typically , using standard assessment criteria , approximately 50 to 100 channel targets are detected in chondrocytes .
not surprisingly the dataset from each study are rather different to each other , but there is a common set of 7 probes that are detected in all 10 of these studies .
it is important to note that this analysis does not select particular ion channel genes to study , but reveals all the genes commonly expressed in the above microarray datasets ( note that there are over 300 ion channel gene probes encoded on each of the cited affymetrix chips ; accessory proteins such as the channel  tetramerization domain  proteins are excluded ) .
with such a large number of ion channel transcripts on the affymetrix chips one would expect the random co - detection of transcripts across datasets . to quantify this we used the following statistical analysis .
the probability of more than n transcripts being co - localised in d datasets is given by : where p is the probability of a given gene appearing in a datasets , and there are a total of g genes on each array .
this gives a p - value for 7 ( or more ) transcripts appearing in all 10 datasets by chance as p < 1e-14 .
it should be noted that these microarray datasets were derived from different species ( 3 rat , 3 mouse , 3 human and 1 bovine ) and there are potential differences in chondrocyte isolation protocols . constraining analysis to just rodent ( 6 datasets ) returns a set of 23 commonly expressed ion channel genes ( table 2 ) .
figure 1 quantitatively illustrates both the overlap of genes commonly expressed between species ( fig .
1a ) and the overlap between each of the transcripts from human , mouse and rat ( fig .
it is evident that far more transcripts were detected in all 3 of the mouse datasets than in all 3 of the human datasets .
this could be for three reasons ; firstly , it is possible that the sensitivity of the mouse chips is greater , but we have seen no specific evidence for this . secondly , each of the protocols requires manual dissection and separation of chondrocytes from the subchondral bone and adnexa .
samples are inherently contaminated with non - chondrocyte tissue . in an electrophysiological or immunohistochemical study such contamination
would be relatively easy to detect , but in a biochemical protocol , where harvested tissue is macerated and then processed , it could be missed .
thirdly , it is conceivable that there are genuine phenotypic differences between chondrocytes in mice and other animals .
these represent transcripts present in each of the 3 datasets for each of the human , mouse , and rat datasets ( i.e. , includes 9 datasets total ) .
the human studies used tissue harvested from either adolescents receiving limb length correction surgery ( e - geod-1277 ) , adults receiving acl surgery ( e - geod-16464 ) , or post mortem ( e - geod-10024 ) .
samples were taken from knee ( e - geod-16464 ) , distal femur ( e - geod-1277 ) , or shoulder ( e - geod-10024 ) and focussed on articular ( e - geod-10024 ) , mixed ( e - geod-16464 ) , or growth plate ( e - geod-1277 ) chondrocytes .
chip ids ; e - geod-10024 used hg - u133a and e - geod-16464 used the slightly newer hg - u133a_plus_2 , but e - geod-1277 used the u95av2 genechip .
all 3 human studies used expanded chondrocytes , but e - geod-10024 and e - geod-16464 re - constituted those into 3d cultures .
extraction enzymes were collagenase p ( e - geod-10024 ) , clostridial collagenase and deoxyribonuclease i ( e - geod-16464 ) and trypsin ( e - geod-1277 ) .
rodent studies suffer from inherent difficulties in extraction of tissue since cartilage is thinner than larger animals .
tissue used from the microarray studies analysed in this letter came from a variety of joints from immature mice and are likely to include mixed chondrocyte phenotypes . where stated explicitly , chondrocytes were expanded in monolayer cultures following collagenase based isolation ( e - geod-8052 and e - geod-7683 ) .
all 3 studies ( e - geod-10556 , e - geod-18052 and e - geod-7683 ) used the same affymetrix mouse430_2 chips .
the rat femoral head ( e - geod-6119 , e - geod-14402 ) or knee ( e - geod-8077 ) tissue was harvested from a range of ages from one day old neonates ( from which  only the outer two - thirds of cartilage  was used to select for articular chondrocytes , e - geod-14402 ) to several month old rats ( 300 - 320 g , e - geod-8077 ) .
strain was either wistar ( e - geod-6119 ) , sprague - dawley ( e - geod-8077 ) or not stated .
e - geod-6119 and e - geod-14402 both used monolayer expanded chondrocytes following collagenase ii based isolation .
e - geod-6119 also included pronase , but e - geod-8077 used direct rna extraction from macerated tissue .
one bovine dataset derived from chondrocytes 3d cultured from carpal bones of 3 to 6 mo old calves was also analyzed ( e - geod-18394 , affymetrix bovine chip , annotated with version 
since there was only 1 bovine chondrocyte dataset on ebi ( albeit including a number of replicates ) this is not included in the venn diagrams .
note that each of the 3 species sets in ( a ) is equivalent to the commonly expressed regions of the venn diagrams in ( b , c , and d ) .
the remainder of this letter focuses on whether the microarray data provides useful clues as to which channels are expressed in chondrocytes , and whether , therefore , initial processing of microarray data will improve the rate of channel detection in chondrocytes , or potentially other tissues .
the chloride channel superfamily is huge and includes the large clc family , cftr protein , calcium - activated , volume - activated , p64 related chloride channels ( clns ) , and intracellular chloride channels ( clic ) . in many systems chloride channels
have been less well studied than cation channels , although they were some of the earliest ion channels identified in chondrocytes .
analysis of the current datasets reveals that both p64-related ( clic1 , clic4 ) and clns1a ion channels were detected in all 10 microarray experiments .
the function of the corresponding channels are unknown , complex , controversial , or a combination of all these .
clic1 , clic4 , and other members of the clic family of proteins appear to be legitimate anion channels .
they are often referred to as  p64-related  simply because their earliest characterization appeared to be of a 64kda protein .
they appear to be of relatively low conductance for a chloride channel ( 8 - 40ps , depending on experimental conditions ) .
whilst , as their name implies , these channels can localize to intracellular compartments , in some cell types they also appear to be in the plasma membrane and could serve a role in secretion .
one possibility is that the channel shuttles to and from the membrane in a cell cycle dependent way .
the pcln channel ( sometimes referred to as p - glycoprotein , picln ) , also detected in 10 out of 10 datasets , was first identified by paulmichl et al as a putative rather ubiquitous volume - sensitive chloride channel .
more recently , this volume regulator role has been refuted and roles in gene - regulation and development have been proposed .
the controversy surrounding the nature of this channel is discussed in detail by strange and furst .
the particular issue is that it can not be clearly determined whether this is a volume - sensitive channel , another type of channel , or is a regulator of a channel endogenous in the various expression systems in which it has been studied .
even the fundamental property of ion selectivity is controversial , since recent studies have shown a rather higher permeability of pcln to cations than would be expected for a chloride channel .
it is possible that one or other of these channels does constitute the chloride channel identified by tsuga et al in chondrocytes , but it is unlikely since that channel is more typical of a classical maxi - type chloride channel in electrophysiological and pharmacological terms .
the associated channels are thought of , generally , as mitochondrial ion channels , found in the outer membrane of this organelle .
the proteins have also been detected in the plasma membrane , where they exhibit voltage - gated anion channel activity ( based on data in the ncbinr and uniprot databases ) .
vdac channels are also implicated in apoptosis and , as such , they will be of profound importance to all cells in which they are expressed .
it has also now been suggested by a number of authors that some vdac protein expression is also of plasma membrane ion channels .
this would be very much in line with the original chondrocyte chloride channel work of tsuga and sugimoto .
the channel identified in these studies was the maxi - chloride channel , which is remarkably similar to the maxi - cl / vdac channel . in our own unpublished work , we see clear expression of a large conductance , niflumic acid and sits - sensitive chloride channel which appears likely to be maxi - chloride .
it is no surprise that kcnma1 ( bk ) has been detected in all of the 10 datasets analyzed here .
currents either broadly , or specifically , identified as being through bk channels have been described in a number of papers . in our own work
we have shown not only that bk currents are present by electrophysiology , but also that the kcnma1 was detectable by immunohistochemical methods .
interestingly , whilst the bk function modifying -subunit kcnmb1 was detected in our own immunohistochemical studies it was not detected in any of the 10 microarray studies discussed here .
there was detection of kcnmb2 and kcnb4 in 2 and 3 out of the 10 studies respectively .
activation of bk results in such large currents that it is likely to be involved with regulation of intracellular osmolarity and volume .
early work did not show whether this activation was direct or indirect via an increase of intracellular calcium ions , but our more recent studies show rather convincingly that trpv4 is activated by stretch and that this results in the opening of a potassium channel .
the detection of the sodium 1-subunit scn1b in all 10 studies is interesting , since the -subunits were undetectable in most studies .
this subunit has been shown , in neurons to convey subtle changes to expression patterns and functional properties of voltage - gated sodium channels .
there has been 1 electrophysiological study , which identified voltage - gated sodium channels in chondrocytes .
this work has not been followed up on and , since chondrocytes do not fire action potentials , it is difficult to see what the role of a voltage - gated sodium channel might be . for completeness : scn7a ( nav2.1 ) , a somewhat atypical na channel was detected in 4/10 experiments , and the classical sodium channel scn2a1 in 3/10 , and the transient type sodium channel scn11a and scn10a in 2/10 datasets .
this list includes trpc1 , of the canonical trp , and trpv4 , a vanilloid channel , both of which were identified in 9/10 microarray studies . in traditional protein and pharmacological studies ,
however , three trp channels have been identified in chondrocytes trpv4 , trpv5 , and trpv6 but not trpc1 .
trpv5 appears important for setting the membrane potential , crucial to maintenance of cell volume in chondrocytes .
trpv4 , however , appears to be critical for allowing entry of ca and activation of bk channels during imposed volume increase , and thus the process of regulatory volume decrease in epithelial cells .
also , however , we find that 2 transient receptor potential channels are commonly detected .
to use traditional methods to identify changes in channel expression between populations of cells from different tissues is probably not feasible unless high throughput automated ion channel recording equipment can be adapted for this purpose .
however , observation of changes in cellular properties and mathematical models may provide clues as to which channels have changed in oa . an alternative approach would be to use microarray comparisons between normal and oa tissue , detect changes in channel mrna abundance and then follow - up with functional or histochemical experiments . generally , differential expression patterns from microarray studies are used to identify changed pathways , however , this process could miss changes which take place in individual ion channels not associated with an established pathway . the next microarray study considered in this review specifically compared transcript abundance from chondrocytes in human normal and oa cartilage .
the authors focused on ( and verified ) changes in many cartilage phenotypic markers , but did not specifically consider changes in other proteins such as ion channels .
probing this list for ion channels and porins significantly changed ( greater than 2-fold change ) produces the data in table 4 .
the data clearly show an approximately 3-fold change in enac , tmem16a and bk ( kcnma1 ) transcript abundance and a 38-fold change in the aquaporin aqp1 channel transcript abundance .
interestingly each of these channels are important for chondrocyte cell volume control . from this ,
by way of proof of principle we verified bk channel changes with immunohistochemistry and aquaporin channel changes with a functional assay .
negative is fold decrease in abundance ratio , positive is fold increase in abundance ratio .
note that whilst there were probes for trpv4 present on the chip , the huge variability between abundance scores between samples for this particular probe set make it unlikely that any change would be detectable , even if there was one .
tissue was taken from stifle joints of horses with and without oa ( see fig .
we investigated the expression of both - and -subunits of bk ( only kcnma1 was included in karlsson 2010 , there were no probe sets for kcnmb1 on the chip ) .
semi - quantitative analysis of protein expression density shows that both bk subunits were significantly increased in oa , in the middle zone ( fig .
immunohistochemical identification of kcnma1 ( bk -subunit ) and kcnb1 ( bk -subunit ) in sections of healthy and oa equine cartilage .
macroscopically normal articular cartilage samples were obtained from weight - bearing regions of the metacarpophalangeal joints of horses of mixed breed , age , and sex .
animals were euthanized for non - research purposes having been stunned before slaughter for meat in accordance with welfare of animals ( slaughter or killing ) regulations 1995 .
sections of normal ( n=6 ) and oa ( n=3 ) equine cartilage were probed for channel expression by immunohistochemistry essentially as previously described .
sections were incubated overnight at 4c with rabbit polyclonal antibodies to the kcnma1 and kcnb1 .
antibody dilutions used ranged from 1:200 to 1:1500 in tris - buffered saline containing 1% bovine serum albumin .
slides were incubated with horseradish peroxidase - labelled polymer conjugated to affinity - purified goat anti - rabbit immunoglobulins .
photomicrographs of immunostained tissue sections captured using nikon digital sight ds-5 m camera driven by nikon eclipsenet image capture software ( nikon ) .
positive staining is indicated by brown staining and particular evident at middle / superficial zones .
the largest increase in expression density ( from data such as that illustrated in fig .
2 ) is in the middle zone , for both kcnma1 ( c ) and kcnmb1 ( d ) ( - and - subunit respectively ) .
cells are challenged with a hypotonic solution , causing them to swell , and the rate of swell can be measured to determine the aquaporin expression .
the bioinformatics showed a 38-fold increase in aqp1 transcript abundance in oa and we see a significant increase in functional aquaporin expression in tissue from oa joints ( fig .
3a ) , although the increase is much smaller than the change in aqp1 transcript abundance .
current pharmacological tools do not allow categorical determination of aquaporin subtype , however , here , water permeability of chondrocytes was blocked by concentrations of bumetanide , tea , and hgcl2 consistent with that expected for aqp1 ( fig .
water permeability can be calculated from the initial slope of the relative volume ( v / vo ) against time curve . where v is the volume at time
( a ) permeability is 303% ( p<0.05 , n=4 ) greater in chondrocytes from dogs with osteoarthritis ( oa ) .
tea ( a blocker of aqp1 ) , bumetanide ( pic50 5.170.11 m , n=6 ,  bumex  , a blocker of aqp1 and 4 ) , and mercuric chloride ( hgcl2 a non - specific aqp blocker reversed by 2-mercaptoethanol ) me ) are included to determine aqp type ( b and c ) .
solution including 120 mm sucrose ( osmolarity 300mosm ) , then moved to an identical physiological saline without the sucrose .
live cell imaging was achieved with a nikon diaphot microscope equipped with a sony icx098qb high sensitivity ccd .
images were analysed offline with imagejvolume was calculated from the 2d surface area ( a ) of the cell disc by assuming the cell is approximately spherical as described previously , using the following equation : vol=43(a)3 ( equation 2 ) . except where stated
, data are presented normalized for starting volume ( v0 ) as v / v0 , where v is the volume at time t. visual data were analyzed with imagej and anova performed with spss ( spss inc . ) .
note that canine tissue was harvested from clinical waste tissue with local ethical approval , no dogs were harmed for the study . in summary , there appears to be considerable agreement between transcriptomic studies and physiological or immunohistochemical studies .
it would seem that most channels common to all 10 datasets can be identified by these other techniques .
there are examples , however , of proteins which have been identified in chondrocytes yet show up in few datasets .
for example , the asic channel ( accn2 gene ) has been shown by immunohistochemistry and rt - pcr yet shows up in only one of the three rat datasets discussed here .
therefore , combining these approaches should massively speed up the rate of discovery of ion channels in cell types which can be isolated in sufficient quantities to perform such studies .
there are tissues , such as the brain , where cell types are too intermingled to allow identification of unique cell types , but for many tissues in the musculoskeletal system ( or cell lines ) , the combination of transcriptomics and protein studies seems ideal . with regard to oa
, this strategy has allowed us to very rapidly pinpoint some phenotypic changes in the expression of two important channels in oa : an aquaporin and the bk channel .
further protein and functional experiments will be necessary to establish whether the other kca channels are also altered , and in particular whether these changes contribute to or result from progression of oa .
 to date , a range of ion channels have been identified in chondrocytes using a number of different techniques , predominantly electrophysiological and/or biomolecular ; each of these has its advantages and disadvantages . 
 here we aim to compare and contrast the data available from biophysical and microarray experiments . 
 this letter analyses recent transcriptomics datasets from chondrocytes , accessible from the european bioinformatics institute ( ebi ) . 
 we discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes . 
 the ion channels which appear most frequently across these microarray datasets are discussed , along with their possible functions . 
 we discuss whether functional or protein data exist which support the microarray data . 
 a microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes , namely the genes encoding large calcium - activated potassium ( bk ) and aquaporin channels . 
Commonality between Datasets
Chloride Channels
The Voltage Dependent Anion Channels
The Large Calcium-Activated Potassium Channel
The Sodium Channel -Subunit
Transient Receptor Potential (TRP) Channels
Changes of Transcript Levels with Onset of Osteoarthritis