Ion channels in osteoarthritis: emerging roles and potential targets

Abstract

Osteoarthritis (OA) is a highly prevalent joint disease that causes substantial disability, yet effective approaches for disease prevention or for the delay of OA progression are lacking. Emerging evidence has pinpointed ion channels as pivotal mediators in OA and as promising targets for disease-modifying treatments. Preclinical studies have assessed the potential of a variety of ion channel modulators, to modify disease pathways involved in cartilage degeneration, synovial inflammation, bone hyperplasia and pain and to provide symptomatic relief in OA models. Some of these modulators are now being evaluated in clinical trials. This review explores the structures and functions of transient receptor potential channels (TRPs), piezo channels, voltage-gated sodium channels, voltage-dependent calcium channels, potassium channels, acid sensing ion channels, and the ATP-dependent P2XR channels in the osteoarthritic joint. The discussion spans channel-related drug discovery and potential clinical applications, emphasizing opportunities for further research, and underscoring the growing clinical impact of ion channel biology in OA.

Introduction

Osteoarthritis (OA) is a highly prevalent joint disease characterized by joint pain and disability, substantially impacting quality of life and conferring a large economic burden ¹. OA manifestations include joint pain, stiffness and swelling, and bone outgrowths, which all undermine stability and daily functioning ². At the pathophysiological level, OA is characterized by articular cartilage degeneration, synovial inflammation, and bone hyperplasia, with cartilage wear leading to irreversible joint damage and reduced mobility (Fig. 1) ^3-5. Pain is a primary clinical symptom, closely associated with articular cartilage loss ^6-8. Synovitis, resulting from inflamed synovial membranes, contributes to pain and joint dysfunction ^9,10, whereas disruptions in bone cell metabolism and imbalanced osteoclast and osteoblast activity contribute to bone structure changes ¹¹. During the development of OA, an imbalance in the differentiation of osteoclasts and osteoblasts results in pathological changes such as local bone loss, which is associated with excessive osteoclast activity, and the formation of osteophytes, which is linked to overactive osteoblasts ¹². Mechanical factors are important for diarthrodial joint health and articular cartilage. Proper loading maintains cartilage, while excessive stress can precipitate OA ¹³. Mechanosensing is not exclusive to sensory cells; chondrocytes also respond to mechanical stimuli, with abnormal stress leading to cartilage degeneration. Despite therapeutic advances, OA treatment remains challenging owing to the continuously evolving nature of the disease. Currently available pharmacological and non-pharmacological interventions yield only symptomatic and, in many cases partial, relief with considerable side-effects ^3,14. There is, thus, urgent need for more effective and safer treatments targeting the underlying mechanisms of OA.

Fig. 1. Overview of ion channels' role in the pathological process of OA.

Mechanical imbalance post-joint injury leads to abnormal joint loading, triggering the release of alarmins that act as danger signals. These alarmins bind to pattern recognition receptors (PRRs) on immune cells, thereby promoting M1 macrophage polarization and fibroblast activation through ion channels such as TRPV4, TRPA1, and ASIC1a. The regulated release of pro-inflammatory mediators, including TNF-α, IL-1β, IL-6, and IL-18, and matrix-degrading enzymes, such as MMPs and ADAMTSs, mediated by ion channels, amplifies the inflammatory response. Consequently, the release of pro-inflammatory mediators like TNF-α, IL-1β, IL-6, and cartilage-degrading enzymes MMPs and ADAMTSs, mediated by ion channels including Nav1.7, TRPV4 and Piezo, amplifies the inflammatory response. This cascade results in cartilage degradation, ectopic ossification, synovial inflammation, and joint pain. Protective ion channels are denoted in blue fonts, destructive ones in black fonts. K_ATP, ATP-sensitive K⁺ channel; K_Ca, Ca²⁺-activated K⁺ channel; Kv, voltage-gated K⁺ channel; Nav, voltage-gated Na channels; Cav, voltage-dependent calcium channels; ASICs, acid sensing ion channels; P2X7R, P2X purinoceptor 7 receptor; ClCs, chloride channels; MMPs, matrix metalloproteinases; ADAMTS, A Disintegrin and Metalloproteinase with Thrombospondin motifs. This figure was created with BioRender.com.

Ion channels are porous proteins located on cell membranes that regulate the flux of ions and are crucial for maintaining ion balance, membrane potential and cell volume ¹⁵. These channels have pivotal roles in joint cells, and particularly in chondrocytes. They regulate the resting membrane potential of chondrocytes and protect them from osmotic shock, ensuring chondrocyte survival and function under the variable osmotic conditions within the cartilage matrix ^16-18. The complex chondrocyte 'channelome' supports not only osmoregulation, but also mechanotransduction, chemotransduction and metabolic function ^18,19.

Abnormal expression or dysregulation of ion channels contributes to OA pathophysiology, with evidence linking them to synovial proliferation, cartilage destruction, endochondral ossification, inflammatory responses and pain (Fig. 1)^20-25. Some ion channels have been highlighted as therapeutic targets for OA, considering their emerging roles in cartilage integrity and pain signaling ²⁶.

OA-related alterations in synovial fluid can impair ion channel function, perturb electrochemical gradients, and compromise joint integrity ²⁷. During OA progression, excessive mechanical stress, inflammation, and the altered synovial fluid microenvironment activate transient receptor potential (TRP) channels, piezo channels, and voltage-gated channels, instigating abnormal Ca²⁺ and Na⁺ movements that exacerbate OA pathology ^24,26,28,29. Dysregulated ion channel activity in osteoarthritic cartilage disrupt chondrocyte–extracellular matrix (ECM) homeostasis, leading to cartilage erosion (Fig. 1) and pain (Fig. 2). In addition, ion channels modulate inflammation by affecting the release of proinflammatory and anti-inflammatory cytokines, which contribute to the inflammatory response and associated pain ^30-35.

Fig. 2. Mechanisms of OA pain transduction mediated by ion channels.

Pain transduction in OA is triggered by various stimuli that activate specific ion channels. Proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 initiate pain signaling. Concurrently, mechanical pressure on joints and temperature fluctuations also activate ion channels including TRP, Nav, ASIC, Piezo, Cav, Kv, and P2X3R. The joint microenvironment, characterized by acidosis and osmotic pressure, along with ATP, plays a role in pain transduction as well. These ion channels facilitate the flow of calcium, sodium, and potassium ions, which regulate intracellular signal transduction and membrane potential changes. When the depolarization threshold, voltage-gated sodium and calcium channels are activated, generating an action potential. Nociceptive signals are transmitted at a central synapse in the spinal cord through the release of excitatory neurotransmitters such as calcitonin gene related peptide (CGRP), or substance P, which can excite second-order nociceptive projection neurons. These channels are essential in activating nociceptive terminals and contributing to OA pain by responding to diverse stimuli. This figure was created with BioRender.com.

In this review, we examine how ion channels, including TRP channels, piezo channels, voltage-regulated sodium and calcium channels, potassium channels, acid sensing ion channels, purinergic P2X receptors, and chloride channels, might impact gene expression and cell signalling in joints to maintain joint tissue homeostasis. Importantly, we discuss the specific contributions of ion channels to OA pathology and the potential of ion channel modulators for improved treatment approaches in OA.

Transient Receptor Potential channels

TRP proteins were established as an ion channel superfamily, initially notable for their Ca²⁺ permeability ³⁶, although it was later discovered that they also transport other cations, including Na⁺ and Mg^{2+ 37}. Mammals express 28 TRP channels, categorized into seven subfamilies by amino acid sequence homology: TRPA; TRPC; TRPM; TRPML; TRPN; TRPP; and TRPV. These channels are activated by diverse stimuli and are integral to sensory responses to pain and stress, emerging as promising drug targets (Table 1). TRP channels are characterized by six transmembrane spanning domains and a pore-forming loop, crucial for regulating cellular responses to various stimuli and implicated in pathological conditions. TRPV, TRPM, TRPA, and TRPC subfamilies are particularly noteworthy for their roles in pathogenesis of OA (Fig. 3).

Table 1. Effects of ion channel modulation in OA.

Substances	In vitro effects (model)	In vivo effects (model)
TRPV1 agonist
Capsaicin	Chondrocyte ferroptosis↓, ROS↓, GPX4↑ (primary mouse chondrocytes) 44	Cartilage injury↓, OARSI score↓ (DMM) 44
	M1 macrophage polarization↓, p-CaMKII↑, Nrf2 nuclear translocation↑ (RAW264.7 macrophages)47	Joint swelling↓, synovitis↓ (DMM) 47
	Neuronal mechanically evoked response↑ (Human synovium, DRG neuron) 22	Pain behavior↑ (MIA) 22
TRPV1 antagonists
JNJ-17203212	Neuronal mechanically evoked response↓ (Human synovium, DRG neurons) 22	Pain behavior↓ (MIA) 22
APHC3		Cartilage injury↓, Joint swelling↓, Mechanical pain↓ (MIA) 48
Capsazepine	ADAMTS9↑, NF-κB↑ (OUMS-27 chondrocyte line) 30	Male mice pain behavior↓ (DMM) 50
TRPV2 KO		Hypertrophic differentiation of chondrocytes↑, Prg4↓(DMM) 25
TRPV4 agonists
36·HCl		Cartilage degeneration↓ (MT) 67
GSK1016790A	Chondrocyte pyroptosis↑, Drp1 mitochondrial translocation↑, CaMKII↑ (Primary mouse chondrocytes) 63	Cartilage degeneration↑, Joint inflammation↑ (ACLT) 63
	Intracellular Ca2+↑ (Human chondrocytes) 13
TRPV4 antagonists
GSK2193874		Pain behavior↓, MWT↑ (ACLT) 73
HC067074	M1 macrophage polarization↓, ROS↓, NLRP3↓ (RAW 264.7 macrophages) 78	Synovitis↓ (DMM) 78
TRPV4 KO	Osteogenic differentiation potential↓ (Bone marrow-derived stem cells) 70	Synovitis↓, Cartilage degeneration↓, Periarticular bone volume↓ (DMM) 64
		ORASI score↑, bone injury↑ (High-fat diet induced OA) 70
si-TRPV4	Chondrocyte apoptosis↓, Ca2+ influx↓, Caspase-3↓ (Primary chondrocytes) 29	Cartilage injury↓ (ACLT) 29
TRPV5 antagonist
Oxoglaucine	Chondrocyte apoptosis↓, TNF-α↓, IL-6↓, MMP-13↓, Beclin-1↑, CAMK-II↓ (Human chondrocytes) 79	Cartilage injury↓(DMM) 79
TRPM2 KO		Pain behavior↓ (MIA) 84
TRPM7 antagonist
Low intracellular Mg2+	Beclin-1↓, Autophagy↓, Wnt↑ (Primary chondrocytes) 86	Cartilage injury↑, ORASI score↑ (DMM) 86
TRPM8 agonist
Menthol	MMP-1↑, MMP-3↑, MMP-13↑, iNOS↑ (Human primary chondrocytes) 90
TRPA1 antagonists
HC-030031	MMP-1↓, MMP-3↓, MMP-13↓, IL-6↓, PGE2↓, COX-2↓ (Primary human chondrocyte) 94,97
	IL-6↓, IL-11↓ (Mouse chondrocytes) 35
TCS 5861528	IL-6↓, IL-11↓ (Mouse chondrocytes) 35	Cartilage injury↓, Acute pain↓, substance P↓, MWT↑ (MIA 97, ACLT 73)
CHEM	IL-1β↓, TNF-α↓, MMP-1↓ (OA-FLS) 96	Synovitis↓, Cartilage degeneration↓ (LPS induced inflammatory arthritis) 96
TRPA1 KO		Acute inflammation↓, Joint pain↓, Cartilage degeneration↓ (MIA) 97
TRPC5 antagonist
ML204		IFN-γ↑, TNF-α↑, Joint inflammation↑, Hyperalgesia↑ (CFA) 104
TRPC5 KO		Synovitis↑, Joint inflammation↑, Hyperalgesia↑ (CFA) 104
Piezo1 agonist
Yoda1	Intracellular Ca2+↑, MMP-13↑, TIMP-2↓ (Human chondrocytes) 13
Piezo1 antagonist
GsMTx4	Chondrocyte apoptosis↓, MMP-3↓, MMP13↓, COL-2↑, ACAN↑, NFAT1↓, Calcineurin↓ (Primary rat chondrocytes) 113	Cartilage injury↓ (ACLT) 113
	p38 MAPK↓, Intracellular Ca2+↓ (Primary porcine chondrocytes) 33	Chondrocyte mechanical response↓33
si-Piezo1	Chondrocyte apoptosis↓, MMP-3↓, MMP13↓, COL-2↑, ACAN↑, NFAT1↓, Calcineurin↓ (Primary rat chondrocytes) 113	Cartilage injury↓ (ACLT) 113
Nav1.7 antagonists
PF-04856264	MMP13↓ (Primary mouse and human chondrocytes) 26	Cartilage injury↓, OARSI score↓, Pain behavior↓ (MIA, DMM) 26
Carbamazepine	MMP13↓, COL-2↑, Intracellular Ca2+↑ (Primary mouse and human chondrocytes) 26	Cartilage injury↓, Pain behavior↓ (MIA) 26
ProTx II	MMP13↓, COL-2↑, Intracellular Ca2+↑ (Primary mouse and human chondrocytes) 26	Cartilage injury↓ (MIA, DMM) 26
Nav1.7 KO		Cartilage degeneration↓, ORASI score↓, Pain hypersensitivity↓ (MIA, DMM) 26
Nav1.8 antagonist
A-803467		Cartilage degeneration↓, ORASI score↓, Pain hypersensitivity↓ (ACLT) 154
α2δ ligand
PD-0200347	PKCα↓, ERK1/2↓, Raf↓ (Chondrocytes) 166	Cartilage injury↓ (ACLT) 166
		Cartilage injury↓, MMP-13↓, iNOS↓ (ACLT) 167
Cav2.2 antagonist
TROX-1		Mechanically evoked neuronal responses↓, Heat-evoked neuronal responses↓ (MIA) 176
T-Type Cav antagonist
NNC 55-0396	Col10a1↓, MMP-13↓, chondrocyte hypertrophy↓ (MC3T3-E1 osteoblast; Primary rat chondrocytes) 178	Cartilage injury↓ (Mechanical loading induced OA) 178
L-Type Cav antagonist
Nifedipine	Apoptosis↓, MMP-13↓, IL-6↓, ROS↓, Nrf-2/HO-1↑ (Human chondrocytes) 164
KATP agonist
Diazoxide	ERS↓, Caspase-3↓, Bax↓ (Primary rat chondrocytes) 186	Cartilage degeneration↓ (H2O2 stimulated surgical induced OA) 186
Kv7/KCNQ/M agonist
Flupirtine	Mechanical pain threshold↑ (Rat DRG neuron) 190	Mechanical allodynia↓, Thermal hyperalgesia↓ (MIA) 190
Kv7/KCNQ/M antagonist
XE-991	Mechanical pain threshold↓ (Rat DRG neuron) 190	Mechanical allodynia↑, Thermal hyperalgesia↑ (MIA) 190
ASIC1a antagonist
Psalmotoxin-1	Beclin-1↓, Lamin B1 degradation↓, β-galactosidase↓ (C28I2 chondrocyte line) 208,209	Cartilage degeneration↓ (ACLT) 208,209
	M1 polarization↓, IL-18↓ (Macrophages) 210	Chondrocyte senescence↓, Cartilage damage↓ (DMM) 210
ASIC3 antagonist
APETX2		OA pain behavior↓, Secondary hyperalgesia↓ (MIA) 213
A-317567		Pain behavior↓, Secondary hyperalgesia↓ (MIA) 214
P2X7R agonist
BzATP	NF-κB-NLRP3↑, Chondrocyte viability↓ (Primary rat chondrocytes) 231	Cartilage degeneration↑ (MIA) 231
P2X7R antagonists
A740003	NF-κB-NLRP3↓, IL-1β↓, MMP-13↓ (Primary rat chondrocytes) 231	Cartilage degeneration↓ (MIA) 231
	NLRP3↓ (Chondrocytes) 235	Cartilage injury↓, Joint inflammation↓ (TMJOA) 235
AZD9056	NF-κB↓, MMP-13↓, IL-6↓ (Chondrocytes) 232	Joint pain↓ (MIA) 232
P2X3R antagonists
TNP-ATP	ERK↓, MEK↓ (Rat DRG neuron) 242	Pain behavior↓, Mechanosensitivity↓ (CFA) 242
MK-7264		PWTs↑, Pain behavior↓ (MIA; CFA) 226
A-317491		Hyperalgesia↓, TNF-α↓, IL-6↓, MPO activity↓ (Carrageenan induced OA) 34
Cl− channel antagonists
DIDS; NPPB; Niflumic acid	Caspase3/7↓, Apoptosis↓, Apoptotic volume decrease↓ (C28/I2 chondrocyte line) 223

↑: increase; ↓: decrease; ACLT: anterior cruciate ligament transection; CFA: complete Freund's adjuvant; DMM: destabilization of the medial meniscus; ERS: endoplasmic reticulum stress; H₂O₂: Hydrogen peroxide; KO: knockout; MIA: sodium iodoacetate; MMP: matrix metalloproteinase; MPO: myeloperoxidase; MT: medial meniscal tear; MWT: mechanical withdrawal threshold; NFAT: Activated T-cell intranuclear factor; PWT: Mechanical paw withdrawal thresholds; PWL: Thermal paw withdrawal latencies; ROS: activated oxygen; TMJOA: temporomandibular joint osteoarthritis.

Fig. 3. Mechanisms of action of TRP channels in OA.

Ion channels, such as TRPV1, TRPV2, TRPV4, TRPV5, TRPM7, and TRPA1 are expressed in chondrocytes, macrophages, fibroblast-like synoviocytes (FLSs), and osteoblasts. Mechanical stimuli, inflammation, and changes in the synovial microenvironment can activate these ion channels, leading to the influx of ions like Ca²⁺ and Mg²⁺. These channels respond to stimuli by modulating membrane potential, regulating intracellular calcium, and initiating cell contraction and other Ca²⁺-dependent processes. For instance, TRPV1 activation leads to Ca²+ influx and subsequent activation of CaMKII and Nrf2, influencing macrophage M1 polarization, while TRPV2 activation induces calcineurin and NFATC1, promoting osteoclast differentiation ^46,47. Other TRP channels have been implicated in pathological processes including chondrocyte death, cartilage anabolism and catabolism, macrophage polarization, and synovial inflammation, primarily through the regulation of Ca²⁺-mediated downstream signaling molecules, such as calcium/calmodulin-dependent kinase cascades, NF-κB, PKC, PI3K/AKT, Wnt/β-catenin and other pathways ^{63,66-68,86-88,94,95}. This figure was created with BioRender.com.

TRPVs

TRPV channels TRPV1–TRPV6 are vanilloid receptors that bind molecules like capsaicin and vanillin. TRPV5 and TRPV6 are Ca²⁺-selective channels, and manage Ca²⁺ levels mainly in the intestine and kidneys, whereas TRPV1–TRPV4 are non-selective cation channels reacting to thermal and mechanical stimuli³⁸. TRPV1, is prevalently expressed in sensory neurons that relay information to the somatosensory cortex through the spinal cord ³⁹, and decreased TRPV1 activity lessens pain perception in skin and internal organs ^40,41. TRPV4 is involved in chronic inflammatory pain by influencing inflammatory mediators ³¹. Beyond their role in nociception, TRPV channels are implicated in cartilage mechanosensitivity, with evidence linking them to chondrocyte apoptosis under excessive mechanical stress ^29,42. Initial studies highlighted TRPV expression in chondrocytes, suggesting their involvement in OA-induced cartilage damage ⁴³. These roles of TRPV channels in pain perception, mechanotransduction, and inflammation highlight their potential relevance in disorders like OA.

TRPV1 and TRPV2

TRPV1 and TRPV2 activation appears to have both protective and pathogenic roles in joint tissues through their effects on chondrocytes, bone, and immune cells. Single-cell RNA sequencing of osteoarthritic chondrocytes recently showed that TRPV1 activation can safeguard chondrocytes from ferroptosis by upregulating glutathione peroxidase 4 (GPX4) ⁴⁴, although the interplay between TRPV1-mediated ferroptosis and other cell death pathways in OA requires further exploration. Activation of TRPV1 by mechanical stress has been indicated to suppress expression of catabolic enzymes in chondrocytes by hindering the nuclear translocation of nuclear factor κB (NF- κB), and this effect was reversed by the TRPV1 antagonist capsazepine ³⁰. Moreover, TRPV1 activation in chondrocytes has been shown to alleviate subchondral bone sclerosis and osteophyte formation in a mouse model of destabilization of the medial meniscus (DMM)-induced OA ⁴⁵. In addition, TRPV2 has been implicated in osteoclast differentiation ⁴⁶. High TRPV2 expression in cartilage has been observed in mouse and human heterotopic ossification lesions ²⁵ Mice with chondrocyte-specific TRPV2 knockout displayed increased articular cartilage degradation and periarticular heterotopic intracartilage osteogenesis in OA ²⁵. Finally, TRPV1 activation, but not TRPV1 inhibition, was shown to prevent M1 macrophage polarization in the synovium through the Ca²⁺–CaMKII–NRF2 signalling pathway, indicating a potential for TRPV1 agonists to counteract OA synovitis ⁴⁷. However, results from other preclinical studies have suggested an OA-promoting role for TRPV1 activation. For example, the TRPV1 antagonist APHC3 partially reduced joint inflammation in a model of monosodium iodoacetate (MIA)-induced OA⁴⁸. The contrasting reports on the effects of TRPV1 and TRPV2 activation or blockade in OA inflammation and ossification, underscore the need for further study of these channels in the pathobiology of OA.

Pain, a common symptom in OA, has also been linked to TRPV1 channel activity. The TRPV1 Ile585Val variant has been associated with increased pain in patients with OA ⁴⁹, and the TRPV1 antagonists JNJ-17203212, capsazepine, or APHC3 could relieve pain in animal models of OA ^{22,50, 48}.

Clinical trials have underscored the therapeutic relevance of targeting TRPV1 for pain relief in OA. A 2017 trial documented that a single dose of the TRPV1 antagonist Mavatrep markedly relieved OA pain ⁵¹, and further research indicated that repeated Mavatrep doses ranging from 2–50 mg reduced pain without inducing tolerance⁵². Moreover, clinical evidence suggests that intra-articular injection of trans-capsaicin, a potent TRPV1 agonist, alleviates knee pain in OA in a dose-dependent manner ⁵³. However, the clinical application of TRPV1 modulation is challenged by side-effects like hyperthermia ⁵⁴. These contrasting findings underscore the complexity of TRPV1 channel involvement in OA pathology and highlight the necessity for further investigation to reconcile these discrepancies. Future research should aim to elucidate the contextual factors governing the dual roles of TRPV1 and TRPV2 in OA pathogenesis, thereby facilitating the development of targeted therapeutic strategies with enhanced efficacy and specificity.

TRPV4

TRPV4 activity has been shown to promote chondrogenic differentiation in vitro and regulate cell survival ^19,55. Blockade of TRPV4 dampens chondrocyte sensitivity to hypo-osmotic stress ^56,57. Calcium signaling downstream of TRPV4 activation enhances anabolic gene expression and suppresses catabolic gene expression in chondrocytes ^{58,59, 60}. TRPV4 activation enhances TGF-β signaling ⁶¹ and GSK3β phosphorylation, thereby increasing chondrocyte sensitivity to ECM viscoelasticity ⁶².

TRPV4 activation has also been reported to contribute to mitochondrial dysfunction, pyroptosis, and matrix degradation in chondrocytes ⁶³, whereas cartilage-specific TRPV4 knockout ameliorated age-related OA in mice⁶⁴. Additionally, TRPV4 inhibition reduced mechanical stress-induced chondrocyte apoptosis and cartilage degeneration in an anterior cruciate ligament-transected rat OA model ²⁹. Further research linked TRPV4 to chondrogenesis, particularly in hyaluronic acid's presence, where its deficiency impaired TRPV4-induced expression of chondrogenic markers SOX9 and Aggrecan ⁶⁵. However, TRPV4 activation in chondrocytes has also been reported to be protective against inflammatory cartilage damage and OA via the CaMKK/AMPK/NF-κB pathway ^66-68, and global TRPV4 deficiency in mice increased subchondral bone volume and OA severity ^69,70.

TRPV4 has been shown to have a role in Ca²⁺ influx and induce osteoclast differentiation ⁷¹, setting a precedent for studying TRPV channels in bone cell metabolism. TRPV4-deficient mice demonstrated increased susceptibility to OA and increased osteogenic response to a high-fat diet, indicating TRPV4's involvement in adipogenic differentiation and osteoblast metabolism ⁷⁰.

Global and microglia-specific knockout of TRPV4 in mice or pharmacological TRPV4 inhibition significantly reduced neuropathic pain behaviors in a spared nerve injury mouse model and in a mouse model of OA, and the TRPV4 antagonist GSK2193874 also reduces knee OA-induced mechanical hyperalgesia in rats, suggesting that TRPV4 inhibition might relieve pain ^72-74. TRPV4 activation in dorsal root ganglia (DRG) neurons with the TRPV4 agonist GSK1016790A exacerbated pain behaviors in a rat model of OA, and increased levels of the endogenous TRPV4 ligand 5,6-EET in OA synovial fluid and rat knee joint capsule lavage fluid might act as a coactivator of mechanical stimulation to activate TRPV4 and enhance OA pain ⁷⁵. These findings emphasize the importance of further assessing the safety and efficacy of TRPV4 inhibitors for OA pain management in large animals and humans.

TRPV channels are expressed in human fibroblast-like synoviocytes (FLSs), mediating Ca²⁺ influx ⁷⁶, with TNF increasing its expression in FLSs ⁷⁷. In OA, a specific TRPV4 inhibitor, HC067074, when intra-articularly injected, slowed OA progression in rats and reduced synovial macrophage M1 polarization, with a decrease in reactive oxygen species in M1 macrophages ⁷⁸. The diversity of the reported roles of TRPV4 might be due to differences in animal species and strains, disease models, drug specificity, and/or stages of OA. While well-controlled research is necessary to delineate the precise functions of TRPV4 in OA and cartilage damage, TRPV4 emerges as a potential therapeutic target in OA treatment.

TRPV5 and TRPV6

TRPV5 was detected in superficial and intermediate chondrocytes of equine articular cartilage, suggesting that it may play a role in chondrocyte biology ⁴³. It has been shown that blocking TRPV5 using oxoglaucine can inhibit apoptosis and matrix degradation in osteoarthritic chondrocytes via the Ca²⁺/CaMK-II/calmodulin pathway ⁷⁹ . The combination of aerobic exercise and glucosamine hydrochloride has been found to suppress chondrocyte apoptosis in a rabbit model of knee OA via down-regulation of TRPV5 expression ⁸⁰. Although these findings indicate that TRPV5 inhibition might counteract mechanisms involved in OA progression, further in vivo studies are required.

In osteoclasts, TRPV5 is expressed and localizes in cellular folds, with TRPV5-deficient mice exhibiting diminished osteoclast function despite an increase in osteoclast numbers. However, there are reports that TRPV5-mediated Ca²⁺ influx inhibits osteoclast bone resorption in vitro ⁸¹. Like TRPV5, TRPV6 is implicated in the regulation of chondrocyte function in OA. Global TRPV6 knockout mice show pronounced OA changes, including cartilage fibrillation and proteoglycan loss ⁴². TRPV6 deficiency correlates with impaired chondrocyte function and increased apoptosis, indicating that TRPV6 is likely to beinvolved in OA pathogenesis ⁴². The detailed molecular mechanisms, however, warrant further study.

TRPMs

The TRPM subfamily consists of eight members (TRPM1–TRPM8) ⁸². TRPM channels exhibit distinct ion permeabilities and regulatory mechanisms. For instance, TRPM1, TRPM3, TRPM6, and TRPM7 are highly permeable to divalent cations and their activities are modulated by PI(4,5)P2 and intracellular Mg²⁺. TRPM2 and TRPM8 can conduct both divalent and monovalent cations, whereas TRPM4 and TRPM5 are restricted to monovalent cations. As a result of this variability, TRPM channels seem to have distinct roles in joint cellular function and in OA mechanisms.

TRPM2 channels are highly expressed in immune cells, and TRPM2 channel activity contributes to the modulation of inflammatory responses. In global TRPM2-deficient mice showed reduced mechanical allodynia, thermal hyperalgesia, and neutrophil infiltration in response to carrageenan-induced pain ⁸³. Similarly, these mice demonstrated diminished mechanical allodynia in response to MIA-induced OA ⁸⁴. Collectively, these findings suggest a role for TRPM2 in OA inflammatory pain. Current research on TRPM2 focuses on pain, yet its involvement in other OA pathologies merits exploration. FLSs contribute to joint swelling and pain in progressive OA ^6,9. Recent studies reveal that TRPM3 expression in FLSs inversely affects hyaluronic acid secretion ⁸⁵, indicating a potential regulatory role of TRPM3 in OA synovitis that warrants further in vivo investigation.

Emerging evidence has elucidated the roles of TRPM7 and TRPM8 in chondrocyte death and hypertrophy. TRPM7, as a Mg²⁺ sensor, has decreased expression in chondrocytes when dietary Mg²⁺ is deficient, thereby triggering Wnt-β-catenin signaling, reducing autophagy, and correlating with OA exacerbation ⁸⁶. TRPM7 blockade with the known nonselective inhibitor 2-APB mitigates sodium nitroprusside-induced apoptosis in rat primary chondrocytes by inhibiting Ihh signaling ⁸⁷. Additionally, genetic and pharmacological inhibition of TRPM7 reduces erastin-induced ferroptosis in rat primary articular chondrocytes and human C28/I2 chondrocytes via the PKCα–NOX4 axis ⁸⁸. TRPM7 has also been reported to maintain Ca²⁺ homeostasis during chondrocyte hypertrophy through PI3K-Akt signaling in vitro ⁸⁹.

TRPM8 is the most prominently expressed of the 19 TRP genes that are expressed in human osteoarthritic chondrocytes ⁹⁰. Menthol, a TRPM8 agonist, increases the expression of MMPs and IL-6 in primary human OA chondrocytes ⁹⁰.

TRPA

Currently recognized as the only TRPA channel in humans, TRPA1 channel is a tetrameric molecule composed of four subunits ³⁷. TRPA1 detects exogenous stimuli potentially harmful to cells, responding to thermal, mechanical, and chemical signals ⁹¹. The TRPA expression pattern in sensory neurons and some non-neuronal cells such as chondrocytes and FLSs, indicates a potential role in pain perception³⁵. Indeed, TRPA1 has been implicated in various pain modalities, including neuropathic and inflammatory pain^92,93. Primary human OA chondrocytes were found to express TRPA1, and stimulation by inflammatory cytokines IL-1β and IL-17 further increased this expression ⁹⁴. Genetic or pharmacological TRPA1 inhibition reduced MMP and IL-6 production in OA chondrocytes, indicating a role for TRPA1 in cartilage degradation ^35,94. Enhanced TRPA1 expression downstream of stimulation with IL-1β, was found to induce chondrocyte apoptosis in vitro through calcium overload and mitochondrial dysfunction ^{90, 95}, although in vivo studies within OA models are required for further validation of this link. TRPA1 antagonist CHEM was also found to decrease the expression of inflammatory cytokines and MMPs in osteoarthritic FLSs, suggesting potential therapeutic benefits in attenuating OA synovitis ⁹⁶. In relation to pain, global TRPA1 knockout mice exhibited reduced nociception in MIA-induced OA, which might be linked to lower levels of substance P ⁹⁷. Subsequent research supports an analgesic effect of TRPA1 inhibition in OA ^73,98. However, the exact mechanisms by which TRPA1 is involved in pain perception are not fully understood, highlighting the need for more in-depth studies.

TRPCs

The TRPC channels structurally resemble other TRP channels family members, forming tetrameric structures with a central ion-conducting pore that is non-selectively permeable to cations ⁹⁹. TRPC channels import Na⁺ and Ca²⁺ and export K⁺ ions, crucially maintaining intracellular Ca²⁺ balance. TRPC channels respond to various stimuli such as mechanical forces and exhibit voltage sensitivity. Although their activation mechanisms remain largely unclear, TRPC modulation via G protein-coupled receptor pathways indicates a sophisticated gating mechanism. TRPC channels have been implicated in multiple pathological conditions, and are, thus, considered as promising therapeutic targets ¹⁰⁰.

The mechanosensitive TRPC1 channel is expressed in articular chondrocytes from patients with OA and in cultured chondrocytes¹⁰¹. Mechanical unloading was shown to downregulate TRPC1 during chondrocyte dedifferentiation ¹⁰², but the role of TRPC1 in chondrocyte mechanotransduction remains to be explored.

TRPC5 activation has been associated with pain, and shows altered expression in the DRG neurons in rat models of neuropathic pain ¹⁰³. TRPC5-deficientmice or mice treated with the TRPC5 antagonist ML204 exhibit increased hyperalgesia in complete Freund's adjuvant (CFA)-induced arthritis, suggesting a pain modulating role for TRPC5 ^104,105. Moreover, ML204 treatment was found to increase synovial inflammation and vascular swelling in CFA-induced arthritis, indicating that TRPC5 might have anti-inflammatory functions and serve as a therapeutic target in OA¹⁰⁴. More work is needed to delineate the roles of TRPC channels in OA.

Piezo channels

The Piezo channels are trimers composed of 114 transmembrane helices that resemble a three-bladed propeller and have a crucial role in mechanotransduction^106,107. Mechanical stimuli influence cation permeability of Piezo channels, which differs in the order of Ca²⁺ > K⁺ > Na⁺ > Mg²⁺, with Piezo1 being more sensitive to mechanical regulation than Piezo2 ^108,109. The inactivation characteristics of Piezo1 and Piezo2 also differ slightly, with Piezo2 exhibiting faster inactivation kinetics than Piezo1^110,111. Mutations of residues L2475 and V2476 in the inner helices and of residues M2493 and F2494 in the C-terminal domains could completely eliminate the inactivation of Piezo1 but not Piezo2, indicating distinct regulation ¹¹². Further exploration of these physiological and pathological differences of Piezo channels is needed. Piezo channels have been linked to various physiological and pathological conditions including OA (Table 1, Fig. 4).

Fig. 4. Involvement of Piezo, Nav, Cav, and K⁺ channels in OA pathophysiology.

Ion channels, including Piezo, Nav, Cav, K_ATP, Kv, and K_Ca channels, are expressed in chondrocytes and fibroblast-like synoviocytes, regulating various cellular functions and playing key roles in the pathophysiology of OA. These channels facilitate the entry of Ca²⁺ and Na⁺ in response to various extracellular stimuli, significantly impacting cellular functions such as chondrocyte metabolism, chondrocyte death, and fibroblast inflammation, influencing anabolic and catabolic processes via the PI3K, PKC, and Wnt/β-catenin signaling pathways. Specifically, blocking Nav1.7 can reduce intracellular sodium ions, promoting sodium-calcium exchange and consequent increase of intracellular calcium ions. The activation of Piezo1/2 channels leads to Ca²⁺ influx, activating calcineurin and NFAT1, which in turn regulates chondrocyte metabolism and death ^113,116. Cav channels facilitate calcium influx affecting pathways like Wnt/β-catenin and COX2^{115,165,166,178}, while potassium channels (K_ATP, Kv, K_Ca) regulate processes like endoplasmic reticulum stress (ERS), PI3K, AKT, and ECM production. Collectively, these ion channels integrate signaling that balances anabolic and catabolic activities, apoptosis, and inflammation in the joint environment. This figure was created with BioRender.com.

Articular chondrocytes detect and respond to mechanical stresses via the activation of Piezo channels³³. Expression of piezo channels has been identified in primary chondrocytes and cartilage ^{58, 33}. Whereas Piezo1 transcript is measurable in mouse chondrocytes ⁵⁹, inconsistent detection of Piezo2 transcript suggests variable expression. Importantly, Piezo1 expression is upregulated in the cartilage of patients with as well as of rats with anterior cruciate ligament transection-induced OA ^33,113. However, no upregulation of Piezo expression was identified in joint tissues or DRG neurons of rats with MIA-induced knee OA ¹¹⁴.

Mechanical strain activates Piezo-mediated calcium signalling in chondrocytes, where Piezo1 functions independently of Piezo2 to protect articular chondrocytes from mechanically induced cell death ^{24,58,115,116}. The selective Piezo1 agonist Yoda1 was found to increase intracellular Ca²⁺ levels in osteoarthritic human chondrocytes and to upregulate expression of MMPs, TIMP metallopeptidase inhibitor 2 (TIMP2), bone morphogenetic protein 2 (BMP2), collagen type Iα1 (Col1A1), and interleukins ¹³. In addition, Yoda1 inhibited TRPV4 activation by GSK1016790A, and this finding suggests that a functional interplay between Piezo1 and TRPV4 occurs in chondrocytes, possibly through direct or indirect physical interactions ¹³. By contrast, the Piezo inhibitor GsMTx4, has been shown to reduce chondrocyte apoptosis and alterations in the cartilage matrix, alleviating OA progression via the Calcineurin–NFAT1 pathway¹¹³. Contrary to these findings, conditional knockout mice lacking Piezo1 and Piezo2 exhibited normal joint development, and piezo deficiency did not affect OA progression ¹¹⁷. Thus chemical activation of Piezo channels might elicit effects that differ from those resulting from mechanical activation, and the involvement of Piezo in OA merits further detailed investigation.

In addition to their functions in articular cartilage, Piezo channels might affect OA progression by adaptive immunity. Piezo1 channels facilitate Ca²⁺ influx in T cells, and might thereby affect T-cell activation threshold ¹¹⁸. Indeed, knockdown of Piezo1 in CD4⁺ T cells has been associated with the expansion of regulatory T (Treg) cellsin a model of autoimmune encephalomyelitis, where it reduced disease severity ¹¹⁹.

Understanding the distribution of Piezo1and Piezo2 in the infrapatellar fat pad-synovial membrane unit, might provide targets for OA pain treatments ¹²⁰. Indeed, single-cell RNA sequencing demonstrated a subset of nociceptors expressing Piezo2, highlighting Piezo2 as a target for pain therapy in OA²³. Piezo2 conditional knockout in nociceptors protected against mechanical sensitization in joint pain models ²³.

Voltage-gated sodium channels

Voltage-gated sodium channels (VGSCs) are composed of a predominant α subunit (Nav) and auxiliary β subunits, with the Nav α-subunit directly regulating ion permeability ^121,122 to control sodium currents. These sodium currents are required for action potential generation, and are also involved in degeneration of injured axons ^123,124, or in the control of effector functions of some non-excitable cells ^125-128. Nine genes (SCN1A–SCN11A) encode nine Nav channel α-subunits (Nav1.1–1.9) ¹²². Nav1.1 and Nav1.2 sequences share a 93% similarity, and sequence similarity ranges between 85–90% for Nav1.1 and Nav1.6 ¹²⁹ —but Nav channels still seem to be functionally diverge. Nav channels are found in high density in neurons, muscle cells and cardiac myocytes, where their rapid activation and inactivation in response to membrane depolarization enable Na⁺ inflow, producing transient all-or-none action potentials ¹³⁰.

VGSCs are implicated in numerous physiological and pathological states, including pain ¹³¹. Nav1.7, Nav1.8, and Nav1.9, preferentially expressed in DRG sensory neurons, and are particularly involved in peripheral pain signaling ^132-134. VGSCs are crucial for neuronal excitability and cellular function but as noted above their presence in multiple types of non-excitable cells ¹³⁵, contributes to various pathologies, including OA (Table 1, Fig. 4) ²⁶.

Nav1.7

Nav1.7 plays a key role as a volume knob on pain signaling by DRG neurons. Gain-of-function Nav1.7 mutations produce excruciating pain ^136-138, while carriers of loss-of function mutations exhibit a syndrome of profound pain insensitivity, experiencing painless burns, tooth extractions, or fractures ^139,140. Intriguingly, individuals with non-functional Nav1.7 retain normal sensation to most other stimuli, except for olfaction, as Nav1.7 is also expressed in olfactory sensory neurons ¹⁴¹. The clear link between these pain conditions and Nav1.7 gain-of-function and loss-of-function mutations highlights the therapeutic potential of targeting this channel for pain relief. Notably, mutations in Nav1.7 have been associated with increased pain sensitivity in certain patients with OA ¹⁴², and mice with global or DRG-specific Nav1.7 knockouts have low inflammatory pain ^143,144. Spinal delivery of ProTx II, a Nav1.7-selective antagonist, attenuates pain in a rat model of OA, further supporting the therapeutic potential of Nav1.7 inhibition for OA pain relief ^145,146.

Excitingly, our research group has discovered that Nav1.7 channels, previously thought to be preferentially expressed in peripheral neurons like those in the DRG and sympathetic ganglia, are also present and functional in osteoarthritic human chondrocytes ^26,147,although Nav1.7 expression levels in chondrocytes are several orders of magnitude lower than in neurons. Genetic ablation studies in mouse models have revealed a dual role for Nav1.7: it mediates pain via its action in DRG neurons and influences OA progression via its action in chondrocytes ²⁶. Inhibiting Nav1.7 with selective blockers like PF-04856264 or the pan-Nav blocker carbamazepine significantly improved joint health and reduced pain in a mouse model of OA ²⁶. At a mechanistic level, Nav1.7 blockers modulate intracellular Ca²⁺ signaling through sodium/calcium exchanger, promoting the secretion of pro-anabolic HSP70 and anti-catabolic midkine by chondrocytes, which in turn impacts OA progression (Fig. 4) ²⁶.

Despite their sparse distribution in chondrocytes, Nav1.7 channels were found to regulate chondrocyte biology, and potentially also other joint cells, through paracrine effects ²⁶. ¹⁴⁸.

Nav1.8

Nav1.8 is selectively expressed in peripheral sensory neurons ¹⁴⁹ and has a complex structure, comprising transmembrane segments, voltage sensors, and ion-selective pores, that is essential for the regulation of sodium ion flow across cell membranes ¹⁵⁰. Nav1.8 is relatively resistant to inactivation by depolarization ¹⁴⁹ and recovers rapidly from inactivation ¹⁵¹. As it contributes about 80% of the sodium current underlying the depolarizing phase of the action potential ¹⁵², Nav1.8 is responsible for repetitive firing of DRG neurons when they are depolarized, as, for example, in many disease processes. Recent clinical trials have demonstrated that a Nav1.8-selective blocker can reduce acute postoperative pain ¹⁵³, clearly demonstrating the essential role of Nav1.8 for pain signaling in humans and providing proof-of principle that block of a peripheral Nav channel can reduce pain ¹³⁴. Studies in a mouse model of OA revealed increased Na_v1.8 expression in response to prostaglandin E2 (PGE2), suggesting that inhibition of Nav1.8 in the DRG neurons might reduce OA pain ¹⁵⁴. Targeted acute inhibition of sensory neurons that express Nav1.8 neurons with designer receptors exclusively activated by a designer drug (DREADD) technology has been shown to block knee hyperalgesia and mechanical allodynia in mice with early-stage DMM-induced OA¹⁵⁵, and the Nav1.8-blocker A-803467 has been found to reduce joint afferent nerve firing rates during simulated joint rotation ¹⁵⁶. Peripheral administration of A-803467 also alleviated hindlimb dysfunction and secondary allodynia in mice with DMM-induced OA, highlighting the potential of Nav1.8 channel targeting in joint nociceptors as an OA pain treatment strategy.

A complexity arises from the observation that pan-VGSC blockers like lidocaine can adversely affect chondrocyte numbers and viability, impacting chondrocyte homeostasis ^157-159. The development of more specific blockers, guided by the structural insights of channels such as Nav1.7 ^160-162, might mitigate this issue. Given that VGSCs have actions beyond pain signaling in OA and contribute to pathological mechanisms, including cartilage matrix degradation, and chondrocyte function alterations ^26,157-159, a more thorough investigation into these roles and molecular mechanisms will enhance our understanding and help identify the full therapeutic potential of sodium channels as therapeutic targets in OA.

Voltage-dependent calcium channels

Calcium ions are essential for intracellular signal transduction, acting primarily through release from internal stores or influx from extracellular spaces. Voltage-dependent calcium (Cav) channels are critical for the entry of extracellular Ca²⁺ into cells, as they open in response to membrane depolarization, allowing Ca²⁺ to move down its electrochemical gradient. Cav channels consist of five subunits—α1, α2, β, δ, and γ—and are pharmacologically classified into T-type, L-type, P-type, Q-type, and R-type ¹⁶³. Research indicates that L-type calcium channel blockers like nifedipine can safeguard OA cartilage, underscoring the significance of Cav channels in OA pathophysiology (Table 1) ^164,165. The non-porous subunits of Cav channels have also been implicated in cartilage damage processes (Table 1). The α2 subunit ligand PD-0200347 inhibits the Erk1/2 pathway, which is crucial in OA cartilage degradation, suggesting its potential utility in slowing OA progression ¹⁶⁶. Oral PD-0200347 administration also reduces metalloproteinase and inducible nitric oxide synthase expression in chondrocytes, indicating that α2 activation could lessen OA-related cartilage degeneration ¹⁶⁷. These findings highlight the therapeutic promise of targeting non-channel Cav subunits. Yet, further in vitro research is needed to clarify these mechanisms. Additionally, the pore-forming function of Cavα1 subunit has been implicated in pain ¹⁶⁸, and the α2 subunit has also been associated with pain mechanisms in OA ¹⁶⁹. CACNA2D1, the gene encoding the Cavδ1 subunit, modulates pain sensitivity; its knockdown in DRG neurons raises pain thresholds in OA rats, while its overexpression lowers them in normal rats ¹⁷⁰. This regulation may occur through DRG calcitonin gene-related protein alteration and the adenylyl cyclase-PKA/PKC/MAPK pathways. Considering the pivotal role of Cav inhibitors in multiple pathological processes, further evaluation of their potential effects as potential OA treatments is needed.

L-Type Cav channels

L-Type Cav channels are expressed in chondrocytesand contribute to cartilage diseases ¹⁷¹. The pathogenic link between Cav channels and OA has been investigated through studies on basic calcium phosphate crystals, which affect cytoplasmic Ca²⁺ levels and oscillations correlating with OA phenotypes in bovine chondrocytes and cartilage explants ¹⁷². Inhibition of L-type Cav channels blocks these crystal-induced Ca²⁺ oscillations and cartilage degradation, underscoring their role in OA ¹⁷². Furthermore, verapamil, an L-Type Cav inhibitor, effectively mitigates OA progression and cartilage damage in a rat model ¹⁶⁵ and reduces hyaluronic acid secretion and protein loss in IL-1β-treated chondrocytes ¹⁷³. These findings suggest an integral role for Cav channels in OA pathogenesis and highlight their potential as therapeutic targets. Additionally, Piezo channels raise intracellular cation concentrations, enhance cell membrane potential, and activate voltage-gated calcium channels (VGCCs)¹⁷⁴. Inhibiting L-type VGCCs reduces mechanical sensitivity to compression, tension, and shear stress, which protects cartilage from degradation and slows the progression of OA, highlighting the importance of managing ion channel interactions ^115,165. Beyond cartilage, L-type Cav channels are implicated in nociceptive transmission ¹⁷⁵, with OA's mechanical and inflammatory stimuli leading to pain. TROX-1, a Cav2 antagonist, notably attenuates mechanically evoked neuronal responses in OA rats ¹⁷⁶, suggesting the involvement of Cav2 channels in OA pain. However, as TROX-1 has low selectivity within the Cav2 family, further investigation is needed to clarify the functional roles of the various Cav2 subtypes in normal nociception and pathological pain.

T-type Cav channels

T-type Cav channels have become a focus in OA research ¹⁷⁷. Local Cav3.2 inhibition with NNC 55-0396 has been shown to downregulate chondrocyte mechanical response genes and prevent mechanical load-induced cartilage damage ¹⁷⁸. Furthermore, T-type Cav channels are implicated in intracellular calcium oscillations and NFATC2 signaling activation in osteoarthritic chondrocytes, highlighting another potential mechanism contributing to OA cartilage degeneration ¹⁷⁹. The analgesic effects of Cav3.2 inhibition have been explored in OA models, where an AAV-delivered Cav3.2 inhibitory peptide aptamer 2 that selectively targets this channel was shown to reduce pain ¹⁸⁰. While the analgesic mechanisms of Cav3.2 inhibition in OA warrant further investigation, targeted delivery strategies could enhance the safety and efficacy of treatments aimed at Cav channels.

Potassium channels

Potassium (K⁺) channels are encoded by a diverse gene family comprising over 90 genes in humans ¹⁸¹. K⁺ channels are tetrameric molecules categorized into three main classes based on their structure and functional characteristics: (1) inwardly rectifying K⁺ channels (Kir channels) are composed of four subunits; (2) two-pore domain K⁺ channels (K2P channels) have fifteen subunits; and (3) the single pore K⁺ channels Kv channels, KCa channels and Slo channels ¹⁸². K⁺ channels are expressed in OA joint tissue and play a key role in OA pathogenesis (Fig. 4).

Adenosine triphosphate (ATP)-sensitive K⁺ (K_ATP) currents have been observed in chondrocytes^183,184, and the K_ATP channels of osteoarthritic chondrocytes have been implicated in glucose sensing and OA pathogenesis ¹⁸⁵. K⁺ channels within mitochondria regulate metabolism and apoptosis in chondrocytes ¹⁸⁶. Diazoxide, which is known to open mitochondrial K_ATP (mitoK_ATP) channels, has been shown to protect chondrocytes from H₂O₂-induced apoptosis and cartilage degeneration in rats with OA, possibly via a mechanism that involves regulation of ER stress and ERK1/2 signaling ¹⁸⁶. The protective effects of K_ATP channels against cartilage degeneration in OA highlight the need for future research with more specific drugs.

Kv channels are expressed in chondrocytes ¹⁸⁷, and have been shown to support ECM synthesis and chondrocyte proliferation ¹⁸⁸. Downregulation of KCNQ1OT1, a member of the lncRNA voltage-gated channel subfamily Q, might contribute to poor chondrocyte survival in OA, while its upregulation enhances OA chondrocyte viability and suppresses inflammation by upregulating PIK3C2A and thereby activating the PI3K/AKT/mTOR pathway, highlighting the need for further research to fully elucidate the underlying mechanisms ¹⁸⁹. In addition, Kv channels have been associated with pain in OA, as DRG neurons from rats with OA displayed reduced mRNA levels of Kv7 (KCNQ) channels, whereas KCNQ activation in this model increased pain threshold and prolonged withdrawal latency ¹⁹⁰. KCa channels respond to internal Ca²⁺ and were initially identified in growth plate chondrocytes from chicken¹⁸⁴. They are involved in histamine-induced hyperpolarization in cartilage inflammation, with large conductance Ca²⁺-activated K⁺ (BK) channel α subunits modulating chondrocyte histamine responses ^191,192. BKCa channels also participate in osmotic stress responses in chondrocytes and are implicated in mechanical loading-induced OA ⁵⁷. In addition, BKCa channels modulate inflammatory pain as evidenced by the ability of BKCa channel openers, such as NS1619, to significantly inhibit mechanical hypersensitivity induced by inflammation in CFA-induced arthritis ¹⁹³. However, NS1619 does not impact thermal hypersensitivity or neuropathic pain behaviors in this model, suggesting a specific modulatory role for BKCa channels in certain types of inflammatory pain. Collectively, all above findings suggest that K⁺ channels play roles in OA, yet further investigation is needed to fully understand their mechanisms in OA pathophysiology.

Acid sensing ion channels (ASICs) and Cloride channels

Cells sense extracellular acid signals through ASICs, proton-gated channels that were initially identified in the nervous system ¹⁹⁴. There are seven ASICs isoforms: ASIC1a; ASIC1b; ASIC1b2; ASIC2a; ASIC2b; ASIC3; and ASIC4 ¹⁹⁵. ASIC1a is also expressed in cartilage, monocytes, and osteoblasts ^196-198, and has been implicated in bone diseases. Structurally, ASICs feature a large extracellular domain for ligand binding, two α-helices forming a funnel-shaped transmembrane domain, and intracellular N- and C-termini ¹⁹⁵. Except for ASIC2b and ASIC4, which are auxiliary subunits, ASIC subunits assemble into functional homotrimeric or heterotrimeric channels ^199,200. As ASICs channels are proton sensors, acidification triggers a rapid, transient inward current, mediating Na⁺ and Ca²⁺ influx ²⁰¹. ASIC3 also responds to mechanical stress ²⁰². Predominantly, ASIC activation by acidosis mediates calcium influx, affecting downstream signaling associated with ischemia and inflammation ^203,204.

ASIC1a

The accumulation of inflammatory metabolites in the joint cavity often causes acidosis, which is a distinctive feature of OA ^205,206. Inflammatory responses increase oxygen consumption and metabolic activity, leading to lactic acid accumulation and localized acidification ¹⁹⁵. This acidification contributes to OA progression, as it promotes osteoclast survival, adhesion, and migration ²⁰⁵. Elevated H⁺ levels in synovial fluid during inflammation suggest that ASIC channels might be involved in arthritis development. Specifically, ASIC1a is expressed in chondrocytes and can be upregulated by the pro-inflammatory cytokines IL-1β, IL-6, and TNF ^195,197,207. ASIC1a activation by extracellular acidification can lead to apoptosis or senescence in articular chondrocytes, thereby accelerating cartilage degradation and OA progression ²⁰⁸. In rat models of OA, ASIC1a expression increased in the osteoarthritic cartilage, and inhibition by the ASIC1a-specific blocker psalmotoxin-1 reduced expression of the senescence markers p16, p21, and p53 in chondrocytes. ASIC1a has been suggested to contribute to chondrocyte senescence in OA through autophagy-dependent Lamin B1 degradation (Fig. 5) ²⁰⁹. Additionally, ASIC1a activation might induce chondrocyte senescence indirectly, as it has been linked to M1 macrophage polarization and the associated secretion of IL-18 which promotes senescence ²¹⁰. These findings highlight ASIC1a as a regulator of chondrocyte function and a potential therapeutic target in OA. Future studies using cell-specific gene knockout techniques should focus on delineating its precise role in OA.

Fig. 5. Roles of ASIC1a, P2X, and chloride channels in OA progression.

These channels, activated by changes in the extracellular environment and mechanical stimuli, mediate ion influx, which subsequently activates signaling pathways such as NF-κB, AMPK, and those involved in apoptosis. These channels play a role in OA progression by influencing processes including macrophage polarization, chondrocyte apoptosis, pyroptosis, and cellular senescence. ASIC1α activation results in Ca²+ influx, triggering pathways involving Bcl2 and Bax that lead to the activation of caspase-3 and caspase-9, PARP ^197,207, and autophagy via Lamin B1, culminating in chondrocyte senescence and apoptosis. P2X7R facilitates calcium influx that activates NF-κB, caspase-3, caspase-7, Runx2, ADAMTS-5, NLRP3 inflammasome, and COX-2, which contribute to IL-1β production ^230-234, chondrocyte catabolism, and apoptosis, regulated by miRNA-373 and miRNA-186. P2X3R also mediates calcium influx, leading to the production of inflammatory cytokines IL-6 and TNF-α, and affecting neutrophil migration. Chloride channels promote Cl⁻ efflux, activate NLRP3 inflammasome and caspase-3 ^220-223, promoting macrophage inflammation and chondrocyte apoptosis. This figure was created with BioRender.com.

ASIC3

The role of ASIC3 in pain perception and transmission is distinct from that of ASIC1a. The ASIC3 antagonist APETx2 has been reported to reduce both acid-induced muscle pain and CFA-induced inflammatory pain ^211,212. ASIC3 has been implicated in weight-bearing pain and secondary hyperalgesia in MIA-induced OA, with ASIC3 levels in knee joint afferent nerves being downregulated by APETx2. Early APETx2 administration also appeared to prevent cartilage damage ²¹³. Additionally, the ASIC3 inhibitor A-317567 alleviated mechanical hypersensitivity in a rat model of OA pain ²¹⁴. Clinical findings have indicated that lysophosphatidylcholineacts via ASIC3 channels, to contributes to chronic pain in joint conditions, highlighting new avenues for pain management in OA ^215,216. By contrast, aspirin injections have been shown to decrease ASIC3 expression in DRG neurons and thereby mitigate secondary hyperalgesia after OA induction ²¹⁷. The emerging relevance of ASIC1a and ASIC3 for chondrocyte senescence and pain in OA highlights the need to further investigate the expression and function of ASICs in the physiological and osteoarthritic joint.

Cloride channels

Chloride ion channels (ClCs) contribute to the maintenance of osmotic balance, cell volume, and cellular potential homeostasis, by facilitating the movement of chloride ions (Cl⁻) across cell membranes ²¹⁸. ClCs are functionally diverse and include voltage-dependent, kinase-activated, Ca²⁺-activated, and ligand-gated channels. The detection of ClC messengers in articular cartilage indicates a role for ClCs in chondrocyte function and OA (Fig. 5) ²¹⁹. Hypotonic conditions and acidification in osteoarthritic cartilage, along with inflammatory factors like IL-1β, can regulate Cl⁻ currents through channels such as ClC-7, affecting chondrocyte function, apoptosis, and macrophage inflammatory responses ^220-222. ClC inhibitors, such as DIDS, NPPB, and Niflumic acid, have been suggested to prevent chondrocyte apoptosis and associated cell volume reduction ²²³. Given these regulatory roles, the involvement of hypoosmotic conditions and acidification in osteoarthritic cartilage in the regulation of Cl⁻ currents suggests that further study of ClCs in OA is essential.

P2X receptors

P2X receptors are ligand-gated cation channels that rapidly response to extracellular ATP, thereby influencing physiological and pathological processes including inflammation, pain perception, and neuromuscular transmission ²²⁴. Recent research has highlighted the involvement of P2X receptors in OA pathogenesis and, particularly, in cartilage degradation, inflammatory responses, and nociception ^225,226.

P2X7R

P2X7 receptors (P2X7Rs), are trimeric ligand-gated cation channels implicated in inflammation ²²⁷. Structurally, P2X7Rs have short N-termini and long C-termini, as well as two transmembrane domains flanking a glycosylated ATP-binding domain ²²⁷. ATP binding to P2X7Rs promotes channel opening and the ensuing Na⁺ and Ca²⁺ influx leads to depolarization of resting membrane potential ²²⁸. P2X7R activation has been shown to trigger the release of inflammatory mediators, cell proliferation, and apoptosis ²²⁷.

P2X7Rs are expressed in chondrocytes ²²⁹, and a study in rabbits has linked P2X7R expression in chondrocytes with apoptosis and OA ²³⁰. P2X7R activation in osteoarthritic chondrocytes has also been associated with pyroptosis, MMP-13 expression, and inflammatory signalling (Fig. 5)^231-233. In addition, P2X7R-targeting microRNAs miR-186 and miR-373 were reported to mitigate knee OA in mice ²²⁵ and inhibit chondrocyte proliferation ²³⁴, respectively. These in vitro studies suggest a regulatory role for P2X7R in OA chondrocytes, yet further in vivo verification and clarification of its mechanism are necessary.

P2X7R activity was also implicated in exacerbation of matrix degradation caused by overexpression of Pannexin 3 in a rat model of temporomandibular OA ²³⁵. Activation of P2X7 on lipopolysaccharide-activated monocytes and macrophages releases and matures IL-1β and IL-18, while P2X7 knockdown reduces IL-1β's effects on macrophage polarization, migration, and inflammatory responses, suggesting a role for P2X7R in inflammatory signaling ^236,237. Supporting these findings, L-carnitine and probenecid have been shown to reduce inflammatory cytokines IL-1β, IL-18, IL-6, and TNF-α, thereby attenuating MIA-induced OA in rats, possibly via P2X7R inhibition²³⁸. Furthermore, other P2X receptors, such as Pannexin 1, involved in ATP release and P2X7R activation, have been associated with OA-induced joint pain ²³⁹. Studies on the intra-articular MIA model in rats indicated that spinal ATP release and microglial P2X7R upregulation are related to OA pain ²³⁹. Collectively, these findings highlight the need to study the function of P2X7R in OA in further detail.

P2X3R

P2X3 receptor (P2X3R) is expressed in chondrocytes ²⁴⁰, and its role in inflammatory pain transmission has been implicated in OA progression ²⁴¹. Intra-articular P2X3R blockade with A-317491 has been shown to significantly reduce nociceptive hypersensitivity and inflammation in rats with inflamed knee joints (Fig. 5)³⁴, and diminished ERK phosphorylation in DRG neurons ²⁴². Moreover, in a rat model of OA, the P2X3R antagonist MK-7264 notably improved nociceptive hypersensitivity, with an effectiveness comparable to naproxen ²²⁶. These discoveries underscore P2X3R as a viable target for OA pain management, laying the groundwork for new analgesic approaches in OA therapy.

Clinical implications and future directions

Several clinical trials currently test the therapeutic potential of ion channel targeting in OA (Table 2). Most trials primarily address the effect of ion channel agonists or antagonists on knee joint pain, often neglecting assessment of articular cartilage degradation, localized inflammation, or effects on other weight-bearing joints such as hips and ankles. Investigations in these areas are likely to both reveal new biomarkers and broaden the scope of the search for more effective treatments for OA.

Table 2. Clinical updates of potential ion channels modulators for OA.

Target channel	Drug	Indications	CT Identifier	Study phase	Status	Mechanism of action
TRPV1	CNTX-4975/ Trans-Capsaicin	OA knee pain	NCT02558439	Phase 2	Completed/CNTX-4975 treatment was associated with dose-dependent improvement in knee OA-associated pain	A potent agonist for the TRPV1/ taking advantage of the selective long-term analgesic effects of capsaicin 53
TRPV1	CNTX-4975-05/ Trans-Capsaicin	OA knee pain	NCT03660943	Phase 3	Completed/ To test efficacy and safety of repeat doses of CNTX-4975-05 in patients with OA knee pain	A potent agonist for the TRPV1
TRPV1	AZD1386	OA knee pain	NCT00878501	Phase 2	Terminated/ Phase IIa/b trial of AZD1386 was terminated as no improvement in pain was demonstrated	TRPV1 antagonist 246
TRPV1	JNJ-39439335/mavatrep	OA	NCT01343303	Phase 1	Completed/ JNJ-39439335 demonstrated sustained pharmacodynamic effects and an efficacy signal in participants with OA pain.	TRPV1 antagonist 52
TRPV1	NEO6860	OA knee pain	NCT02712957	Phase 2	Completed/NEO6860 did not statistically significantly outperform placebo but showed an analgesic trend	TRPV1 antagonist 247
TRPV1	GRC-6211	OA knee pain	/	Phase 2	Terminated/ Reasons not disclosed	TRPV1 antagonist
TRPV1	Civamide/ Zucapsaicin	OA knee pain	NCT00995306	Phase 3	Completed/ Demonstrated the efficacy of civamide cream for up to 1 year of continuous use	TRPV1 agonist
TRPV1	Resiniferatoxin	OA knee pain	NCT04044742	Phase 3	Withdrawn/ To be replaced by a different protocol	TRPV1 agonist
TRPV1	V116517	OA knee pain	NCT01688934	Phase 2	Completed/No Study Results Posted	TRPV1 antagonist
TRPV1	Capsaicin	OA	NCT00471055	Phase 3	Completed/ Capsaicin has an effect on symptom control in patients with knee OA	TRPV1 agonist
TRPA1	LY3526318	OA	NCT05080660	Phase 2	Completed/ Compared with placebo, it has an effect on the pain of arthritis patients	TRPA1 antagonist
ASICs	LPC16:0	OA	NCT01867840	Observational	Completed/ A specific increase in LPC16:0 in OA patients	Involved in chronic joint pain through the activation of ASIC3 248
VGCC-α2δ	Gabapentin	OA knee pain	NCT03334903	Phase 4	Completed/ To determine the best strategy of administering gabapentin in connection with our current approach to perioperative pain management.	VGCC-α2δ antagonist
Cav1.2	Verapamil	OA	NCT01645709	Phase 2	Terminated/ Sponsor decision to terminate study	Cav1.2 antagonist
VGCC-α2δ	Pregabalin	OA knee pain	NCT05447364	Phase 4	Recruiting	VGCC-α2δ antagonist
Nav1.7	iN1011-N17	OA knee pain	NCT05496205	Phase 1	Completed/ Oral administration of iN1011-N17 has safety, tolerability, and favorable pharmacokinetic profiles in healthy volunteers	Nav1.7 antagonist 249
Nav1.7	TV-45070	OA	NCT02068599	Phase 2	Completed/ Compared with placebo, failed to improve the patient 's pain	Nav1.7 antagonist
Nav1.7/Nav1.8	Lidocaine	OA	NCT00904462	Phase 2	Completed/ Lidocaine patch 5% reduces the intensity of pain qualities	Antagonist for the Nav1.7/ Nav1.8
Nav1.7	PF-05089771	OA	NCT01529671	Phase 1	Completed/ No Study Results Posted	Nav1.7 antagonist
P2X7	CE-224535	OA	NCT00418782	Phase 2	Terminated/ Results of interim analysis indicate lack of efficacy when compared to placebo	P2X7 antagonist
P2X3	Gefapixant/ AF-219/MK-7264	OA	NCT01554579	Phase 2	Completed/ Compared with placebo, it can improve the pain of patients measured by NPRS	P2X3 antagonist

OA, Osteoarthritis; LPC, lysophosphatidylcholine; NPRS, Numeric Pain Rating Scale; NA, Not Applicable

Despite a growing understanding of the functions of ion channels in OA, the path towards their therapeutic exploitation is not without challenges. One crucial barrier is the lack of the specific inhibitors or activators for selected ion channels. Developing such specific modulators is essential to ensure safety and tolerability for long-term treatments. Furthermore, a better understanding of the mechanisms through which ion channels interact with each other as well as with other molecular pathways involved in OA progression, is crucial for the creation of treatments that are not only efficacious but also safe for individuals living with this widespread degenerative joint condition. In concert with these considerations, there is a pressing need for biomarkers of disease progression and specificity of treatment that can be used in clinical studies.

Numerous unknowns about ion channel biology in OA persist and require further exploration. The electrophysiological properties of ion channels in joint cells like chondrocytes and synovial cells are not fully elucidated. Known activators such as mechanical and osmotic pressures, temperature, and acidification do not fully account for the reported contribution of ion channels to OA, and the precise factors influencing their activity warrant further study. Some ion channel types have a functional impact in articular chondrocytes but are moderately expressed in these cells. The small number of channels per cell presents a challenge to their study.

Ion channel inhibitors, commonly prescribed for cardiovascular and neurological disorders such as epilepsy and some pain syndromes, have effects that extend beyond the myocardium, vascular smooth muscle, and neuronal tissues, also modulating ion channels in other organs such as subchondral bone, synovium, and articular cartilage ²⁴³. This modulation can influence metabolism, cartilage ECM renewal, and inflammatory responses, or can result in pain, suggesting that the implications of these drugs in the pathogenesis of OA should be carefully evaluated in long-term treatment strategies to discern their potential exacerbating or ameliorative effects on OA progression ²⁴⁴. Additionally, the broader systemic impacts of ion channel blockers when used in OA treatment warrant consideration. Beyond specific small-molecule drugs targeting ion channels, siRNA, antisense oligonucleotides, antibodies, and mRNA targeting might present alternative approaches for OA treatment and prevention ²⁴⁵.Tailoring intra-articular injections to specific osteoarthritic tissues could also be used, to enhance drug bioavailability while reducing systemic side effects, especially on the nervous and cardiovascular systems. The systemic use of adeno-associated virus (AAV) vectors for gene therapy has, at least at high doses, raises questions about possible toxicity, but this might be mitigated by intra-articular administration. In this regard, the application of biomaterials like nanomaterials that promote cartilage penetration, and hydrogels could improve drug delivery control. These methods aim for targeted and extended drug release, potentially reducing side effects, decreasing dosage frequency, and increasing treatment durability, thus potentially prolonging patient lifespans and curtailing treatment expenses.

Conclusions

The pursuit of novel OA treatments is an urgent scientific endeavor, as traditional management approaches have seen limited advancement over the past several decades. This review examines the potential of ion channels as therapeutic targets within the multifaceted landscape of OA pathology and associated pain. While the path forward is complex and requires a nuanced understanding of ion channel biology and pathophysiology, the targeting of ion channels holds significant promise for revolutionizing the treatment of OA. This therapeutic direction, while still in its nascence, offers hope for more effective and targeted future interventions in OA management.

Key points.

• Ion channels have key functions in the joints and emerge as important contributors to pathogenic processes in osteoarthritis (OA).

• Dysregulation of mechanical and biochemical stimuli activates ion channels like TRPV4 and Piezo channels, leading to Ca²⁺ and Na⁺ influx, which contributes to cartilage destruction, inflammation, and pain in OA.

• Some ion channels typically associated with peripheral neurons are also expressed in chondrocytes and immune cells, and play a crucial role in the pathophysiology of OA.

• Exploration of ion channel expression, interactions, and properties in joints, along with development of specific channel agonists and antagonists has helped accelerate research on their potential roles in OA.

• Several ion channel modulators have been recently tested in animal models and patients with OA with encouraging results.

• Targeting of ion channels, thus, holds promise for the development of new and more effective OA treatment strategies.

Glossary

Ferroptosis

Ferroptosis is an iron-dependent form of cell death characterized by lipid peroxidation, driven by iron accumulation and depletion of antioxidant enzymes, distinguishing it from apoptosis, necrosis, and autophagy.

Glutathione Peroxidase 4 (GPX4)

This enzyme is crucial for protecting cells from ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols; inhibition or depletion of GPX4 results in lipid peroxide accumulation, thereby triggering ferroptosis.

All-or-none action potentials

Neuronal signaling follows the all-or-none principle, whereby an action potential is generated and propagated along the axon without amplitude decrement when a neuron's membrane potential reaches a stimulus-induced threshold, and no action potential occurs if the threshold is not reached.

Designer Receptors Exclusively Activated by Designer Drugs (DREADD) Technology

A technology in neuroscience that remotely controls specific neurons by genetically engineering receptors unresponsive to endogenous ligands but activated by synthetic, designer drugs.

Purinergic receptors

Responsive to purine nucleotides and nucleosides such as ATP and adenosine, these receptors are classified into P1, P2Y, and P2X types, with P1 and P2Y functioning as GPCRs activated by adenosine and nucleotides like ATP and adenosine diphosphate, respectively.