROIsaround Mito Tracker Green FM perimeter and Rhod-2 colocal-ization were used to identify the mitochondrial network. Foradditional insights, colocalization of the two stains was measuredusing the ImageJ [JACop (Just Another Colocalization Plugin)software [], using Pearson’s correlation coefficient (Pearson’sr) and Manders’ Colocalization Coefficients (MCC). Pearson’scorrelation coefficient was calculated on a pixel-by-pixel basisto assess the linear correlation between fluorescence intensitydistributions in the two channels, yielding values ranging from −1to +1, where higher positive values indicate stronger covarianceof signal intensities. Manders’ coefficients (M1 and M2) werecomputed following intensity thresholding to determine theproportion of one fluorophore’s signal overlapping with the other,independent of signal proportionality, thereby providing a mea-sure of spatial co-occurrence. M1 = red, fraction of Rhod-2 over-lapping green (Mitotracker Green); M2 = fraction of MitotrackerGreen overlapping red (Rhod-2). Fluorescence intensity raw dataoutput was presented in excel spreadsheet format, then copiedand analysed using programmable spreadsheets as illustrated inFigure 1 [baseline fluorescence: recorded from 0–60 s in 0 mMCa2+ solution; F0 : minimum fluorescence; Fmax : fluorescence atmaximum peak; T1 : time at minimum fluorescence intensity; T2 :time at maximum fluorescence intensity; relative fluorescence= Fmax /F0 ; slope = [(Fmax− F0 )/(T2 −T1 )]; half-time (T1/2 ) of Ca2+influx = T1/2 max]. ### 2.6 Biological and Chemical Reagents EDTA Tubes (product code: 367839) for blood collection werepurchased from BD Biosciences. Ficoll paque plus (productcode: GEHE17-1440-03), used for PMBCs isolation and Cell-Tak cell adhesion (product code: BDAA354240, Bio-Strategy),was purchased from Bio-Strategy, Australia. EasySep human NKcell enrichment kits (product code: 19055) and EasySep buffer ### Page 5 (product code: 20144) were purchased from Stem Cell Tech-nologies. The following reagents were purchased from ThermoFisher Scientific: RPMI 1640 (product code: 11835030), HEPES,Mito Tracker TM Green FM (M7514), Rhod 2 AM (R1245MP),Hoechst 33342 (H3570) and Pluronic F-127 (P3000MP). A 24-well glass-bottom plate with high-performance #1.5 cover glass(product code: P24-1.5H-N) was purchased from Cellvis, USA.NaHCO3 (product: S5761-500g), NaCl (product code: 793566-500G), KCl (product code: 793590-500G), CaCl2 (product code:C5670-100G), MgCl2 (product code: 63069–100ML), NaBH4 (prod-uct code: 452882-100g), FCCP (product code: C2920), Thapsigar-gin (product code: T9033-1MG), DMSO (product code: D2650-5×10 mL) were purchased from Sigma-Aldrich. PregS (productcode: RDS537650) and Ononetin (product code: RDS514350)were purchased from In Vitro Technologies. Stock solutions forPregS, Ononetin, Thapsigargin, Fluo-8 AM, Mitotracker Green,and Rhod-2 AM were prepared in dimethylsulfoxide (DMSO)(product code: D2650, Sigma-Aldrich). ### 2.7 Statistical Analysis Participant demographics data were analysed using StatisticalPackage for the Social Sciences (SPSS) v26 (IBM Corp, Version 29,Armonk, NY, USA). Flow cytometry data were exported from theBD X20 flow cytometer and analysed for purity using GraphPadPrism, version 9 (GraphPad software Inc., CA, USA). Extractedtime-lapse fluorescence intensity imaging data were analysedusing a programmable Excel sheet, Origin software version2024b (OriginLab Corporation, Massachusetts, USA) for qualityanalysis check, and GraphPad Prism for statistical analyses. Therobust regression and outlier removal (ROUT) method was usedto identify and remove outliers from the analysis. For statisticalcomparison, the normality of distribution of parameters wasassessed using the Shapiro–Wilk test. The Mann–Whitney Utest was performed on nested nonparametric data. The dataare presented as median (Mdn) values and mean ± standarddeviation (SD) for descriptive statistics. Significance was set at p< 0.05. ## 3 Results ### 3.1 Participants Demographics Sixteen participants were included in this study: N = 8 HC and N= 8 PCC patients who met the WHO clinical case definition []and did not report other fatigue-related illnesses that explainedtheir symptoms. Demographic data are presented in Table. ForPCC patients, the average age and SD were 42.50 ± 15.57 years and45.00 ± 14.83 years for HC. Three HC and three PCC were femaleparticipants. Five HC and five PCC were male participants. Therewas no significant difference in gender, age, and BMI amonggroups.WHO DAS and SF-36 surveys were used to assess the QoL anddisability for all participants. Data are shown in Table. Therewas a significant difference identified between HC and PCCpatients in all domains of SF-36, including physical functioning,physical role, social functioning and vitality (p = 0.001), generalhealth (p = 0.001), pain (p = 0.001), emotional wellbeing (p = 0.004) and emotional role (p = 0.008). WHO DAS scoreswere significantly different between the two groups in alldomains except work/school participation (p = 0.132). Commu-nication and understanding (p = 0.022), mobility (p = 0.004),life activities (p = 0.001), self-care (p = 0.035), interpersonalrelationships (p = 0.023) and participation in society (p =0.008). #### 3.1.1 Participant Demographics Quality of Life andDisability Scores (SF-36 and WHODAS) #### 3.1.2 Symptoms and Onset Details FromPost-COVID-19 Group The average age and SD of illness onset for PCC patients were40.13 ± 15.45 years, with 2.68 ± 0.51 years as the average andSD for disease duration. All 8 (100.0%) PCC patients reportedexperiencing fatigue and sleep disturbances, while 7 (87.5%)reported experiencing cognitive difficulties and pain. Sensory,immune, gastrointestinal, and cardiovascular disturbances weredeclared by 6 (75.0%), respiratory problems were reported by4 (50.0%) and thermostatic intolerance by 5 (62.5%) of PCCpatients. Only 1(12.5%) PCC patient reported experiencing urinarydisturbances. All symptoms and frequency data are detailed inTable. #### 3.1.3 Full Blood Count Parameters A full blood count was performed for all participants. Theresults were within the normal range for all cases, as definedby pathology service providers that were a NATA-accreditedlaboratory. However, PCC patients had significantly elevatedhaematocrit (p = 0.027) and haemoglobin (p = 0.031) levels.We concluded this could have been due to dehydration insome of the participants. Full blood count results are shown inTable. ### 3.2 Changes in Cytosolic Calcium Influx inResponse to PregS Stimulation in NK Cells inPost-COVID-19 Condition Patients Fluorescence live cell imaging measured the relative amount ofCa2+ influx and rate of response using Fluo-8 AM Ca2+ indicator,as illustrated in Figure. Results are presented in Figure.Cytosolic Ca2+ influx data are presented as the relative changein fluorescence intensity (Fmax/F0) of Fluo-8 AM, as shown inFigure. The average Ca2+ influx amplitude into the cytosol wassignificantly reduced in NK cells from PCC patients (Mdn = 5.26,3.31–8.16) compared with HC (Mdn = 8.62 (7.11–17.0) (Figure,p < 0.0001) after depletion of Ca2+ stores using 1 μM thapsigargin,followed by addition of 1.8 mM Ca2+ buffer. Although therewas no significant difference in the rate of response (slope, p= 0.2209, Figure) to 1.8 mM Ca2+ buffer, the time to half-maximal (T1/2 ) response was significantly longer in PCC patientscompared with HC (Figure, p = 0.0016). Stimulation of theTRPM3 ion channel with 50 μM PregS successfully induceda significantly higher rate of response (Figure, slope, p <0.0001) in HC compared with PCC patients. In contrast, following ### Page 6 | HC | PCC | p-value | | - | - | - | | Age (years) | 45.00 ± 14.83 | 42.50 ± 15.57 | 0.951 | | Gender | Male | 5 (62.5) | 5 (62.5) | | Female | 3 (37.5) | 3 (37.5) | | BMI (kg/m2 ) | 25.12 ± 5.29 | 24.38 ± 3.73 | 0.957 | | SF-36 (%) | | Physical functioning | 98.57 ± 2.44 | 49.36 ± 26.25 | 0.001 | | Physical role | 100.00 ± 0.00 | 16.41 ± 25.65 | 0.001 | | Pain | 94.29 ± 5.35 | 44.38 ± 33.29 | 0.001 | | General health | 73.81 ± 20.37 | 34.38 ± 17.92 | 0.001 | | Social functioning | 92.86 ± 18.90 | 25.00 ± 21.13 | 0.001 | | Emotional role | 100.00 ± 0.00 | 67.71 ± 37.12 | 0.008 | | Emotional wellbeing | 80.71 ± 9.32 | 53.75 ± 27.22 | 0.004 | | Vitality | 70.54 ± 8.63 | 21.09 ± 19.75 | 0.001 | | WHODAS (%) | | Communication and understanding | 6.77 ± 9.94 | 34.90 ± 31.34 | 0.022 | | Mobility | 3.13 ± 8.84 | 36.88 ± 27.25 | 0.004 | | Self-care | 0.78 ± 2.21 | 20.31 ± 26.46 | 0.035 | | Interpersonal relationships | 7.03 ± 7.04 | 35.94 ± 34.19 | 0.023 | | Life activities | 3.13 ± 8.84 | 51.56 ± 33.53 | 0.001 | | Work/school participation | 10.94 ± 12.83 | 37.50 ± 40.78 | 0.132 | | Participation in society | 5.04 ± 6.58 | 35.98 ± 28.68 | 0.008 | Note: High SF-36 scores reflected good QoL, and low scores suggested poor QoL (0 = severe symptoms and 100 = no symptoms). Low WHO DAS scores wereassociated with no functional limitations, and high scores reflected severe functional impairments (0 = no symptom, 100 = very severe). Data is presented as mean± SD or N (%). Values of p < 0.05 are bolded.Abbreviations: BMI, body mass index; HC, healthy controls; PCC, post-COVID-19 condition; SF-36, 36-item short-form health survey; WHODAS, World HealthOrganization disability assessment schedule. PregS stimulation, the slope (Figure) and cytosolic Ca2+ influxamplitude (Figure, p < 0.0001) were significantly reduced inPCC (Mdn = 1.06, 1.00–1.19) compared with HC (Mdn = 1.11, 1.01– 1.39), indicating altered channel function. The T1/2 response toPregS stimulation was comparable between groups (Figure,p = 0.1017). Representative time-lapse fluorescence intensityresponses of NK cells in HC are shown in Figure, respectively. ### 3.3 Changes in Calcium Mobilization in theMitochondria in Response to PregS Stimulation inNK Cells in Post-COVID-19 Patients The downstream effect of TRPM3 ion channel impairmenton Ca2+ influx into the mitochondria was examined usingmitochondrial Ca2+ indicator Rhod-2. Results are illustrated inFigure. Mitochondrial Ca2+ influx data are expressed as therelative change in fluorescence intensity (Fmax/F0) of Rhod-2as presented in Figure. Following depletion of internal Ca2+stores, addition of 1.8 mM Ca2+ buffer resulted in a significantlyfaster rate of response (Figure, slope, p < 0.0001) and highermitochondrial Ca2+ influx amplitude (Figure, p < 0.0001 inPCC (Mdn = 2.30, 1.76–2.83) compared with HC (Mdn = 1.77,1.42–2.49). However, the T1/2 response (Figure, p = 0.2845) was comparable. Meanwhile, although the slope for PregS responsewas significantly higher (Figure, slope, p = 0.0001) in PCC,the TRPM3-dependent Ca2+ influx amplitude (Figure, p =0.0036) into the mitochondria was significantly reduced in PCC(Mdn = 1.21, 1.07–140) compared with HC (Mdn = 1.24, 1.11–1.50).The T1/2 response (Figure, p = 0.0042) to PregS stimulationwas significantly shorter in PCC patients, indicating alteredchannel function. Figure shows representative time-lapsefluorescence intensity responses of NK cells from an HC anda PCC, respectively. As a monitor to indicate mitochondrialuncoupling signals, the mitochondrial Ca2+ influx response inboth groups was significantly attenuated when NK cells weretreated with the mitochondrial uncoupling agent FCCP (1 μM).To demonstrate a potential downstream signalling of TRPM3-dependent Ca2+ influx into the mitochondria, NK cells stainedwith Fluo-8 and Rhod-2 were imaged simultaneously (Figure). We successfully detected TRPM3-dependent Ca2+ influxresponse and the downstream signalling into the mitochondriain HC, whereby the responses in PCC were highly altered. Toconfirm Ca2+ influx via TRPM3 ion channel mediation, ononetinsuccessfully inhibited TRPM3 activity, which was successfullyactivated again by PregS stimulation (Figure). TRPM3 inhibi-tion and activation in NK cells were apparent when cytosolic andmitochondrial Ca2+ responses were plotted against each other. ### Page 7 ![image]() The Ca2+ responses from single dye wells (Fluo-8 alone and Rhod-2 + Mitotracker Green) were consistent with combined dye wells(Fluo-8 + Rhod-2) responses. ### 3.4 Colocalization of Mitotracker Green andRhod-2 Signals Over Time in NK Cells inPost-COVID-19 Patients For additional insights into a downstream signal into the mito-chondria, Ca2+ influx into the mitochondria was confirmedusing scatterplots demonstrating Mitotracker Green and Rhod-2overlay, and changes in Rhod-2 fluorescence intensity over-time. Pearson’s r provided a pixel-by-pixel linear correlationbetween fluorescence intensity distributions in the two channels,while Manders’ coefficients (M1 and M2) provided a measureof spatial co-occurrence overtime. Changes in the distributionand colocalization of Rhod-2 with Mitotracker Green in the mitochondria were visible overtime, as illustrated in Figurefor PCC and Figure for HC. The degree of colocalizationbetween Rhod-2 (M1 = red) and Mitotracker Green (M2 = green)over time is presented in Figure. The scatterplots (Figure) display the intensity range of Mitotracker Green and Rhod-2.At resting conditions, the distribution of the two stains waspositively mapped, with a Pearson’s r of 0.799, M1 = 0.517,and M2 = 0.705 (Figure). Increased Rhod-2 fluorescenceintensity and overlapping of the two stains were observedfollowing stimulation of NK cells with PregS (Pearson’s r =0.847, M1 = 0.821, and M2 = 0.762) (Figure), indicatingPregS induced Ca2+ influx into the mitochondria. Hence, achange in slope by the fitted line, movement of more dottedpoints toward the fitted line, and rotation of the dotted cloudtoward the Rhod-2 axis as shown in the scatterplot in Figure.The Ca2+ signal of Rhod-2 was attenuated when NK cellswere treated with the mitochondrial uncoupling agent FCCP(1 μM) (Pearson’s r = 0.799, M1 = 0.536 and M2 = 0.742). ### Page 8 ![image]() ### Page 9 ![image]() ### Page 10 | PCC | | - | | Age of onset (years [mean ± SD]) | 40.13 ± 15.45 | | Disease duration (years [mean±SD]) | 2.68 ± 0.51 | | Fatigue | YesNo | 8 (100.0%)0 (0.0%) | | Cognitive difficulties | YesNo | 7 (87.5%)1 (12.5%) | | Pain | YesNo | 7 (87.5%)1 (12.5%) | | Sleep disturbances | YesNo | 8 (100.00%)0 (0.0%) | | Sensory disturbances | YesNo | 6 (75.0%)2 (25.0%) | | Immune disturbances | YesNo | 6 (75.0%)2 (25.0%) | | Gastrointestinal disturbances | YesNo | 6 (75.0%)2 (25.0%) | | Cardiovascular disturbances | YesNo | 6 (75.0%)2 (25.0%) | | Respiratory | YesNo | 4 (50.0%)4 (50.0%) | | Thermostatic intolerance | YesNo | 5 (62.5%)3 (37.5%) | | Urinary disturbances | YesNo | 1 (12.5%)7 (87.5%) | Note: Data presented as mean ± SD or N (%).Abbreviations: N, number of participants; PCC, post-COVID-19 condition; SD,standard deviation. Consequently, a change in the slope and separation of thedotted cloud was observed (Figure). A summary of Pearson’sr indicating strong coordinated signal variation and Manders’colocalization coefficients showing changes in spatial overlapbetween fluorescence signals overtime, respectively, is presentedin Figure. Together, these complementary metrics enabled arobust quantitative assessment of both signal correlation and sub-cellular colocalization overtime. Collectively, despite evidence ofTRPM3 dysfunction in NK cells from PCC patients, PregS mayfacilitate compensatory mitochondrial Ca2+ uptake, potentiallythrough activation of residual TRPM3 channels or via unknownpathways. 4 Discussion This recent study investigated TRPM3-dependent Ca2+ influxinto the mitochondria in NK cells freshly isolated from patientswith PCC and HC. We report for the first time the significantdecrease in TRPM3-dependent Ca2+ influx into the mitochondriain NK cells from PCC. Results from this study further highlightthe role of ion channel dysfunction and consequential decreasedCa2+ influx into the mitochondria, suggesting further evidenceof TRPM3 dysfunction and Ca2+ influx in the pathomechanismof PCC and their adverse impact on cellular organelles, in thisinstance, the mitochondria. Importantly, these current findingssupport recent electrophysiological findings of TRPM3 ion chan-nel dysfunction ex vivo in NK cells from PCC patients [] andfurther support a common pathomechanism identified in PCCand ME/CFS [].Immune dysregulation is reported as a key feature in patientsrecovering from COVID-19 disease, with various studies report-ing altered immune cell populations, increased presence ofinflammatory mediators and reduced cytotoxicity by NK cells[]. The function and cytotoxicity of NK cellsare dependent upon a stable supply of Ca2+ ions, with SOCErepresenting the principal pathway for Ca2+ entry []. Accord-ingly, the distribution and functional states of Ca2+ permeableion channels, receptors, pumps and Ca2+ storage organellesfundamentally impact Ca2+ mobilization across cells []. Hence, we initially assessed NK cell responses to the additionof 1.8 mM extracellular Ca2+ following depletion of ER Ca2+stores using 1 μM thapsigargin. The addition of 1.8 mM Ca2+led to a slow, sustained, and higher cytosolic Ca2+ rise in NKcells from HC compared with PCC, successfully demonstrating areintroduction of Ca2+ to intracellular stores expected in healthycells. Depletion of intracellular Ca2+ stores and inhibition of thesarco/endoplasmic reticulum calcium-ATPase (SERCA) pump bythapsigargin activate the SOCE and subsequently additional Ca2+permeable channels to replenish the Ca2+ stores []. Notably,TRP channels, including TRPM3, are proposed to contribute toSOCE, acting as modulators of intracellular Ca2+ homeostasis []. Thus, a significant reduction in cytosolic Ca2+ influx observedin NK cells from individuals with PCC suggests impaired SOCEresponses.The ability of cells to maintain a stable Ca2+ homeostasis acrosscells is vital for activation and initiation of both the innateand adaptive immune response; therefore, the efficiency andspeed of Ca2+ signalling is crucial for maintaining homeostasis[]. Over the last few years, various studies have reportedpersistent immune dysregulation such as compromised B-cellfunction, impaired memory T cell responses and reduced NK ### Page 11 ![image]() ### Page 12 | HC | PCC | p-value | | - | - | - | | Full blood count | White Cell Count(4.0–11.0 × 109 /L) | 5.69 ± 1.15 | 5.81 ± 1.26 | 0.844 | | Lymphocytes(1.0–4.0 × 109 /L) | 1.75 ± 0.52 | 1.67 ± 0.34 | 0.754 | | Neutrophils(2.0–8.0 × 109 /L) | 3.15 ± 0.54 | 3.42 ± 1.04 | 0.527 | | Monocytes(0.1–1.0 × 109 /L) | 0.51 ± 0.21 | 0.45 ± 0.15 | 0.556 | | Eosinophils(< 0.6 × 109 /L) | 0.24 ± 0.17 | 0.23 ± 0.08 | 0.954 | | Basophils(< 0.2 × 109 /L) | 0.05 ± 0.02 | 0.03 ± 0.02 | 0.319 | | Platelets(140–400 × 109 /L) | 244.00 ± 29.77 | 253.38 ± 56.51 | 0.674 | | Red Cell Count(3.8–5.2 × 1012 /L) | 4.79 ± 0.44 | 5.14 ± 0.44 | 0.086 | | Haematocrit(0.33–0.47) | 0.42 ± 0.03 | 0.45 ± 0.03 | 0.027 | | Haemoglobin(115–160 g/L) | 140.13 ± 12.52 | 150.00 ± 10.47 | 0.031 | Note: Data are presented as mean ± SD. Reference ranges for full blood count parameters have been included in the table.Abbreviations: FBC, full blood count; HC, healthy controls; N, number of participants; PCC, post-COVID-19 condition. cell cytotoxicity in PCC []. The reduced reintroduc-tion of Ca2+ into the cytoplasm of NK cells in PCC afterdepletion of ER stores found in this study could potentiallycontribute to previously reported reduced cytotoxicity by NKcells []. As Ca2+ ions are vital for many vitalbiological processes, a disturbance to Ca2+ signalling couldcontribute to pathological outcomes involving other body sys-tems, including the immune system, metabolism and neurolog-ical dysfunction exhibited in PCC, thus supporting the aboveclaim [].Recently, TRPM3 ion channel impairment in NK cells fromPCC and ME/CFS was demonstrated using the patch-clamptechnique []. TRP channels function as critical sensors of phys-ical and chemical stimuli, regulating inflammatory responsesand immune cell function through Ca2+ -dependent signallingpathways, thereby shaping and maintaining intracellular andextracellular Ca2+ homeostasis []. Notably, these chan-nels are expressed in virus-susceptible host cells, where viralpathogens can alter their expression and activity to exploit TRP-mediated Ca2+ influx for key stages of the viral life cycle, includingentry, replication, and egress []. This hijacking of host Ca2+signalling machinery frequently results in dysregulated intracel-lular Ca2+ concentrations, contributing to cellular dysfunction[]. Mutations within TRP channel genes are associated witha spectrum of channelopathies characterised by either gain orloss-of-function phenotype, including neurological, metabolic,and immunological disorders []. In this context, impairedTRPM3 activity in NK cells from individuals with PCC wasfirst reported using the patch clamp technique, highlightingpotential dysfunctional similarities with ME/CFS []. Hence, we examined the effect of TRPM3-dependent Ca2+ influx intothe cytoplasm. Activation of the TRPM3 ion channel with 50 μMPregS successfully evoked a significantly higher response rate(p < 0.0001) in HC, triggering a significantly higher cytosolicCa2+ influx. In contrast, the amplitude (p < 0.0001) and rateof Ca2+ influx were significantly reduced in PCC comparedwith HC. These results support previous findings that reportedTRPM3 dysfunction in PCC using the electrophysiology tech-nique []. The decreased TRPM3 activity observed in NK cellsfrom individuals with PCC may arise from multiple convergingmechanisms involving isoform dysregulation and inflammatorymodulation. TRPM3 functional integrity is highly dependenton its primary amino acid sequence and structural domains;specifically, alterations within exon 24, which encodes the pore-forming region of TRPM3, can significantly alter ionic selectivityand reduce Ca2+ permeability by modifying electrostatic prop-erties within the channel pore []. Additionally, deletions ormutations within exon 13, encoding a critical TRPM3 structuraland functional domain known as the region indispensable forchannel function (ICF), result in the generation of nonfunctionalTRPM3 isoforms []. These altered isoforms can exert dominant-negative effects, interfering with the activity of fully functionalchannels and thereby attenuating overall TRPM3-mediated Ca2+ influx [].TRPM3-mediated Ca2+ mobilization engages broader signallingnetworks, including phosphorylation and activation of phos-phatidylinositol 3-kinase (PI3K) and mitogen-activated proteinkinases (MAPKs) such as rapidly accelerated fibrosarcoma(Raf), extracellular signal-related kinases (ERK1/2) and c-Jun N-terminal kinase (JNK), which are implicated in the regulation ### Page 13 of NK cell activation, degranulation and inflammatory cytokinerelease []. In parallel, ligand binding on natural killer group(NKG) family or natural cytotoxicity receptors (NCR) familyreceptors evokes Ca2+ entry through the SOCE pathway andCa2+ -permeable channels, including TRPM3, activating Ca2+ -dependent immune responses []. Thus, reduced Ca2+ influxrate correlates with lower cell activation and diminished effectorfunctions. Accordingly, impaired TRPM3 function may bluntCa2+ signalling, weaken MAPK/PI3K pathway activation andimpair Ca2+ -dependent processes required for effective NK cellcytotoxicity. While outside the scope of the current investigation,future research should aim to elucidate the downstream con-sequences of protein kinase phosphorylation following NK cellactivation.In addition to their role as active Ca2+ ion channels, TRP ionchannels in the plasma membrane contribute to changes incytosolic Ca2+ by modulating alternative Ca2+ -permeable ionchannels and driving force pathways, including the SOCE pro-cess, voltage-gated Ca2+ channels and changing the membranepotential []. Therefore, alteration to their optimal func-tioning has a potential downstream cumulative effect leadingto detrimental cellular consequences, is this instance decrementin Ca2+ mobilization in NK cells, potentially contributing toreduced cytotoxicity properties and other disturbances reportedin PCC. Further, since TRPM3 is highly expressed in varioustissue organs and cells, including the brain, spinal cord, eye,pituitary gland, kidney, adipose tissue and pancreatic beta cells[], the impaired TRPM3-dependent Ca2+ mobilization maypotentially be present in other tissues, contributing to cognitivedysfunction, chronic fatigue and other disturbances exhibitedin PCC.Further, attributable to their dynamic function, the mitochondriahave the ability to accumulate and store Ca2+ ions, contributingto a stable intracellular homeostasis []. Recently, TRPM3activation was found to trigger mitochondrial Ca2+ loading innociceptive dorsal root ganglion (DRG) neurons, highlightingdynamic mitochondrial activity downstream of TRPM3 activation[]. Given that these proteins and organelles are functionallycoupled, it was deemed relevant to investigate TRPM3-dependentCa2+ influx into the mitochondria in NK cells from PCC patients.Following depletion of internal Ca2+ stores, addition of 1.8 mMCa2+ led to significantly higher Ca2+ influx amplitude into themitochondria, response rate (slope) and T1/2 response in PCCcompared with HC (p < 0.0001). Although stimulation of NK cellswith PregS pronouncedly activated the TRPM3 ion channel (p <0.0001) in HC, and triggered significant Ca2+ influx, Ca2+ loadinginto the mitochondria and T1/2 response (p = 0.0001) was signifi-cantly reduced in PCC patients compared with HC. On the otherhand, NK cells from PCC had a faster rate of Ca2+ influx (slope,p = 0.0001) into the mitochondria, and the TRPM3 activationT1/2 was shorter compared with HC. The rapid and higher Ca2+rise into the mitochondria in NK cells from PCC contrasts withthe slower and lower Ca2+ rise observed in the cytoplasm. Basedon our current results, given that Ca2+ influx into the cytoplasmwas significantly reduced in NK cells from PCC patients, it ispossible that Ca2+ storage amount in the mitochondria is reduced,consequently causing rapid (increased slope) Ca2+ uptake fora longer time (longer T1/2 response) to replenish Ca2+ stores.Indeed, a compensatory mechanism to rapidly replenish Ca2+ stores and buffering by the mitochondria is proposed [].In addition, alterations in the Ca2+ flux pathways and speedwere found to subsequently increase the Ca2+ influx speed intothe mitochondria and amount via the mitochondrial calciumuniporter (MCU), even at relatively low cytosolic Ca2+ , poten-tially leading to mitochondrial Ca2+ overload []. Althoughthe mitochondria possess the ability to accumulate substantialamounts of Ca2+ in their matrix, stockpiling high levels of Ca2+has detrimental consequences, particularly the collapse of energyhomeostasis []. Disturbances in mitochondrial Ca2+ handlingare linked to neurodegenerative diseases such as Alzheimer’sdisease, Parkinson’s disease and Huntington’s disease []. Under conditions that disturb cytosolic Ca2+ homeostasis,the MCU rapidly load Ca2+ into the matrix at a rate that exceedsthe mitochondrial sodium/calcium/lithium exchanger (NCLX)Ca2+ clearance []. This excessive rise in Ca2+ concentrationin the mitochondria leads to increased permeability of the innermitochondrial membrane (IMM), provoking IMM depolarisationand matrix swelling, eventually leading to compromised cellfunction and cell death [].Ca2+ modulates the activity of TRP ion channels, which generatespositive or negative feedback, moulding overall intracellularCa2+ concentration []. As mentioned above, changes in theactivity of ion channels and pumps that regulate Ca2+ have acentral role in some disease onset and progression, includingAlzheimer’s, Parkinson’s, Huntington’s, and myopathies; hence,maintaining Ca2+ at a homeostatic balance is very importantfor optimal cell function []. Given the vitalrole Ca2+ ions play in various important biological processes,more research focusing on exploring the potential implicationsof TRP channels and Ca2+ dysregulation to PCC onset andprogression is required. The upstream disruptions, in this case,TRPM3 ion channel impairment in both PCC and ME/CFS [],are believed to potentially lead to downstream disturbances inbioenergetics, such as the mitochondria, thus contributing toloss of cell activity. Accordingly, future investigations shouldinvestigate the functional consequences of impaired TRPM3activity and associated downstream Ca2+ dysregulation in NKcells from individuals with PCC. In particular, it will be essentialto determine the mechanistic link between TRPM3 dysfunc-tion, Ca2+ signalling abnormalities, mitochondrial impairment,and reduced NK cell function reported in PCC. Given thecentral role of Ca2+ in regulating mitochondrial bioenerget-ics and immune cell effector functions, this may be furtherexplored through assessment of mitochondrial reactive oxygenspecies (ROS) production, intracellular ATP levels, mitochon-drial membrane potential, metabolic activity, and respiratorycapacity using approaches such as Seahorse extracellular fluxanalysis.The use of patient-derived NK cells in this study enhances thebiological and clinical relevance of the findings; however, thelimited yield of primary cells per participant constrained thenumber of experimental conditions that could be performed.Moreover, in vitro expansion was not feasible due to the limitedproliferative capacity of primary NK cells relative to immortalisedlines, as prolonged culture is associated with cellular stress,phenotypic drift, senescence, and reduced viability, therebylimiting large-scale expansion without compromising biologicalintegrity. ### Page 14 This study used an extremely strict participant selection criterion;for instance, participants were excluded if diagnosed with otherchronic illnesses or were extensively using medications thatdirectly or indirectly influence TRPM3 or Ca2+ activity, henceour sample size was small. To mitigate this limitation, method-ological steps were taken to increase the population of NK cellsanalysed per participant, strategically increasing the total numberof cells analysed per group. Additionally, to mitigate potentialtechnical limitations, protocol optimizations were performed,and six separate wells were imaged per participant, includingsingle dye control wells. Further, previously optimized concen-trations of PregS and ononetin were applied to limit potentialoff-target effects. Inhibition of TRPM3 activity using ononetin,sequentially followed by activation using PregS, was performed toconfirm TRPM3 channel activity. Further, to monitor and confirmmitochondrial uncoupling, FCCP was used at the end of eachexperiment.In conclusion, research from this study provided new evidencethat demonstrate that TRPM3 ion channel dysfunction has asignificant impact on cytoplasmic and Ca2+ mobilization inthe mitochondria in PCC. In addition, this study highlightedpotential overcompensatory mechanisms in the mitochondria,possibly arising from altered Ca2+ signalling processes. AlteredCa2+ signalling can lead to broader systemic issues, impactingboth the immune system and bioenergetic processes. Collectively,these findings provide an avenue for further studies on thephysiological functions of the TRPM3 ion channel and its role inPCC and similar chronic illnesses. Further, the combination ofelectrophysiology and Ca2+ imaging protocols provides a compre-hensive characterisation and analysis of Ca2+ influx via TRPM3 inNK cells of PCC and similar illnesses, such as ME/CFS patients.Recently, research has reported considerable overlap in symp-tom presentations predominantly associated with neurological,metabolic, and immunological disturbances between PCC andME/CFS patients. In addition, a proportion of PCC patients meetthe diagnostic criteria for ME/CFS. For this reason, it is crucialto employ a multidisciplinary approach for the investigation ofboth conditions. Further, considering both conditions have asignificant impact on quality of life and an enormous impacton public health, more research focusing on understanding thepathomechanisms involved is required. ## Author Contributions C.T.M. performed all the experiments, data analysis and writing ofthe manuscript. S.M.-G. conceived the project and study coordination.C.T.M., N.E.-F., K.M., and S.M.-G. contributed to the experimental design.C.T.M., N.E.-F., K.M., and S.M.-G. contributed to the critical analysis ofthe data. All the authors critically reviewed this manuscript and approvedthe final version. ## Acknowledgements The authors are grateful to those who volunteered their time and bloodsamples for this project. We acknowledge the NCNED team membersProf Sonya Marshall-Gradisnik, Dr Natalie Eaton-Fitch, Kay Schwarz,Dr Kiran Thapaliya, Dr Leighton Barnden, Dr Etianne Martini Sasso,Tania Manning, Jessica Dwyer, Jacob Batham, Maira Inderyas, SofiaJimenez Sanchez, Breanna Weigel, Tuong Huynh, Urooj Ishrat andTanoj Singh. The authors would like to further extend their gratitude to the funding bodies. We thank the Steering Committee Members, MrRoss Humphreys (Chair), and Mrs Christine Hunter. We appreciate thespecialist clinical collaborators at GU, Dr Peter Smith, MBBS FRACPPhD and Dr Jennifer Ng, MBBS MRCP FRACP. We are grateful to theCHIME Collaborators Prof Katsuhiko Muraki, Aichi-Gakuin University,Japan, Prof John Stenos, ARRL, Geelong, Australia, Prof James Baraniuk,Georgetown University, USA, and Prof Pawel Zalewski, Nicolaus Coper-nicus University, Poland. Our sincere gratitude to the funding bodies: theStafford Fox Medical Research Foundation (489798), National Health andMedical Research Council (1199502), McCusker Charitable Foundation(49979), Henty Community (4879), Henty Lions Club (4880), BlakeBeckett Trust Foundation (4579), Alison Hunter Memorial Foundation(4570), Talei Stewart (0000)Open access publishing facilitated by Griffith University, as part of theWiley – Griffith University agreement via the Council of AustralasianUniversity Librarians ## Resource Availability Requests for further information and resources should be directedto and will be fulfilled by the lead contact, Chandi Tabeth Magawa(chandi.magawa@griffithuni.edu.au; c.magawa@griffith.edu.au). Thisstudy did not generate new, unique reagents. ## Ethics Approval and Consent to Participate This project was approved by Griffith University Human Research EthicsCommittee (GU: 2022/666). All participants provided written consentbefore participation. ## Consent for Publication All participants provided informed and written consent before participa-tion. ## Conflicts of Interest The authors declare no conflicts of interest. ### Data Availability Statement All relevant data are present within the manuscript. The datasets fromthis study are not publicly available due to confidentiality agreements, butcould be available on reasonable request. Any request should be submittedto the Chair, Griffith University Human Research Ethics Committee, byemail at research-ethics@griffith.edu.au. ### Peer Review For transparency, the peer review documents associated with this articleare available at https://doi.org/10.1002/eji.70240. ## References - 1. J. B. Soriano, S. Murthy, J. C. Marshall, P. Relan, and J. V. Diaz,“A Clinical Case Definition of Post-COVID-19 Condition by a DelphiConsensus,” The Lancet Infectious Diseases 22, no. 4 (2022): e102–e107. - 2. Y. 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