Perspective Article published: 14 October 2010 doi: 10.3389/fphys.2010.00135 The emerging chondrocyte

Richard Barrett-Jolley1, Rebecca Lewis1, Rebecca Fallman1 and Ali Mobasheri 2*

1 Musculoskeletal Research Group, Department of Comparative Molecular Medicine, School of Veterinary Science, University of Liverpool, Liverpool, UK 2 Musculoskeletal Research Group, Division of Veterinary Medicine, School of Veterinary Medicine and Science, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, Leicestershire, UK

Edited by: Chondrocytes are the resident cells of articular cartilage and are responsible for synthesizing Antonio Felipe, Universitat de a range of collagenous and non-collagenous extracellular matrix macromolecules. Whilst Barcelona, Spain chondrocytes exist at low densities in the tissue (1–10% of the total tissue volume in mature Reviewed by: Carmen Valenzuela, Instituto de cartilage) they are extremely active cells and are capable of responding to a range of mechanical Investigaciones Biomédicas and biochemical stimuli. These responses are necessary for the maintenance of viable cartilage CSIC-UAM, Spain and may be compromised in inflammatory diseases such as arthritis. Although chondrocytes Peter Larsson, University of Miami are non-excitable cells their plasma membrane contains a rich complement of ion channels. School of Medicine, USA This diverse channelome appears to be as complex as one might expect to find in excitable *Correspondence: Ali Mobasheri, Faculty of Medicine and cells although, in the case of chondrocytes, their functions are far less well understood. The ion

Health Sciences, School of Veterinary channels so far identified in chondrocytes include channels (KATP, BK, Kv, and SK), Medicine and Science, University of sodium channels (epithelial sodium channels, voltage activated sodium channels), transient Nottingham, Sutton Bonington receptor potential calcium or non-selective cation channels and chloride channels. In this Campus, Sutton Bonington, Nottingham, Leicestershire LE12 5RD, review we describe this emerging channelome and discuss the possible functions of a range UK. of chondrocyte ion channels. e-mail: ali.mobasheri@nottingham. Keywords: chondrocyte, K channel, K channel, BK (MaxiK) channel, ENaC ac.uk v ATP

Introduction at the synovial joint and absorbs shock. Friction would be unde- Chondrocytes are metabolically active cells found in mature articu- sirable because it would damage the joint and also generate heat, lar cartilage (Iannotti, 1990; Archer and Francis-West, 2003). The thereby causing pain (Tatari, 2007). Articular cartilage is avascular extracellular matrix (ECM) of cartilage is composed of elastic and without a perichondrium connective tissue surround. In human collagen fibers (mainly type II collagen), which provide tensile articular cartilage, chondrocytes may be as far away as 3 mm from strength with embedded proteoglycans forming a gel-like ground the nearest artery. Therefore, synovial fluid supplies chondrocytes substance that provides elasticity and the ability to resist compres- in adult articular cartilage with oxygen and nutrients, and removes sive forces (Buckwalter and Mankin, 1998). Chondrocytes occur carbon dioxide and metabolic waste products, by diffusion (Lee singularly or in groups or clusters of three or more cells within and Urban, 1997; Allan, 1998). Synovial fluid is periodically washed spaces called lacunae in the ECM (Stockwell, 1975). Articular car- over the surface of the articular cartilage by the movement of the tilage has a high matrix to cell ratio, with chondrocytes occupy- joint (Lee and Urban, 1997). Oxygen and substrate concentra- ing only 10% of the total tissue in mammals (Carney and Muir, tions within cartilage reduce near to the cartilage-bone margin to 1988). Articular cartilage is a type of hyaline cartilage that cov- almost zero (Otte, 1991). Therefore, chondrocytes generate ATP ers the surface of bones which meet at a synovial joint (Mankin, by substrate-level phosphorylation during anaerobic respiration, 1982). Synovial joints include a cavity between the bones within leading to the accumulation of lactate and lowering of the pH the articular capsule in order to allow free movement (Edwards through the production of H+ ions, which can continue in anoxic et al., 1994). The synovial cavity contains synovial fluid, which conditions (Lee and Urban, 1997). The extracellular pH affects the acts as a lubricant to decrease friction between the bones meeting chondrocyte metabolism and its ability to synthesize matrix. Low pH reduces lactate production, but also slows down the synthesis of glycosaminoglycans. However, the rate of collagen synthesis Abbreviations: ASIC, acid sensing ; BK, calcium-activated potassium appears to be independent of pH (Wu et al., 2007). Chondrocytes channel, high conductance; CFTR, cystic fibrosis transmembrane conductance re- gulator; ClC, ; DEG, degenerin; ECM, extracellular matrix; ENaC, embedded within the ECM have an unusual ionic environment epithelial sodium channels; IC50, concentration causing 50% inhibition; KATP, ATP because they are surrounded by negatively charged proteoglycans, dependent ; K , calcium activated potassium channels; Kir, + (Ca) which attract large numbers of cations, such as Na ions, creating inwardly rectifying potassium channel; KV, voltage-gated potassium channel; MIP, major intrinsic ; NMDA, N-methyl d-aspartate; PCR, polymerase chain re- a high extracellular osmolarity and contributing to the low pH action; RMP, resting ; SERCA, sarco/endoplasmic reticulum (Urban et al., 1993). Ca2+-ATPase; SITS, 4-acetamido-4′-isothiocyanatostilbene-2,2-disulfonic acid; Chondrocyte primary function is to synthesize and secrete SK, calcium-activated potassium channel, low conductance; SUR, sulfonylurea proteoglycans, collagen and non-collagenous to ­maintain receptor; TEA, ; TRP, transient receptor potential channel; TTX, tetrodotoxin; VGCC, voltage-gated calcium channels; VGSC, voltage-gated the cartilage ECM (Fassbender, 1987). Chondrocytes maintain cartilage by establishing a balance between replacing degraded

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­macromolecules and increasing synthesis in response to injury (Wu and Chen, 2000), proteins and sulfated glycosaminoglycans (Martin and Buckwalter, 2000). Proteoglycans contribute to carti- (Mouw et al., 2007). Chondrocyte proliferation is also inhibited lage rigidity, stability and durability during compression (Redini, by channel blockers lidocaine and verapamil (Wohlrab et al., 2001, 2001). Types II, IX, and XI collagen form the tensile fibril networks 2005) and apoptosis increased (Grishko et al., 2010). As with other within cartilage. Type VI collagens form adjacent to chondrocytes cells, the chondrocyte RMP is determined by the balance of positive and may be involved in attachment of the chondrocyte to the ECM and negative ion permeabilities in the cell membrane (Hodgkin (Bruckner and van der Rest, 1994). Non-collagenous proteins, and Huxley, 1952a). These permeabilities are, in turn, controlled such as anchorin CII, are also involved in chondrocyte anchorage by the chondrocyte channelome (the complement of expressed ion (Fernandez et al., 1990). The cartilage matrix protects chondro- channels and porins). cytes from mechanical stress placed on the joint (Buckwalter and Ion channels are the essential components that control ion Mankin, 1998; Martin and Buckwalter, 2000). Chondrocyte meta- movement in and out of the cell (Hodgkin and Huxley, 1952a). They bolic activity is directly correlated with the weight of mechanical are embedded within the plasma membrane and usually consist of stress placed on the cartilage; increased activity when the cartilage one or more proteins with a central aqueous pore, which opens by is heavily loaded provides maximum proteoglycan content (Urban, conformational change (Neher and Sakmann, 1992). The stimulus 1994). The ability of articular cartilage to withstand and respond for opening (gating) is specific to each ion channel, and may be volt- to pressure and shearing forces is vital for it to fulfill its function. age, chemically or mechanically induced (Hille, 2001). A number Accumulating evidence suggests that the resting membrane poten- of studies have now shown the presence of an ever-expanding list tial (RMP) is vital for fulfilling this function. The RMP has been of ion channels in chondrocytes (Figure 1), and this review will shown to be central to the secretion and synthesis of substances summarize the data to date, both on the variety of expression and in a variety of other cell types (Breittmayer et al., 1996; McCarty, the proposed roles of these channels. 1999; Penyige et al., 2002). It therefore seems likely that if the

RMP of chondrocytes is changed by ion channel manipulation, Kv channels their ability to produce ECM will be compromised. This conjec- One of the first discovered ion conductances in was the ture is indeed supported by experiments where RMP modifying potassium delayed rectifier Hodgkin( and Huxley, 1952b; Ramage ion channel blockers reduced the production of matrix mRNAs et al., 2008). The ion channels underlying this are now known to be

Figure 1 | Summary of the chondrocyte channelome. Many studies have potassium cannel, high conductance; ClC, chloride channel; ENaC, epithelial

now identified ion channels and porins in chondrocytes. Frequently the sodium channels; KATP, ATP dependent potassium channel; Kv, voltage-gated function of these channels is either unknown or controversial. This figure potassium channel; NMDA, N-methyl D-aspartate; SK, calcium-activated illustrates some of the major channel proteins identified to date, either by potassium channel, low conductance; TRP, transient receptor potential electrophysiological, immunological or molecular biological techniques. Note in channel; VGCC, voltage-gated calcium channels; VGSC, voltage-gated sodium

this figure, K(Ca) is taken to be equivalent to any calcium activated potassium channel; This data is summarized more fully in Table 1. For references please channel including BK and SK. AQP, channel; BK, calcium-activated see text and or Table 1.

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members of the Kv potassium channel family. This family is one of and Stanfield, 1978; Barrett-Jolley et al., 1999) and glibenclamide the largest ion channel families with at least 40 members (Gutman is an inhibitor of ATP dependent potassium channels (Tomai et al., 2005) of six transmembrane domains. Interestingly these et al., 1994). So far only KATP channels have been observed in were also one of the earliest ion channels discovered in chondro- chondrocytes (Mobasheri et al., 2007). KATP channels are a widely cytes (Walsh et al., 1992; Sugimoto et al., 1996). Kv channels have expressed subfamily of inwardly rectifying potassium channels. now been reported in chondrocytes by a number of authors and These channels are closed by intracellular ATP and thus serve to have been shown to be archetypal slowly inactivating ion channels couple metabolism to membrane excitability (Quayle et al., 1997; (Walsh et al., 1992; Wilson et al., 2004; Mobasheri et al., 2005a; Ashcroft and Gribble, 1998; Minami et al., 2004). Structurally these Ponce, 2006). In essence, these channels are very similar to those channels exist as heteromultimers. Each functioning protein con- channels found in skeletal muscle (Pallotta and Wagoner, 1992) sists of four ATP binding cassette proteins (SUR) surrounding four and in neurones (Barrett-Jolley et al., 2000) where, in those cell inwardly rectifying potassium channel subunits (Kir 6.x) (Babenko types, they are critical for of the membrane fol- et al., 1998). Of particular interest to investigators of chondrocyte lowing an . The role of a delayed rectifier channel function is the fact that, in addition to being opened by decreas- in the chondrocyte plasma membrane is far less clear. Since the ing intracellular ATP (Figure 2), KATP channels are also frequently chondrocyte exists at far more depolarized levels than neurones or observed to be opened by low oxygen tension and hypoxia (Dart skeletal muscle (Wright et al., 1992; Wilson et al., 2004), logic would and Standen, 1994). This suggests that these channels are impor- suggest that these channels would be constantly inactivated. Close tant in hypoxia-mediated cell signaling (Phillis, 2004). We showed study of the mathematical relationship between voltage, time and recently that KATP channels were expressed in articular chondro- fractional inactivation (Hodgkin and Huxley, 1952b) reveals that cytes (Mobasheri et al., 2007). We used polyclonal antibodies raised a certain, albeit small, proportion of these channels will remain against the KATP channel to show expression in both human and active even at the relatively depolarized RMP of a chondrocyte. equine chondrocytes. Expression was largely restricted to the This is supported by the observation by Wilson et al. (2004) and superficial and middle zones of normal cartilage and the superficial Clark et al. (2010b) that TEA inhibition of the potassium channels zone of fibrillated osteoarthritic cartilage in clusters (Mobasheri does have a significantly depolarizing effect on chondrocyte RMP, et al., 2007). In patch-clamp studies we found the biophysical as it does with other non-excitable cells such as those of smooth properties of KATP channels to be broadly similar to KATP channels muscle (Telezhkin et al., 2001; Park et al., 2007). expressed elsewhere (Babenko et al., 1998; Mobasheri et al., 2007).

Relatively few studies have attempted to establish the molecular Several KATP subtypes (i.e., Kir 6.1 and Kir 6.2) are each potentially identity of the delayed rectifier in chondrocytes. However, reports coupled with one of the SUR subtypes; SUR1, 2A or 2B (Babenko suggest that these channels are similar between species (chicken, et al., 1998). Pharmacological properties of KATP channels are thus canine, equine, and elephant) in terms of their steady-state half- very different between tissues. Glibenclamide is sometimes used as activation voltage and slope (Wilson et al., 2004; Mobasheri et al., a functional discriminator between KATP subtypes. It is highly active 2005a; Ponce, 2006). Half activation parameters range from 12 to in pancreatic β-cells (IC50 of <10 nM, Krause et al., 1995), but

25 mV; typical of Kv 1.x or Kv 4.x potassium channels (Coetzee et al., rather less potent in muscle (IC50 25–100 nM, Beech et al., 1993; 1999). Activation time constants are, however, quite fast compared Barrett-Jolley and Davies, 1997; Barrett-Jolley and McPherson, with many Kv channels (Mobasheri et al., 2005a). Such rapid kinetics 1998). In pharmacological studies of chondrocytes, the KATP chan- have been reported for members of the Kv 1.x family and also homo- nel’s IC50 is within the range seen in muscle (Mobasheri et al., meric Kv 3.4 (Coetzee et al., 1999). The inactivation time constant 2007). It therefore seems highly likely that chondrocytes express in the order of seconds (Mobasheri et al., 2005a) is typical of Kv 1.x, at least one subtype of KATP channel and that these may be impor-

Kv 2.x and Kv 3.x channels (Coetzee et al., 1999). Together these data tant for regulation of cartilage metabolism and sensing ATP levels suggested that the potassium channel of chondrocytes is likely to within the cell (Mobasheri et al., 2005b). be a member of the Kv 1.x. Pharmacological data are discussed in

(Mobasheri et al., 2005a) and are not entirely consistent for Kv 1.x Large Calcium-Activated Potassium Channels channels or one particular Kv channel. We therefore feel that the key, Several studies have putatively identified BK channels in chondro- published data identifying the subunit identity of the chondrocyte cytes (Grandolfo et al., 1990, 1992; Long and Walsh, 1994; Martina

Kv channels are the immunohistochemical and RT-PCR data. Such et al., 1997; Mozrzymas et al., 1997; Mobasheri et al., 2010). In our data have unequivocally revealed the presence of Kv 1.4 subunits own study (Mobasheri et al., 2010), the principal stretch-activated in equine chondrocytes (Mobasheri et al., 2005a) and Kv 1.6 in the channel we identified had a slope conductance, reversal potential, mouse (Clark et al., 2010b). Since Kv channels are known to exist and consistent with it being a large calcium-activated as functional heteromultimers (Villalonga et al., 2010) we would potassium channel (BK) (Latorre et al., 1989; Cui et al., 2009). tentatively suggest that articular chondrocytes may express Kv 1.x, We found the sensitivity to iberiotoxin to be statistically signifi- probably as a heteromultimer including the Kv 1.4 or Kv 1.6 subunits cant but weak (Mobasheri et al., 2010). This is interesting because and probably some other, as yet unidentified, vK subunit(s). whilst the BK channel can exist as a standalone six trans-membrane α-subunit, complete with potassium conducting pore and Ca2+ Inwardly Rectifying Potassium Channels sensor (Wang and Sigworth, 2009), the presence or absence of a Study of inwardly rectifying potassium channels is greatly ham- β-subunit determines many of the channel’s functional properties pered by a lack of selective inhibitors. Barium and chloroethyl- (Salkoff et al., 2006; Torres et al., 2007). In particular, low sensi- clonidine are inhibitors of the strong inward rectifiers (Standen tivity to iberiotoxin is highly characteristic of the expression of

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Figure 2 | KATP channels in chondrocytes. Chondrocytes have been shown to ATP (Babenko et al., 1998), increase in ADP (Dunne and Petersen, 1986),

express KATP channels. The function of these is generally accepted to be coupling extracellular hypoxia (Dart and Standen, 1994) or other chemical signals such as metabolic status with membrane potential and thus cell activity. In other cell adenosine (Dart and Standen, 1993; Barrett-Jolley et al., 1996), angiotensin

types, endogenous triggers for activation of KATP include decrease of intracellular (Sampson et al., 2007) etc., KATP, ATP dependent potassium channel.

BK channels consisting of both the α1 and β1-subunits (Lippiat Small Calcium-Activated Potassium Channels et al., 2003). This correlated well with our identification of posi- In addition to the body of work showing the presence of BK chan- tive immunohistochemical staining of normal articular cartilage nels, there have also been a few reports of SK activity in chondrocytes samples with antibodies to both α1 and β1-subunits. (Wright et al., 1996; Lee et al., 2000; Ramage et al., 2008; Funabashi In general terms, there appear to be two possibilities to explain et al., 2010b). In the study by Wright et al. (1996), osmotic shock led to the activation of BK channels by stretch. These could be termed a hyperpolarization, which was largely insensitive to iberiotoxin, but either calcium dependent or calcium independent mechanisms. highly sensitive to the SK channel inhibitor, . Interestingly, in The calcium dependent hypothesis would require that stretch led our own study of stretch activated potassium channels in chondro- to an increase in intracellular Ca2+ and that this activated the BK cytes (Mobasheri et al., 2010), whilst single channel studies clearly channel (Figure 3). Indeed a number of studies show changes in identified BK channels, the hypo-osmotic hyperpolarization was intracellular Ca2+ with osmotic or other mechanical challenge resistant to the low concentrations of TEA which would be expected (Grandolfo et al., 1998; Guilak et al., 1999; Yellowley et al., 2002; to block BK channels. The hyperpolarization was, however, inhibited Sanchez et al., 2003; Sanchez and Wilkins, 2004). The source of by symmetrical 10 mM TEA. This was an observation consistent with such Ca2+ is controversial, but potentially, dogma states that it the original observations of an SK component to the hyperpolariza- must come from either influx (e.g., a channel or other transporter tion shown by Wright et al. (1996), since both SK and BK are rather protein Sanchez et al., 2003; Sanchez and Wilkins, 2004; Phan resistant to extracellular TEA (Latorre et al., 1989). et al., 2009) or from intracellular stores (e.g., Grandolfo et al., 1998). The calcium independent hypothesis would involve either Transient Receptor Potential Channels direct sensing of stretch by the channel itself, or coupling of the Transient receptor potential (TRP) channels are a family of loosely channel to other mechanoreceptors such as integrins (Mobasheri related ion channels that show relatively little selectivity between et al., 2002). The function of BK activation by stretch is still permeable cations such as sodium, calcium, and magnesium1. unknown, but there are a few clear possibilities. Firstly, the BK They were initially proposed to couple hypo-osmotic shock to channel could be acting as an “osmolyte” channel (Hall et al., intracellular Ca2+ mobilization in chondrocytes on the basis of 1996; Kerrigan and Hall, 2008), since activation of potassium ­gadolinium sensitivity (Sanchez et al., 2003), but since then ­several conductances will allow potassium ions to leave, decrease intracel- lular osmotic potential and facilitate regulatory volume decrease. 1 Secondly, it is possible that it is the influence of the BK channel Transient receptor potential channels. Authors: David E. Clapham, Bernd Ni- lius, Grzegorz Owsianik. Last modified on 2010-04-07. Accessed on 2010-06-24. on the membrane potential which is critical, as it is in vascular IUPHAR database (IUPHAR-DB), http://www.iuphar-db.org/DATABASE/Family tissue (Ledoux et al., 2006). MenuForward?familyId=78.

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Figure 3 | Activation of BK by calcium ions. A number of studies have cation permeant ion channels. Both of these pathways could in turn, be activated identified BK channels in chondrocytes (see text), but the function of these by either mechanical or other (e.g., inflammatory) signals. ECM, extracellular

channels is not confirmed. Control of RMP or volume would are two theories. It matrix; Ins-P3-R, inositol trisphosphate receptor; IP3, inositol trisphosphate; K(Ca), is suggested that they are activated by calcium ions, which could be introduced calcium activated potassium channels; PLC, phospholipase C; SERCA, sarco/ to the cytoplasm by either release from stores, or by entry through divalent endoplasmic reticulum Ca 2+-ATPase; TRP, transient receptor potential channel.

TRP ­channels have been identified in osteoarthritic cartilage by and ­cytoskeletal complexes in close proximity. The presence of L-type PCR (Gavenis et al., 2009). TRPV4 has also been identified in both (and T-type) calcium channels in chondrocytes was recently sup- porcine and canine chondrocytes (Phan et al., 2009; Lewis et al., ported by Mancilla et al. (2007), however, (Sanchez and Wilkins, 2010) by PCR. TRPV4 is an established stretch activated channel 2004) found that osmotically induced changes in intracellular cal- and is widely regarded to be a conduit for stretch-activated entry cium ions were not influenced by more selective L-type calcium chan- of calcium ions (Nilius et al., 2004). TRPV4 has been shown to be nel blockers (including verapamil). In contrast aggrecan and collagen a regulator of the chondrogenic SOX9 pathway (Muramatsu et al., synthesis induced by electrical stimulation of cartilage is dependent 2007) and the deficiency of TRPV4 in knockout mice leads to a upon the activity of VGCCs (Xu et al., 2009). Clearly, further evidence loss of Ca2+ response to hypo-osmotic challenge and the onset of for the presence of this channel is needed to clarify these data. osteoarthritic changes (Clark et al., 2010a). Thus, it may be that by linking chondrocyte membrane stretch to calcium mobilization Voltage-Gated Sodium Channels (VGSC) TRPV4 is key to regulation of chondrogenesis, activation of calcium Voltage-gated sodium channels (VGSC) are integral membrane activated potassium channels and volume regulation. proteins that are activated in response to voltage-changes across the plasma membrane (Catterall, 1991, 1992, 1995, 2002). The presence Voltage-Gated Calcium Channels of tetrodotoxin sensitive VGSC in rabbit chondrocytes has been Voltage-gated calcium channels (VGCC) are a group of calcium reported by Sugimoto et al. (1996) and in chondrocytes from oste- permeable voltage-gated ion channels found in excitable cells (e.g., oarthritic cartilage by Ramage et al. (2008). It would be interesting muscle, glial cells, neurones, etc., Goldin, 2001; Dolphin, 2009). The to see how the expression of this channel fits into the control of presence of L-type VGCCs was suggested by Wright et al. (1996) the chondrocyte membrane potential, since current studies have on the basis of pharmacological inhibition of calcium dependent failed to observe sufficient hyperpolarization of chondrocytes for hyperpolarization by somatostatin and cadmium. It should be noted a typical VGSC to be substantially reactivated. Under conditions that whilst this is a plausible hypothesis, both somatostatin and of constant depolarization, for example, these channels would be cadmium affect a range of other ion channels including transient permanently inactivated. receptor potential channels (Carlton et al., 2004; Alexander et al., 2008), which may be present in chondrocytes. Ultrastructural stud- Epithelial Sodium Channels ies have confirmed the presence of L-type VGCCs in mouse limb Epithelial sodium channels (ENaC) have been identified in bud chondrocytes (Shakibaei and Mobasheri, 2003). These channels chondrocytes both immunohistochemically (Trujillo et al., 1999) appear to be organized around β-1 integrin receptors with kinases and functionally (Lewis et al., 2008). They are members of the

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degenerin (DEG) and ENaC superfamily (Mano et al., 2009). ENaC identification will be a tricky task since the ClC family is large and is a heteromeric channel, formed of up to four subunits; α, β, δ, and the available pharmacological inhibitors are rather non-selective γ (Canessa et al., 1994). Using immunohistochemistry, the α and β between each of the family members (Alexander et al., 2008). In our subunits have been shown to be present in chondrocytes (Trujillo own laboratories we have attempted to locate ClC-1 mRNA using the et al., 1999). ENaCs are significantly more permeable to sodium primers based on the sequence already identified for canine skeletal than potassium (Eaton et al., 1995) and are sensitive to the channel muscle (Rhodes et al., 1999). These studies show a lack of ClC-1, inhibitor amiloride (IC50 100–200 nM; Alexander et al., 2008). The but as yet there have been no positive studies on chondrocytes. The ENaCs main function in the kidney, bladder, and colon is control identity was suggested to be the maxi-ClC by Yabu and colleagues of sodium reabsorption (Rossier et al., 2002). They are found in (Sugimoto et al., 1996; Tsuga et al., 2002), but further studies will be lung tissue (Mall et al., 1998) and the taste buds (Lindemann, 2001) needed to clarify this. The ClC identified in rabbit articular cartilage and are known to regulate blood volume and pressure through by Isoya et al. (2009) proved to be swelling activated, but whilst its sodium balance in the cardiac system (Canessa et al., 1993). ENaC molecular identity is unknown, ClC-3 was suggested as a possibil- is known to have roles in various disease states, including cystic ity on the basis of its biophysical and pharmacological properties. fibrosis and Liddle’s Syndrome Snyder( et al., 1995; Stutts et al., In terms of the function of ClCs in chondrocytes, at least two clear 1995). Differential expression and up-regulation of the subunits possibilities exist; the first would be that they are required for set- between normal and disease states is thought to contribute to cel- ting of the membrane potential as implied above, but the second lular changes in disease (Burch et al., 1995; Greig et al., 2004). In would be that they could be important as anionic osmolyte channels. chondrocytes the role of ENaC is less clear; however, it is thought The latter hypothesis arises from the fact that any osmotic loss of to be one of mechanotransduction, possibly where the channel K+ ions as a part of volume regulation would need to be matched contributes to the maintenance of the RMP. This, in turn, may by an effective anion loss. ClCs would be an obvious candidate to regulate signaling pathways that allow chondrocytes to maintain fulfill such a role. their ECM and prevent chondrocyte apoptosis (Wright et al., 1996; Shakibaei et al., 2001; Shakibaei and Mobasheri, 2003). It is thought Aquaporin Channels that the mechanotransduction pathways involving ENaC become (AQP) are a family of small integral membrane pro- progressively defective during osteoarthritis, leading to a loss of teins related to the major intrinsic protein (MIP), sometimes called chondroprotective mechanisms (Salter et al., 2004). It is possible AQP0 (Agre et al., 1993). The first AQP discovered, AQP1, was iden- that ENaC subunits are differentially expressed in chondrocytes, tified during experiments investigating the identity of the rhesus potentially to cope with different mechanical stresses throughout blood group antigens (Agre et al., 1987; Denker et al., 1988; Smith the zones of articular cartilage, and changes in chondrocytic prop- and Agre, 1991). Oocytes from Xenopus laevis microinjected with erties during disease (Trujillo et al., 1999; Shakibaei et al., 2001). in vitro-transcribed mRNA of AQP1 (previously known as CHIP28) exhibited increased osmotic water permeability compared to unin- Chloride Channels jected controls. This observation, combined with the reversible The chloride channel family (ClC) is widely expressed in many tissue inhibition induced by mercuric chloride, provided the first molecu- types. It was first discovered byJentsch et al. (1990) using Xenopus lar evidence for water channels (Preston et al., 1992). Since the oocytes, who isolated and sequenced the channel primary structure identification of AQP1 the field has expanded to now include study using cDNA. Using the same cDNA, ClC-1 was identified in rat of AQP in all types of organisms. In mammals, over a dozen AQP skeletal muscle. In skeletal muscle, ClC-1 is involved in stabilization have been identified. The classical AQP transport water exclusively. of the RMP (Gronemeier et al., 1994). ClCs have been identified in However, a second class of AQP has now been identified (Rojek rabbit articular cartilage (Sugimoto et al., 1996; Tsuga et al., 2002; et al., 2008), these so-called aquaglyceroporins also transport small, Isoya et al., 2009) and in OUMS-27, the human chondrocyte-derived uncharged molecules such as glycerol and urea; examples include cell line (Funabashi et al., 2010a). A commonly used pharmacologi- AQP3, AQP7, and AQP9 (Carbrey and Agre, 2009). Many models cal inhibitor of ClCs is 4-acetamido-4′-isothiocyanatostilbene-2,2- of chondrocyte function involve changes in volume (Hall et al., disulfonic acid (SITS) (Pesente and Signorile, 1979; Lefevre et al., 1996). For this to occur there must be pathways for the movement 1996; Vaca, 1999; Alexander et al., 2008). This and other ClC inhibi- of water into and out of the cell. The discovery of AQP channels in tors were used by Sugimoto et al. (1996) and Tsuga et al. (2002) to chondrocytes would appear to provide an appropriate mechanism show that ClCs are important for control of the RMP. Furthermore, (Mobasheri and Marples, 2004; Mobasheri et al., 2004a,b; Trujillo exposure of chondrocytes to high concentrations of SITS leads to et al., 2004; May et al., 2007). Whilst studies have already shown a signs of necrotic damage (Wohlrab et al., 2004) suggesting that loss of volume regulation with inhibition of AQP channels (May activity of ClCs may be critical to the survival of chondrocytes. et al., 2007) and reductions in migration and adhesion (Liang et al., Chondrocytes may express a number of ClCs. So far the only one 2008), it would be interesting to investigate whether cell survival successfully identified in molecular terms is the cystic fibrosis trans- or progression of osteoarthritis are also affected by AQP block or membrane conductance regulator (CFTR) (Liang et al., 2010). This by AQP knockouts. is particularly interesting since CFTR is known to function both as a channel in its own right, and as a regulator of other ion channels NMDA Channels known to be expressed in chondrocytes (Mall et al., 1998; Nilius There have been a few reports of expression of excitatory neuro- and Droogmans, 2003; Arniges et al., 2004). As yet no studies have transmitter receptor (NMDA) channels in chondrocytes (Millward- successfully identified other ClCs expressed by chondrocytes. Such Sadler et al., 2001; Salter et al., 2004; Ramage et al., 2008). These are

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al. (2010) al. , , May et May Shakibaei Wilson Gavenis et Gavenis Ramage et Ramage al. (2001) al. , , , Lewis , , , al. (1997) al. al. (2007) al. (2010) al. b) (2004a, al. al. (1996) al. (1997) al. Mobasheri et Mobasheri et Lewis

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Walsh et Walsh et (2005a) et Mobasheri (1994) Walsh and Long et Mobasheri et Wright et Phan et et Lewis et Sugimoto et Trujillo (2003) Mobasheri and et Lewis et Sugimoto et Tsuga et Poiraudeau (2003) Mobasheri and Shakibaei (2004) Marples and Mobasheri et Mobasheri et Trujillo et Sanchez et Millward-Sadler et Salter et Knight R eferences

regulation

+ 2

chondrocytes stretch membrane Coupling function; channel BK and osmoregulation known Not known Not Mechanotransduction signaling Calcium volume cell of Regulation regulation pH Intracellular mechanical to Response Mechanocoupling Coupling cellular activity cellular Coupling Control of membrane potential membrane of Control Mechanotransduction Coupling of membrane of Coupling Intracellular Ca Intracellular stretch to RVD to stretch stimulation membrane potential membrane Regulation of Regulation Proposed role in role Proposed state metabolic to known Not pS) 280 ∼

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Na Na P P / / activated activated Ca Ca

+ + P P 2 2 , voltage-gated potassium channel; MIP, major intrinsic protein; NMDA, N-methyl N-methyl NMDA, protein; intrinsic major MIP, potassiumchannel; voltage-gated , V phenotype Ca Large Ca Small divalent and Mono conductor cation selective calcium Highly ( channel selective calcium Highly ( channel sensitive TTX ( conductance Large Voltage-activated small and Water activated Voltage channel ion Ligand-gated hemichannels junction Gap Closed with normal with Closed Low conductance, Low transport solute Voltage activated Voltage G eneral [ATP] intracellular channels Cation sensitive amiloride

Equine Human equine canine, Porcine, equine Canine, equine Canine, Rabbit Rabbit mouse Rabbit, porcine, Equine, Bovine Human Bovine Equine, human Equine, Canine, human Canine, elephant, equine, rat equine, elephant, ovine lines, cell bovine, Canine, chicken, Canine, Species cartilage osteoarthritic Human -subunits β 1.4 - and - v chondrocytes KCNMNB1 KCNMA1, – – – – – Maxi-Cl – AQP-3 AQP-1, – – 43 Kir6.x /SURx Kir6.x α – , ATP-dependent potassium channel; Kir, inwardly rectifying potassium channel; K potassiumchannel; rectifying inwardly Kir, potassiumchannel; ATP-dependent ,

ATP on channels that have been identified in the chondrocyte channelome. chondrocyte the in identified been have that channels I on 1 | 1

iu m c h anne l s tass K v ATP Po io n c h anne l s No n - se l ect iv e cat Sodiu m c h anne l s h e r c anne l s Ot Table family Channel in i dentified K K BK SK TRPV4 TRPV5 TRPV6 TRPC1/3/6, /2 TRPV1 TRPM5/7, VGSC ENaC Chloride VGCC Aquaporins ASIC NMDA Ion either channels ASIC,channel; aquaporin identified acid or sensing proposedchannel; ion to BK, exist calcium-activatedAQP, chondrocytes. in potassium cannel, high conductance; ENaC, epithelial sodium K channels; voltage-gated sodium channel. VGSC, voltage-gated calcium channels; VGCC, TTX, tetrodotoxin; transient receptor potential channel; TRP, receptor; conductance; low SUR, sulfonylurea potassium channel,

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interesting observations however the role of these ligand-gated ion ment was that mechanical stimulation of chondrocyte cilia activates channels in chondrocyte function is not yet understood. It does not the hemichannel which then acts as a conduit for ATP release. This seem likely that they are involved with neurotransmission because, released ATP then acts on chondrocyte membranes (via P2 purino- despite some similarities between neurone and chondrocyte pheno- ceptors) to increase intracellular Ca2+ (Knight et al., 2009). type, no “pre-synaptic” neurones project to the immediate vicinity of the chondrocytes. It again appears likely that NMDA channels are Conclusions in someway involved in the mechanotransduction pathway, since There is growing interest in the expression and function of ion mechanically induced hyperpolarizations are reduced by NMDA channels in chondrocytes. Part of this interest stems from the reali- antagonists (Salter et al., 2004). Furthermore, glycine induces a zation that many ion channels are involved in mechanotransduc- number of changes on chondrocytes in cartilage explants (including tion, chemotransduction and osmoregulation. It is important to accumulation of calcium) and these effects can be reduced with bear in mind that ion channels are also important drug targets an NMDA antagonist as, presumably, glycine acts via the glycine because of their localization in the chondrocyte plasma mem- binding site of the NMDA receptor (Takahata et al., 2008). brane. A number of research groups, including ours, have used , molecular biology and immunohistochemistry Other Ion Channels to study ion channels in articular chondrocytes. Table 1 contains Two further ion channels recently identified in chondrocytes are a summary of the ion channels studied in the chondrocyte chan- the acid sensing channel, ASIC1a and ASIC3 (Kolker et al., 2010; nelome so far. It is likely that some ion channels in chondrocytes Yuan et al., 2010) and the connexin 43 hemichannel (Knight et al., are multifunctional, serving a number of different physiological 2009). ASIC are very small cation selective channels closely related purposes. The processes of mechanical and chemical sensing and to ENaC (reviewed by Wemmie et al., 2006). As their name implies, metabolic regulation may well be intricately linked and make use they are opened by extracellular protons. This is particularly relevant of a number of ion channels as common denominators. In sum- to chondrocyte biology since chondrocytes are routinely exposed to mary, ion channels are important for chondrocyte function and relatively acidic conditions, as low as pH 6.6 for example (Wilkins further investigations are required to explore the full complement et al., 2000). In vitro studies show that these channels mediate an of channels present in the chondrocyte channelome. This knowl- increase in intracellular calcium upon exposure of chondrocytes edge will help us understand the unique biology of chondrocytes to acidic conditions. This intracellular Ca2+ is likely to be a signal and may lead to the development and formulation of therapeutic for production of enzymes and for proliferation. Potentially, inap- strategies to treat arthritis. propriate increases in calcium could result in cell death from either necrosis or apoptosis (Kolker et al., 2010; Yuan et al., 2010). The role Acknowledgments of the connexin 43 is possibly more complex. Knight et al. (2009) The Biotechnology and Biological Sciences Research Council found it to be constitutively active in about 40% of chondrocytes, (BBSRC) and The Wellcome Trust have funded our research on and as such it might be expected to profoundly depolarize the mem- the chondrocyte channelome. We thank Dr Madura Batuwangala brane. In summary, the suggested scheme of connexin 43 involve- for the illustrations in the figures.

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