Is NAPQI part of paracetamol efficacy? Insights from ion channel modulation

Is NAPQI part of paracetamol efficacy? Insights from ion channel modulation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 7 March 2026 V1 Latest version Share on Is NAPQI part of paracetamol efficacy? Insights from ion channel modulation Authors : Sara M. Alicante , Janire Urrutia , Arantza Muguruza-Montero , Miren Revuelta , Ane Arrizabalaga-Iriondo , Alvaro Villarroel 0000-0003-1096-7824 , and Eider Nuñez [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.177287615.55368178/v1 174 views 61 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Paracetamol (acetaminophen, APAP) remains one of the most widely used analgesic and antipyretic drugs, yet its mechanism of action is not fully resolved. A recurring observation in APAP pharmacology is that structural analogues designed to limit formation of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) often show reduced analgesic efficacy, raising the possibility that NAPQI could contribute to certain aspects of the therapeutic effects in addition to its established role in hepatotoxicity. Recent studies indicate that, at concentrations compatible with therapeutic exposure, NAPQI can covalently modify redox-sensitive cysteine residues on specific ion channels. In particular, NAPQI potentiates neuronal Kv7 (KCNQ) channels by altering PIP 2 and Ca 2+ sensitivity, thereby enhancing the M-current and reducing neuronal excitability. NAPQI has also been shown to modulate TRP channels, including TRPA1 and TRPV1, through redox-dependent mechanisms. For TRPV1, this includes channel activation with potential subsequent desensitization under certain conditions. These findings support the view that limited and spatially restricted NAPQI formation is theoretically capable of influencing nociceptive and thermoregulatory pathways. At higher concentrations, however, NAPQI overwhelms detoxification systems, leading to oxidative stress, mitochondrial dysfunction, and tissue injury. A concentration-dependent perspective may therefore help contextualize aspects of paracetamol’s efficacy and toxicity profile. Further experimental clarification of the localization, magnitude, and molecular specificity of NAPQI formation under therapeutic conditions will be essential to determine its contribution to clinical effects. Is NAPQI part of paracetamol efficacy? Insights from ion channel modulation Sara M. Alicante 1 , Janire Urrutia 2 , Arantza Muguruza-Montero 2 , Miren Revuelta 2 , Ane Arrizabalaga-Iriondo, Alvaro Villarroel 1 , Eider Nuñez 2* . 1 Biofisika Institute, CSIC-UPV/EHU, Leioa, Spain 2 Physiology Department, Faculty of Medicine and Nursing, Euskal Herriko Unibertsitatea UPV/EHU, 48940 Leioa, Spain Contact: [email protected] Abstract Paracetamol (acetaminophen, APAP) remains one of the most widely used analgesic and antipyretic drugs, yet its mechanism of action is not fully resolved. A recurring observation in APAP pharmacology is that structural analogues designed to limit formation of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) often show reduced analgesic efficacy, raising the possibility that NAPQI could contribute to certain aspects of the therapeutic effects in addition to its established role in hepatotoxicity. Recent studies indicate that, at concentrations compatible with therapeutic exposure, NAPQI can covalently modify redox-sensitive cysteine residues on specific ion channels. In particular, NAPQI potentiates neuronal Kv7 (KCNQ) channels by altering PIP₂ and Ca²⁺ sensitivity, thereby enhancing the M-current and reducing neuronal excitability. NAPQI has also been shown to modulate TRP channels, including TRPA1 and TRPV1, through redox-dependent mechanisms. For TRPV1, this includes channel activation with potential subsequent desensitization under certain conditions. These findings support the view that limited and spatially restricted NAPQI formation is theoretically capable of influencing nociceptive and thermoregulatory pathways. At higher concentrations, however, NAPQI overwhelms detoxification systems, leading to oxidative stress, mitochondrial dysfunction, and tissue injury. A concentration-dependent perspective may therefore help contextualize aspects of paracetamol’s efficacy and toxicity profile. Further experimental clarification of the localization, magnitude, and molecular specificity of NAPQI formation under therapeutic conditions will be essential to determine its contribution to clinical effects. KEYWORDS: KCNQ channels, TRP channels, NAPQI, redox signaling, paracetamol, analgesia, neuroprotection, hepatotoxicity, perspective Introduction Paracetamol (Acetaminophen, APAP) is one of the most widely used pain and fever medications worldwide. Despite its extensive clinical use, its exact mechanism of action remains incompletely understood and continues to be debated. Initially, paracetamol was classified with non-steroidal anti-inflammatory drugs (NSAIDs) based on the hypothesis that it inhibits cyclooxygenase (COX) enzymes, particularly COX-2 in the central nervous system (Vane et al ., 1998; Ouellet & Percival, 2001). However, this explanation is insufficient, as paracetamol shows only weak COX inhibition in peripheral tissues and lacks the anti-inflammatory effects characteristic of classic NSAIDs (Graham et al ., 2013). Later studies pointed to other mechanisms, particularly involving the endocannabinoid system. A metabolite of paracetamol called AM404, which is formed in the brain when p-aminophenol combines with arachidonic acid, can inhibit anandamide reuptake and modulate TRPV1 channels, activating them at low doses and blocking them at higher doses (Zygmunt et al ., 2000; Ottani et al ., 2006) (Figure 1). While this offers a plausible explanation for some of the central effects of APAP, it does not fully account for its overall pharmacological profile. Notably, direct TRPV1 antagonists have generally failed to reproduce the broad clinical efficacy profile of paracetamol and have been limited by on target adverse effects such as hyperthermia (Gavva et al ., 2008; Szallasi et al ., 2007). A central paradox in APAP pharmacology involves its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is formed by cytochrome P450 enzymes, mainly CYP2E1, and has long been viewed exclusively as a toxic compound responsible for liver failure after overdose (Mitchell et al ., 1973; Hinson et al ., 2010) (Figure 1). According to this classic view, NAPQI depletes glutathione and forms covalent bonds with cellular proteins, leading to mitochondrial damage and cell death (McGill & Jaeschke, 2013). This toxicological framework has guided drug development efforts toward eliminating NAPQI formation through structural modifications of APAP. However, attempts to reduce oxidative metabolism have produced mixed results. Several APAP analogues designed to limit NAPQI formation show reduced analgesic and antipyretic efficacy. At the same time, this apparent paradox requires careful interpretation. Differences in pharmacokinetics, brain penetration, metabolic routing, and local bioactivation can complicate straightforward structure efficacy comparisons across compounds (Holme et al ., 1991; Hadi et al ., 2013) (Table 1). An integrative perspective that has gained attention views NAPQI as a potential redox modulator of ion channel function. According to this framework, low localized levels of NAPQI could produce redox modifications of cysteine residues on ion channels involved in pain sensing and temperature regulation (Ray et al ., 2024; Losgott et al ., 2025; Andersson et al ., 2011; Eberhardt et al ., 2017). Experimental evidence indicates that at concentrations achievable under therapeutic conditions, NAPQI can enhance potassium currents, particularly through Kv7 channels, reducing neuronal excitability in sensory neurons (Ray et al ., 2019; Losgott et al ., 2025). In addition, studies have shown that NAPQI can modulate TRP channels, primarily by activating them, with effects that for TRPA1 have been linked to subsequent desensitization, potentially contributing to analgesia (Andersson et al ., 2011; Eberhardt et al ., 2017). Notably, this framework is biologically plausible because both Kv7 and TRP channels are known to be highly sensitive to redox modulation. Kv7 channels are among the most sensitive proteins to reactive oxygen species, responding to low micromolar concentrations of H₂O₂ (Gamper et al ., 2006; Nuñez et al ., 2023). Likewise, multiple TRP channels, including TRPA1, TRPV1, and TRPV4, act as cellular redox sensors and exhibit high sensitivity to oxidative stress (Piciu et al ., 2023; Kunka et al ., 2024). Thus, NAPQI may mimic an endogenous redox signaling pathway rather than acting solely through a foreign toxic mechanism. In contrast, excessive NAPQI production overwhelms cellular redox defenses, leading to widespread protein damage, oxidative stress, and cell toxicity, consistent with the classic model of liver damage. Analog Name NAPQI Formation (Reported or Predicted) Analgesic Effect Antipyretic Effect Reference 3-Fluoroacetanilide Reduced (metabolic inference; not directly quantified) Reduced (~30-40% of APAP) Not clearly reported Barnard et al ., 1993; Zhao et al ., 2011 3-Trifluoromethylacetanilide Predicted to be strongly reduced Reduced relative to APAP Not clearly demonstrated Zhao et al ., 2011 Adamantyl analogues (6a/6b) Predicted to be strongly reduced by design Retained or enhanced in specific models (TRPA1-dependent) Not reported Fresno et al ., 2014 N-acetyl-meta-aminophenol (AMAP) Strongly diminished relative to APAP Significantly weaker than APAP Present but weaker Holme et al ., 1991; Hadi et al ., 2013 Caprylic acid derivative (21b) Predicted to be low (no direct metabolic data) Moderate (~21.3% mechanical inhibition) Present (eliminates Phase I fever) Lisouskaya et al ., 2024 Palmitic acid derivative (21e) Predicted to be low (no direct metabolic data) Moderate (21.7% mechanical; 42.2% thermal inhibition) Present (eliminates Phase I fever) Lisouskaya et al ., 2024 Caprylic ethanolamide (22b) Predicted to be low (no direct metabolic data) Strong (~48.1% mechanical inhibition) Present (eliminates Phase I fever) Lisouskaya et al ., 2024 Palmitic ethanolamide (22e) Predicted to be low (no direct metabolic data) Moderate (23.1% mechanical; 29.8% thermal inhibition) Present (eliminates Phase I fever) Lisouskaya et al ., 2024 APAP (reference) Present (well established) Full (28.5% mechanical; 53.0% thermal inhibition) Present (reduces fever) Lisouskaya et al ., 2024; Graham & Scott, 2005 Table 1. Summary of APAP Analogues and their effect. This table summarizes selected APAP analogues in terms of their reported or predicted capacity to form the reactive metabolite NAPQI and their pharmacological profiles. When direct metabolic measurements were unavailable, NAPQI formation was inferred from structural or mechanistic considerations. Cross-study comparisons should be interpreted cautiously due to differences in experimental models and pharmacokinetic properties. Metabolism and the Context-Dependent Role of NAPQI: From Hepatic Detoxification to Neuronal Signaling Paracetamol Metabolic Pathways: Balancing Efficacy and Safety After oral intake, APAP is extensively metabolized in the liver through competing pathways that together determine its therapeutic index (Figure 1). Most of a therapeutic dose (around 40-65%) undergoes glucuronidation via UDP-glucuronosyltransferase enzymes (UGT1A1, UGT1A6, UGT1A9), while another 25-35% is sulfated by sulfotransferases (SULT1A1, SULT1A3) (Ouellet & Percival, 2001; Graham et al ., 2013). These Phase II conjugation reactions produce water-soluble, inactive metabolites that are eliminated in urine, representing the main detoxification routes under normal conditions. A smaller but toxicologically important fraction of APAP, about 5-10% of a therapeutic dose, is oxidized by cytochrome P450 enzymes to form NAPQI. This reaction is carried out primarily by CYP2E1, with contributions from CYP1A2 and CYP3A4 (Mitchell et al ., 1973; Chen et al ., 1998). NAPQI is a highly reactive electrophile that readily forms covalent bonds with thiol groups, particularly cysteine residues found in proteins and glutathione (Dahlin et al ., 1984). Under normal conditions, this reactivity is kept in check by rapid conjugation with reduced glutathione (GSH), a reaction catalyzed by glutathione S-transferases. The resulting APAP-GSH adducts are then further processed into mercapturic acid derivatives, which are also eliminated in urine (Muldrew et al ., 2002). The pathological role of NAPQI has traditionally been understood in the context of liver toxicity resulting from overdose. When APAP intake exceeds approximately 150 mg/kg, the Phase II conjugation pathways become saturated. This leads to increased metabolism through the oxidative route and subsequent overproduction of NAPQI (Hinson et al ., 2010). This excess NAPQI depletes glutathione stores in the liver, allowing unconjugated NAPQI to accumulate and form irreversible bonds with critical cellular proteins, particularly those in mitochondria that are essential for energy production and cell structure (McGill & Jaeschke, 2013). This cascade disrupts redox balance, impairs ATP generation, triggers mitochondrial dysfunction, and ultimately leads to liver cell death through necrosis and apoptosis. This is the well-established mechanism behind APAP-induced liver damage. Can NAPQI Form Outside the Liver? A Look at the Evidence The hepatic metabolism of APAP is well established. In contrast, whether NAPQI can be formed outside the liver, particularly in the nervous system, remains unclear. This issue is relevant because even small amounts of locally generated NAPQI in pain pathways could affect neuronal excitability and thereby potentially contribute to paracetamol’s pharmacological effects. Several lines of evidence suggest that such extrahepatic formation may indeed be possible. First, extrahepatic NAPQI formation requires local expression of the relevant CYP enzymes. CYP2E1, the principal enzyme responsible for converting APAP into NAPQI, has been detected in both human and rodent brain tissue, although at lower levels than in the liver and with regional variability (Johansson & Ingelman-Sundberg, 1994; Upadhya et al ., 2000). Available evidence indicates that brain CYP enzymes are catalytically active and capable of contributing to local drug metabolism (Ferguson & Tyndale, 2011). CYP3A4, which can also generate NAPQI, has been reported at the blood–brain barrier and in selected brain regions. Thus, the enzymatic machinery required for NAPQI formation is present in the Central Nervous System (CNS). However, enzyme expression alone does not establish that pharmacologically meaningful amounts of NAPQI are produced in vivo. Direct measurements remain limited, largely because NAPQI is highly reactive and short-lived (McGill & Jaeschke, 2013). Second, indirect evidence supports the possibility of local formation under certain conditions. APAP–protein adducts have been detected in brain tissue from treated animals, although at substantially lower levels than in the liver (Mallet et al ., 2010). In cultured rat cortical neurons, APAP exposure induces CYP2E1-dependent glutathione depletion and apoptotic signaling, effects that are prevented by CYP2E1 inhibition (Posadas et al ., 2010). These findings are consistent with metabolic activation occurring within neurons. Finally, the redox environment of the brain must be taken into account. Glutathione levels are relatively high, and neurons are equipped with additional antioxidant systems (Dringen, 2000). Low, spatially restricted NAPQI formation could therefore be buffered without producing overt toxicity. Under such conditions, limited covalent modification of redox-sensitive proteins, including ion channels, may occur without widespread glutathione depletion. However, more recent in vivo data call for some caution. Nyera and colleagues (2025) looked for signs of NAPQI activity in mouse brain tissue after administering doses high enough to produce liver injury. They detected neither glutathione depletion nor APAP–protein adducts in whole-brain homogenates. These observations suggest that, under these experimental conditions, NAPQI does not accumulate to measurable levels across the brain as a whole. The interpretation of these negative findings nevertheless requires careful consideration. Analyses based on whole-tissue homogenates may dilute highly localized biochemical events occurring in small cellular populations or restricted anatomical compartments. Consequently, modifications confined to specific neuronal populations, dorsal root ganglia, peripheral nociceptive terminals, or discrete spinal circuits could remain below detection thresholds when the entire brain is analyzed as a bulk sample. Thus, while the available data argue against widespread central accumulation of NAPQI, they do not exclude the possibility of transient or spatially restricted formation within defined nociceptive pathways. Taken together, the available evidence does not support widespread central accumulation of NAPQI. At the same time, it remains compatible with restricted and transient formation in anatomically defined regions. The framework proposed here does not depend on sustained or global exposure within the brain. It assumes instead that limited NAPQI generation in specific compartments could modify redox-sensitive cysteine residues, including those in Kv7 channels, without producing measurable whole-brain glutathione depletion. Testing this will require approaches with greater anatomical and cellular resolution. Concentration-Dependent Redox Effects of NAPQI In redox biology, the same molecule can sometimes act as a signal and other times as a toxin. It depends on how much is present, how long it lasts, and what kind of cell it acts on (Forman et al ., 2014). At very low concentrations, NAPQI may induce reversible modification of reactive cysteine residues on target proteins. In many signaling systems, electrophilic modification of cysteines functions as a regulatory switch that can modulate protein activity (Poole, 2015). Whether NAPQI produces comparable regulatory effects under therapeutic conditions remains uncertain and will require direct experimental validation. At intermediate concentrations, which could in some contexts be reached under therapeutic dosing, NAPQI may be able to modify less reactive or less readily accessible cysteine residues, potentially leading to more sustained protein effects. Experimental work demonstrates that NAPQI can produce functionally relevant covalent modification of ion channels (Ray et al ., 2024), but whether such target engagement occurs in a concentration dependent manner in vivo remains unclear. The physiological consequences would likely depend on the specific proteins affected. At present, however, direct evidence linking therapeutic NAPQI exposure to graded, target specific effects in intact systems is still limited (McGill and Jaeschke, 2013). At the high concentrations seen in overdose, there is clearer evidence. NAPQI overwhelms the cell’s ability to detoxify. This leads to widespread protein damage, loss of glutathione, problems with mitochondria, and eventually cell death. This toxicity is not just a liver problem. Posadas and colleagues (2010) showed that paracetamol levels of 0.5 to 2 mM, which can temporarily occur in spinal fluid, trigger cell death in neurons. This happens through CYP2E1 and glutathione depletion, suggesting that too much local NAPQI in the brain can reach toxic levels. Figure 1. Schematic representation of Paracetamol metabolism. Multiple mechanisms have been proposed for paracetamol, including inhibition of cyclooxygenases (COX) and modulation of the endocannabinoid system via AM404. The majority of APAP is metabolized through glucuronidation (40-65%) and sulfation (25-35%), while 5-10% is converted into the metabolite NAPQI via cytochrome P450 2E1 (CYP2E1). At elevated doses, NAPQI can induce hepatic toxicity. Recent evidence suggests that at therapeutic doses, NAPQI may modulate Kv7 channels, affecting neuronal excitability. KCNQ Channels and Neuronal Excitability: A Primary Redox Target KCNQ channels, particularly Kv7.2 through Kv7.5, help control how easily neurons fire. They generate what is called the M-current, which helps keep the resting membrane potential stable and limits repetitive firing (Wang et al ., 1998). When these channels do not work properly, it can lead to problems like neonatal epilepsy (Biervert et al ., 1998; Millichap & Cooper, 2012). Drugs like retigabine and flupirtine, which enhance Kv7 channel activity, have been used clinically for their analgesic effects, confirming the importance of these channels in regulating neuronal activity (Gamper et al ., 2006; Korsgaard et al ., 2005). One feature of KCNQ channels is that they respond to redox changes. Cysteine residues in the intracellular S2-S3 linker act as sensors that make Kv7 channels sensitive to oxidation (Figure 2). Oxidizing agents like hydrogen peroxide (H 2 O 2 ) increase the M-current through thiol oxidation. This process requires Ca²⁺ binding to the EF3 hand of calmodulin, a protein that modulates KCNQ channels and is reversible by reducing agents such as dithiothreitol (DTT), consistent with modification of cysteine residues (Figure 2) (Gamper et al ., 2006; Nuñez et al ., 2023). When this happens, neurons become more hyperpolarized and less excitable. This has been proposed as a potential endogenous protective mechanism to dampen neuronal activity during periods of oxidative stress (Gamper et al ., 2006). NAPQI appears to act through a related redox dependent mechanism. Experimental studies indicate that NAPQI forms covalent adducts with redox sensitive cysteine residues on neuronal Kv7 channels, increasing channel open probability and thereby reducing neuronal firing (Ray et al ., 2019; Ray et al ., 2024). Consistent with this model, Losgott and colleagues (2025) reported that NAPQI counteracts the inhibitory effects of an inflammatory mediator mixture on Kv7 currents in sensory neurons. This effect is selective for certain channel types. Kv7.2 through Kv7.5 are enhanced, but Kv7.1, the cardiac version that lacks the key cysteine motif, is not affected (Ray et al ., 2024). At the molecular level, when NAPQI modifies residues like Cys242 in Kv7.2, it likely changes the shape of the S2-S3 linker. This change in shape may affect the channel interaction with phosphatidylinositol-4,5-bisphosphate (PIP₂), a lipid that KCNQ channels need to open (Zhang et al ., 2003; Hernández et al ., 2008). Losgott and colleagues (2025) found that NAPQI makes Kv7 channels less dependent on PIP₂, which could help explain why they stay open even when PIP₂ levels drop during inflammation. Whether this happens through strengthened PIP₂ binding or some other mechanism is still being studied. Evidence from animal studies is consistent with the possibility that this mechanism matters in living systems. In rats with inflammatory pain, blocking Kv7 channels with a drug called XE991 reduced the pain-relieving effect of APAP (Stampf et al ., 2023). The same study found APAP GSH conjugates in the liver but not in cerebrospinal fluid. This suggests that NAPQI formation and its effects on Kv7 channels may happen mainly in peripheral pain circuits rather than in the CNS, though more research is needed to confirm this. In summary, both H₂O₂ and NAPQI appear to modulate Kv7 channels through oxidative mechanisms that favor membrane hyperpolarization and reduced neuronal excitability. This redox sensitivity has led to the proposal that Kv7 activation may in some contexts contribute to neuronal protection under pathological conditions such as ischemia or neurodegeneration. However, direct experimental evidence supporting a protective role specifically attributable to this mechanism remains limited (Gamper et al ., 2006; Zhong et al ., 2012). Figure 2. Redox regulation of neuronal KCNQ channels. (A) Four identical or different subunits assemble to form a functional pore in a domain-swapped architectural arrangement. (B) One subunit is composed of six transmembrane segments (S1–S6) and the N- and C-termini are located intracellularly. The S1–S4 segments constitute the voltage sensing domain and the S5 and S6 segments form the pore domain. CaM binds to hA and hB in the C- terminus. The cysteine residues in the S2–S3 linker for each channel are indicated. (C) Schematic representation of channel opening induced by cysteine oxidation in the S2–S3 linker (right) or by the absence of Ca²⁺ binding to EF-hand 3 of CaM (left). Other Redox Sensitive Channels: Possible Contributions from TRP Channels Beyond KCNQ channels, NAPQI may also affect other ion channels involved in pain and temperature sensing. Several members of the TRP channel family have been studied in this context (Figure 3). Compared with the relatively strong mechanistic and functional evidence supporting Kv7 channel modulation, the data implicating TRP channels in the therapeutic actions of APAP remain more limited and should be considered preliminary. Notably, several TRP channels, especially TRPA1 and TRPV1, exhibit high sensitivity to redox modification in sensory systems due to cysteine residues that serve as molecular sensors for electrophilic and oxidative stress (Mori et al ., 2016). TRPA1 TRPA1 is a channel that responds to irritants and contains several reactive cysteine residues that can act as redox sensors. Andersson and colleagues (2011) found that NAPQI activates both mouse and human TRPA1, while paracetamol itself does not. When NAPQI activates TRPA1, it reduces calcium and sodium currents in sensory neurons. In animal studies, injecting NAPQI directly into the spine produced pain relief that depended on TRPA1, as this effect was absent in mice lacking the channel (Table 2). How channel activation leads to pain relief is not fully understood. One suggestion is that acute activation is followed by desensitization or internalization of the channel, which could silence pain signals at the spinal level (Akopian et al ., 2007). The proposed mechanism involves NAPQI binding to cysteine residues in the N-terminal ankyrin domain, such as Cys621, Cys641, and Cys665. This covalent modification may change the channel’s shape, opening the pore but potentially also marking it for downregulation. The temporal dynamics of initial activation versus longer-term desensitization in vivo remain an important area for future investigation. TRPV1 TRPV1, the receptor for capsaicin and heat, is sensitive to oxidizing compounds. Eberhardt et al . (2017) reported that NAPQI can activate and sensitize TRPV1 under experimental conditions, whereas APAP itself shows no direct effect (Table 2). This response depends on modification of three intracellular cysteine residues (Cys158, Cys391, Cys767), as reducing agents such as dithiothreitol or N-acetylcysteine suppress channel activation and mutation of these residues markedly reduces the effect. These findings support the view that NAPQI can covalently modify and activate TRPV1 in vitro. In mouse spinal neurons, NAPQI produces a TRPV1-dependent increase in intracellular Ca²⁺ and enhances heat responses. In humans, intradermal NAPQI evokes burning pain and neurogenic inflammation (Eberhardt et al ., 2017), indicating clear pronociceptive potential in experimental settings. This profile differs from the inhibitory effects associated with Kv7 and TRPA1 modulation. TRPV1 is also targeted by AM404, a CNS metabolite of paracetamol formed from p-aminophenol and arachidonic acid. Unlike the redox-dependent action of NAPQI, AM404 activates TRPV1 through a non-covalent mechanism involving the vanilloid binding site (Zygmunt et al ., 2000; Ottani et al ., 2006; Stueber et al ., 2018) and can promote channel desensitization. Consistent with this, AM404 reduces excitatory transmission in spinal cord preparations via TRPV1 at C-fiber terminals (Kawano et al ., 2015). Whether these pathways interact in vivo remains uncertain. NAPQI has been reported to potentiate AM404-evoked currents (Stueber et al ., 2018), raising the possibility that covalent modification might influence subsequent agonist responses or facilitate Ca²⁺-dependent desensitization. However, direct evidence for such cooperative effects under therapeutic conditions is currently lacking. Moreover, given the high reactivity and rapid detoxification of NAPQI, it remains unclear whether sufficient exposure occurs during standard paracetamol use to engage these mechanisms to a meaningful extent. TRPV4 TRPV4 can be activated by reactive oxygen species such as H₂O₂ through oxidation of cysteine residues in its N-terminal region (Hong et al., 2016) (Figure 3). When activated, TRPV4 allows calcium entry into cells, including hypothalamic neurons and vascular endothelial cells. In the hypothalamus, this may contribute to heat loss and temperature regulation, while in blood vessels it can promote vasodilation (Yadav et al., 2017; Goswami et al., 2017). There is currently no direct evidence that NAPQI activates TRPV4 in neuronal systems. However, NAPQI is known to covalently modify reactive cysteine residues in other TRP channels such as TRPA1 and TRPV1, making a similar interaction with TRPV4 mechanistically plausible. Interestingly, acetaminophen itself has been reported to suppress TRPV4-mediated Ca²⁺ entry in heterologous expression systems (Nakagawa et al., 2020), indicating that this channel is pharmacologically sensitive to the APAP system. Consistent with a broader involvement of TRPV4 in APAP responses, the channel has also been implicated in acetaminophen-induced liver injury (Echtermeyer et al., 2019). Although the cellular contexts differ substantially from that of sensory or hypothalamic neurons, these findings support the general concept that TRPV4 can participate in tissue responses to APAP-derived signals. Figure 3. Schematic representation of TRP channels. Each subunit consists of six transmembrane segments (S1–S6), of which S5 and S6 form the pore domain. The lower panels show the differences in the N- and C-terminal regions among the different TRP channel subtypes . KCNQ (Kv7.2–7.5) CNS and PNS neurons (nociceptive fibers). Covalent modification of cysteines in the S2–S3 linker → allosteric potentiation of the M-current. Membrane hyperpolarization, ↓ neuronal excitability. Central and peripheral analgesia, neuroprotection. TRPA1 Sensory neurons (DRG, trigeminal), spinal cord. Covalent modification of cysteines in ankyrin repeats → activation followed by desensitization/internalization. Inhibition of spinal nociceptive transmission. Spinal analgesia (via desensitization). TRPV1 Sensory neurons (DRG, trigeminal), CNS. Covalent modification of Cys158, Cys391, Cys767 → persistent activation and sensitization. Acute burning pain, neurogenic inflammation; possible long-term desensitization. Potential contribution to analgesia at high doses (via desensitization). TRPV4* Hypothalamus, astrocytes, vascular endothelium. Hypothetical (no direct evidence) cysteine modification or indirect sensitization via oxidative stress. ↑ Ca²⁺ influx in hypothalamic neurons and endothelium → heat loss, vasodilation. Antipyresis, mild hypotensive effects. Table 2. Summary of redox-sensitive ion channels potentially modulated by NAPQI and their proposed role in paracetamol action. The table summarizes key ion channels from the KCNQ and TRP families that are sensitive to redox modulation, along with their tissue distribution, proposed mechanism of interaction with NAPQI, functional consequences, and potential contribution to the therapeutic effects of paracetamol. This framework illustrates how NAPQI could engage multiple targets to produce the distinctive pharmacological profile of paracetamol. * Modulation of TRPV4 by NAPQI remains hypothetical at present. Concluding Remarks: What We Know and What We Still Need to Learn The evidence reviewed here suggests that NAPQI, beyond its well-established toxic role, may in some contexts contribute to certain pharmacological effects of paracetamol. This framework could help explain why it has been so difficult to develop safer versions that keep the same effectiveness. Drugs designed to reduce NAPQI formation, like 3 fluoroacetanilide or AMAP, often show better liver safety but weaker pain relief, although cross-study comparisons should be interpreted cautiously (Holme et al ., 1991; Zhao et al ., 2011; Hadi et al ., 2013). More recent compounds show the same pattern: less toxicity often means less effectiveness (Lisouskaya et al ., 2024). Although not proving causality, this inverse relationship is consistent with the possibility that NAPQI formation contributes to the pharmacological profile of paracetamol. Open Questions • Where does NAPQI form after a normal dose? As discussed in Section 2.2, negative findings in whole-brain homogenates (Nyera et al ., 2025) do not rule out highly localized formation in specific pain-processing structures. The key challenge now is technical: developing sufficiently sensitive and spatially resolved methods to detect NAPQI-protein adducts in defined neuronal populations such as dorsal root ganglia or periaqueductal gray after therapeutic dosing. Approaches like proximity ligation assays or imaging mass spectrometry could help address this question. • What concentrations of NAPQI reach Kv7 channels in humans? Direct measurement in humans is not feasible due to the reactivity and short half-life of NAPQI. However, in vitro studies demonstrate that NAPQI potentiates Kv7 currents at low micromolar concentrations (approximately 1-10 µM) (Ray et al ., 2019; Losgott et al ., 2025), well below those associated with cellular toxicity (≥150 µM) (Posadas et al ., 2010). Whether such concentrations are achieved locally in sensory neurons after therapeutic dosing remains unknown. • How specific is NAPQI at therapeutic doses? Proteomic studies using click chemistry or affinity-based probes could identify which proteins are covalently modified in neuronal tissues after paracetamol administration at therapeutic doses. This would reveal whether Kv7 channels are primary targets in vivo and might uncover additional contributors to the analgesic effect. • Where is the boundary between signaling and toxicity? Animal studies comparing graded doses while measuring both channel function (e.g., Kv7 activity in sensory neurons) and early markers of oxidative stress in the same tissues could help define the therapeutic window more precisely. Such experiments would clarify whether the same molecular modification underlies both efficacy and toxicity, or whether these effects engage distinct pathways. • Testable Predictions and Experimental Priorities The hypothesis proposed here generates several experimentally testable predictions. First, redox-insensitive Kv7 mutants should exhibit reduced sensitivity to APAP/NAPQI-mediated analgesia. Second, selective inhibition of CYP2E1 in peripheral versus central compartments could help define the anatomical locus of relevant NAPQI formation. Third, TRPA1-deficient models should display altered components of APAP-induced antinociception if TRPA1 desensitization contributes significantly. Finally, the development of sensitive biomarkers of low-level NAPQI–protein adduct formation in neural tissues would provide critical validation of the proposed signaling framework. Taken together, the evidence reviewed here raises the possibility that NAPQI may contribute to aspects of paracetamol’s effects through ion channel modulation, potentially explaining elements of its pharmacological profile that remain difficult to reconcile. However, the available evidence remains largely indirect and several key aspects require experimental confirmation. Most studies examining ion channel modulation have relied on exogenous NAPQI application or in vitro systems, and it remains unclear whether comparable levels are generated in relevant neuronal compartments under therapeutic conditions. In addition, reports of absent NAPQI activity in whole-brain homogenates highlight the need for approaches with higher anatomical resolution to determine whether localized formation occurs within specific nociceptive pathways. Addressing these gaps will require improved detection methods, a clearer understanding of which channels are modified under physiologically relevant conditions, and strategies to disentangle potential beneficial actions from liver toxicity. Author contributions E.N. conceived the original idea and the conceptual framework of the manuscript. S.M.A. designed and prepared the figures. S.M.A., J.U., A.M.-M., M.R., and A.A.-I. contributed to the analysis and interpretation of the literature and to the development of the central hypothesis. A.V. provided supervision and critical input. All authors contributed to manuscript revision, approved the final version, and agree to be accountable for all aspects of the work. Funding We gratefully acknowledge the Basque Government for its funding and support through the postdoctoral grant awarded to E. Nuñez (POS_2021_1_0017) and Sara M. Alicante, and the project Grupos Consolidados (IT-1707-22). We also thank the Spanish Ministry of Science and Innovation for the project Generación del Conocimiento (PID2022-139230NB-I00). Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Generative AI statement The author(s) declare that no Generative AI was used in the creation of this manuscript. 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Authors Affiliations Sara M. Alicante Instituto Biofisika View all articles by this author Janire Urrutia Universidad del Pais Vasco View all articles by this author Arantza Muguruza-Montero Universidad del Pais Vasco View all articles by this author Miren Revuelta Universidad del Pais Vasco View all articles by this author Ane Arrizabalaga-Iriondo Universidad del Pais Vasco View all articles by this author Alvaro Villarroel 0000-0003-1096-7824 Instituto Biofisika View all articles by this author Eider Nuñez [email protected] Universidad del Pais Vasco View all articles by this author Metrics & Citations Metrics Article Usage 174 views 61 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Sara M. Alicante, Janire Urrutia, Arantza Muguruza-Montero, et al. Is NAPQI part of paracetamol efficacy? Insights from ion channel modulation. Authorea . 07 March 2026. 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