Journal of Cell Science as cldnoiia ypai ydistinct mechanisms by dysplasia oculodentodigital cause recessive Autosomal Article Research a ucincanl opsdo n onxnaetermed are connexin one of selectivity composed various channel channels and result, Connexons in junction 2006). (Laird, a gap resulting constituents or connexin As exist its one to 2006). express unique can to (Laird, hexamers known isoforms connexin are connexin types cell more many and physiology, So 1990; an Revel, and exposed loops, Laird 2004). termini 2006; Willecke, carboxyl (Laird, extracellular and cytoplasm amino the of are two to the consisting which with topology domains, of loop membrane intracellular all transmembrane similar family, a connexin four share human to the are predicted in There 2003). members Paul, and with 21 (Goodenough consisting cluster channels frequently cell of to plaques hundreds junction opposing proceeds of gap an which form to channel from channels junction other connexon gap a a a as exist with known forming can membrane, dock plasma connexon or the within A hemichannel, unit 2006). singular a Laird, proteins, as membrane 1996; either plasma (Cx) the al., connexin to et delivered six (Goodenough is of cells which connexon, oligomerization as a connected an forming produced of two are junctions second result of Gap the and 2003). cytoplasms Goldberg, metabolites and ions, the (Alexander molecules, between for small messengers allow of that channels transfer membrane the specialized are junctions Gap Introduction to than inability burden R33X disease complete words: greater its the Key exhibit with whereas mutant together R33X Cx40, connexin recessive R33X mutant. wild-type or autosomal of in this R76H effect Cx37 harboring reduction the trans-dominant patients Cx32, a harboring the why by Cx26, explain that patients manifested may co-expressed suggest as channel results of Cx40 functional our a and function together, form Cx43 the that Taken on transfer revealed on plaques. effects further dye junction effect trans-dominant cell, studies mutant, gap gap or negative transfer functional R76H the dominant dye form detectable the significant 350 could throughout no of exhibited Alexa mutant conductance. mutant case localized this had channel that the mutant diffusely single revealed In and cells R76H remained channels. N2a macroscopic the functional gap GJIC-deficient reduced mutant in with form either analysis R33X albeit to conductance in channels, the failed the electrical junction expressed to mutant and whereas cells When trafficked R33X HeLa the mutant plaques, (R33X). in expected, that R76H studies mutation junction-like As the reported truncation nucleus. gap cells, mutations premature the NRK form recessive including a Cx43-expressing autosomal to or and two cells membrane (R76H) been HeLa the plasma substitution have (GJIC)-deficient in acid communication there mutations amino intercellular Surprisingly, by junctional single caused (Cx43). a disease connexin43 ODDD: human protein dominant cause junction autosomal gap an the mainly encodes is (ODDD) dysplasia Oculodentodigital Summary 10.1242/jcs.123315 doi: 2857–2866 126, ß Science Cell of Journal 2013 March 27 Accepted ( correspondence for *Author 2 1 Huang Tao eateto hsooyadPamclg,Uiest fWsenOtro odn NNA51 Canada N6A-5C1, ON Canada London, N6A-5C1, Ontario, ON Western London, of Ontario, University Western Pharmacology, of and University Physiology Biology, of Cell Department and Anatomy of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. onxn r eprlyadsailyrgltdi human in regulated spatially and temporally are Connexins x3 a ucin,Ouoetdgtldysplasia, Oculodentodigital junctions, Gap Cx43, 1 igShao Qing , [email protected] 1 nrwMacDonald Andrew , ) GJA1 2 iXin Li , h and ¨hl GJA1 2 oetLorentz Robert , C4)gn mutations gene (Cx43) w R6 n 3X uooa eesv uain ikdto linked However, mutations revealed recessive 2005). autosomal have al., R33X) screens and et genetic (R76H McLachlan and al., two et 2006; presentations Gong co- clinical al., 2005; of recent et al., function Gong et the (Flenniken 2007; on Cx43 channel dominant-negatives wild-type reduce as to expressed act shown dominant been autosomal and have the function date of to known majority examined The 65 2003; mutations al., 2009). over et al., Paznekas been date, et 2008; loss, have Laird, Paznekas To 2006; sequence, (Laird, coding 2009). ODDD enamel to gene al., linked Cx43 (Paznekas et the microdontia, spanning Paznekas abnormalities mutations, 2003; craniofacial digits, and al., and et syndactyly the by defects as characterized ophthalmic such of predominantly is phenotypes ODDD camptodactyly the clinical 2003). common al., in dysplasia several et oculodentodigital (Paznekas results called (ODDD) disease Cx43, dominant autosomal connexin, human many that expressed to find 2011). lead al., to et intercellular GJIC surprising Pfenniger in 2008; all junctional (Laird, changes at pathologies or gap not mutations is termed of connexin it are (GJIC), network a and communication connexins connexins immense of al., those expression distinct ubiquitous et the resulting of (Goodenough whereas result more a respectively As 1996). or heterotypic; respectively; and two heteromeric homotypic, of composed and homomeric uain ntegn noigteams ubiquitously almost the encoding gene the in Mutations 1 ogi Bai Donglin , 2 n aeW Laird W. Dale and GJA1 ee which gene, 2857 1,2, * Journal of Cell Science fteR6 n 3XatsmlrcsieC4 uat.We mutants. Cx43 recessive autosomal R33X and R76H the of is channel it junction Nevertheless, pleiotropic tested. different gap symptoms. cause be disease mutants skeletal to residual Cx43 different yet that that have and has intriguing notion this may the teeth although raises mutant function, This many carry R76H 2004). in al., and also the et result but hypotrichosis (Pizzuti that abnormalities patients stature, syndrome ODDD Hallermann-Streiff small expectancy dominant of autosomal life similarities characteristics the share harboring the to compromise patients appeared to the mutant R76H However, severely recessive 2006). autosomal and al., that et abnormalities (Richardson dental defects many of other syndactyly, symptoms ophthalmological extensive clinical other and severe defects, eyes are deep-set very knockout small past including display patients survive ODDD Unlike do they mutant knockout. these although R33X the birth, Cx43 for that homozygous a patients suggesting mice, to form equivalent channels, to expected essentially of not junction is fragment Cx43 partial R33X of gap domain a the acid transmembrane and amino humans, first 32 N-terminus the In a the 1995). only representing studies al., of segment consisting et Previous mutant, Cx43 (Reaume of recessive birth loss autosomal body. the at that is lethal human and Cx43 development is that heart the revealed normal for mice required of knockout Cx43 organs systemic employing various throughout a and Cx43 provide of function Richardson mutations tissues the 2004; Cx43 study to al., recessive platform et Interestingly, unique Pizzuti 2006). 2009; al., al., et et (Paznekas ODDD 2858 ntecretsuyw sesdtelclzto n function and localization the assessed we study current the In ora fCl cec 2 (13) 126 Science Cell of Journal Results of variety wide a in channel organs. Cx43 innate and with no tissues co-expressed has members frequently family mutant are connexin be inhibit R33X that patients further may the may patients and that why ability R76H fact forming than the reason morbidities in greater reaching the rooted possess far mutant that more and R33X trans- and recessive suggest Cx37 autosomal and the results Cx43, harboring dominant Cx32. co-expressed These or exhibited on Cx40. Cx26 mutant effects Cx37, R33X Cx40, dominant-negative co-expressed the of However, function effects trans-dominant no the had mutant on We R76H channels. the junction that junction gap found functional further gap form failed and functional mutant properly R33X form traffic the to whereas can coupling, mutant reduced with R76H channels the that found ucinlk lqe tcl nefcs(i.1,,arrowheads), 1A,B, gap (Fig. assembled interfaces cell mutant at R76H plaques GFP-tagged junction-like and the and both with R76H R76H that untagged revealed recessive transfected Immunofluorescence autosomal mutants. transiently Cx43 the R33X encoding were constructs cells cDNA HeLa cells Cx43-negative apposition NRK cell to and cell HeLa of in sites to localized mutant R76H The omdgpjnto-iepausa ie fcell-to-cell R33X of the sites whereas cells, at NRK plaques mutant Cx43-positive R76H in the junction-like Similarly, appositions 1A,B). gap (Fig. cells localization formed HeLa nuclear and in plaques cytoplasm any profile homogenous form a to failed exhibited mutant the and both R33X However, GFP-tagged arrowheads). and 1A,B, FLAG (Fig. Cx43 wild-type like 10 a ucin(J lqe a uniid ** quantified. was plaques Cx43 (GJ) immunolabeled junction exhibited gap that R33X-GFP or Cx43-GFP uat T idtp.( R76H the type. and wild Cx43 WT, from mutant. formed Arrowheads plaques junction (red). gap Cx43 that for reveal R33X immunolabeling the to or prior mutant R76H mutant the Cx43, (green) GFP-tagged express 3Xmtn ro oimnlbln o x3adHoechst and Cx43 for ( immunolabeling staining. to the or prior mutant mutant R76H R33X the Cx43, GFP-tagged ( express nuclei. to the constructed FLAG of or staining Cx43 FLAG- for Hoechst the immunolabeling and to or prior mutant mutant R76H R33X the tagged Cx43, express mutants. to Cx43 engineered of Localization 1. Fig. m m. C x3pstv R el eeegnee to engineered were cells NRK Cx43-positive ) D h ecnaeo el expressing cells of percentage The ) B eaclswr further were cells HeLa ) ( A eaclswere cells HeLa ) P , .1 cl bars: Scale 0.01. Journal of Cell Science euetenme fC4 a junctions. to gap acted Cx43 but surface of cell number the the reach whereas R76H even reduce structures, to the failed plaque mutant that junction-like R33X indicated the gap results to Cx43 These localizes that in mutant 1D). NRK reduction (Fig. a distribution in was junctions there Furthermore, gap mutant, intracellular 1C). R33X (Fig. the expressing diffused nucleus cells the a entering included exhibited mutant Fg C.We el oepesn 3XGPadC4 were Cx43 and GFP R33X-GFP or was co-expressing RFP cells this with tagged When and was 2C). of cells mutant (Fig. HeLa function the in whether a the dead. of co-expressed inhibited irrespective forming were functionally both mutant of was when R33X Cx43 capable mutant the was R33X only 2A,B). Interestingly, mutant the (Fig. R76H channel, Cx43 functional the wild-type yellow to Lucifer whereas transferring compared Thus, in directly efficient again as wild-type when as 40% the 350 about pass, assessed. Alexa to passing and were at dye Cx43 well transfer either equally allow dye was to mutant of failed R76H mutant incidents Alexa R33X the the or and Whereas Da) (443 HeLa Da) and yellow (350 expressing (R76H Lucifer 350 mutant with mutants microinjected channels, were recessive functional cells autosomal form can the R33X) if determine mutants To Cx43 of status Functional idnswudsgetta h 3Xmtn ieyinteracts channels. wild-type likely these of mutant function Collectively, the R33X inhibit 3). the to (Fig. Cx43 that with less suggest cells significantly would cells control findings exhibited expressing untransfected mutant mutant low and R33X to expressing compared the cells recovery fluorescence of that mutant fluorescence revealed levels low examined analysis and Cx43- high FRAP high selectively loaded in cells. (FRAP) AM expressing and photobleaching calcein after cells in recovery NRK mutant R33X positive increased expressed we with RFP-tagged hypothesis, this escalate test the would To expression. inhibition mutant of channel levels inhibit that of predicted to we extent acting interaction was physical the mutant a by R33X function the channel If Cx43 2D). was (Fig. mutant down R33X pulled the Cx43, endogenous for immunoprecipitated hw nFg A h vrg arsoi junctional macroscopic average the 4A, Fig. As (g GJIC- recordings. conductance in in patch-clamp ability double forming through shown channel cells R76H their N2a cells, the HeLa assess deficient in that to channels showed proceeded junction studies gap we functional transfer form dye can that mutant fact the Given single reduced with conductance channels channel forms mutant R76H The eesv x3mtnsaddsae2859 disease and mutants Cx43 Recessive j fR6-F hnesws19n ( nS 1.9 was channels R76H-GFP of ) x3 ugsigta hyinteracted. they that length suggesting full Cx43, R33X with the immunoprecipitated that was Note mutant antibody. detected anti-Cx43 were Cx43 the antibody and with chain anti-GFP heavy for Ig control The a labeling. as used Recombinant was Cx43. GFP or GFP for (IB) immunoblotting to subjected were and immunoprecipitates Cx43, were for mutant (IP) R33X-GFP immunoprecipitated the and Cx43 expressing eoetenme fclsijce ihde Scale dye. with injected 20 cells bar: of number the denote dye impaired mutant *** R33X transfer. the of Note expression (right). the quantified that was surrounding cells to expressing dye Cx43 or passing GFP cells of expressing incidences mutant the phase- and under microscopy, and contrast dye injection to dye prior after imaged microinjection, were asterisks) cells by Microinjected (denoted 350. Alexa from fluorescence represent the or images GFP Post-injection the fluorescence. represent RFP cells 350. pre-injected Alexa the of of microinjection Images the to prior or R33X-RFP R33X-GFP R76H-GFP, GFP, with encoding transfected constructs were Cx43 ( transfer. expressing dye cells of capable were mutant R76H quantified. were transfer *** dye and of 350, incidences Alexa the with microinjected were or R33X-RFP R33X-GFP R76H-GFP, Cx43-GFP, expressing h niecso y rnfrwr quantified. were transfer ** dye of and incidences yellow, the Lucifer with microinjected were R33X-GFP mutants or R76H-GFP Cx43-GFP, express the Cx43. while co-expressed functional inhibits is mutant R33X mutant non-functional R76H The 2. Fig. P P , , .1 *** 0.01; ( .0.Nt htclsepesn x3o the or Cx43 expressing cells that Note 0.001. A m .( m. eaclso eaclsegnee to engineered cells HeLa or cells HeLa ) P D , eaclso eaclsco- cells HeLa or cells HeLa ) P .0.Nmesi parentheses in Numbers 0.001. , .0.( 0.001. B ieie eacells HeLa Likewise, ) C n 5 HeLa ) 14), Journal of Cell Science uasa ta. 00.Teerslsdmntae htthe conductance. that unitary cells reduced demonstrated N2a substantially in a results channels 2001; junction with and gap These al., functional 1999) forms 2000). et mutant al., R76H al., (Bukauskas et pS) (Elenes et (110 pS) Bukauskas channels (120 channels Cx43 Cx43 GFP-tagged of that than ic h 3XadR6 uat r nw orsl in result to al., known et are (Paznekas burden mutants disease R76H of levels and different considerably R33X the Since Cx32 on and effects Cx26 trans-dominant co-expressed no have mutants Cx43 gap Cx43 wild-type of (g that than channels lower junction significantly is which or (a) mutant R33X (panel of (b) levels mutant high no expressing Cells (FRAP). photobleaching a GJIC. after calcein impaired with mutant preloaded R33X the were of R33X-RFP levels High 3. Fig. h nlsso elpisyeddcanl iha average an with channels yielded pairs cell 4 of analysis The ntr odcac ( conductance unitary 2860 lutae ersnaiesnl 7HGPgpjunction 4B Fig. gap analysis. V R76H-GFP under channel recorded single currents junction channel representative we conductance, gap channel illustrates junction single gap single performed the of reduction a eemn ftelwrg lower the if determine ihgende cl bar:10 Scale dye. corn green small a with where (B) in shape rectangular out are areas photobleached The mutant. calcei the represents fluorescence Green int recovering calcein and photobleached ora fCl cec 2 (13) 126 Science Cell of Journal A ,o o eeso h 3Xmtn ai panel in (a mutant R33X the of levels low or ), c j 5 j f4 S hc ssbtnilysmaller substantially is which pS, 41 of ) m j m. 85nS, 18.5 nR6-F hneswscue by caused was channels R76H-GFP in ie nrdi A n eie ihnared a within defined and (A) in red in lined ,adrdfursec eoe h R33X the denotes fluorescence red and n, h htbece elwstakd( tracked was cell photobleached the o dsbetdt loecnerecovery fluorescence to subjected nd ro otcigcl ntnl refilled instantly cell contacting a of er n 5 j 27, usso 0ad8 mV. 80 and 40 of pulses P , .1.T further To 0.01). R el expressing cells NRK B ,were ), C ). ersnaiesnl 7HGPgpjnto hne urnswr recorded conductance. were V channel currents under channel single junction for gap R76H-GFP assessed single further Representative were pairs cell expressing F.Da ac-lm hl elrcrigwsue oass the assess to used (g was conductance recording electrical cell macroscopic whole patch-clamp Dual GFP. htotie rmcl ar xrsigC4 (** Cx43 with expressing compared pairs conductance cell electrical from of obtained level that lower substantially a showed o neato martefnto fete x6o Cx32. or Cx26 either of transfer function to the dye appeared impair mutants inhibited the or predict, interact and might not one trans-dominant as Thus, dye a R33X 6A–D). nor 350 (Fig. as R76H Alexa the acted co- neither Subsequent that mutants the 5A–D). revealed of (Fig. further level and studies Cx32 expression transfer Cx32 the or 5C,D). change or Cx26 (Fig. not did either Cx26 mutants Cx32 the expressed of host cells or expression rigorously the HeLa cells that test 5A,B) revealed (Fig. in HeLa To blotting western mutants function. Cx26 and the Immunofluorescence their mutants expressing the co-expressed on that we stably trans-dominants reasoned hypothesis, as further this act we in Cx43, not co-expressed as would rarely cells or are heterotypic same and form differential not the Cx43 do with the their Cx32 channels in and to co-expressed Cx26 heteromeric Since connexins linked type. of cell function mechanistically same the reasoned impair be we to 2006), may abilities al., et this Richardson 2004; that al., et Pizzuti 2009; naverage an i.4 arsoi n ntr hne odcac fteR76H the of conductance channel unitary mutant. and Macroscopic 4. Fig. idtp x0(i.7)adC3 Fg D,adtelvl of of levels co-expression the the and by unaffected 7D), were (Fig. connexins mutant Cx37 7B). wild-type and (Fig. the with 7C) R33X plaques mutants (Fig. the junction Cx40 of the expression wild-type gap robust a and Cx40 revealed blotting less Western Cx40 R76H exhibited or co-expressing 7A) R33X Cx37 (Fig. the or Cells express to 7A–C) transfected (Fig. mutants. this further Cx40 test were 7D) expressing To (Fig. mutants. cells Cx43 HeLa disease-linked the hypothesis, by the affected be of may connexins co-expression these that possibility the raising situ Cx40 In on effect trans-dominant Cx37 a and had mutant R33X The x3i rqetyc-xrse ihC3 n/rCx40 and/or Cx37 with co-expressed frequently is Cx43 , j ( usso 0m n 0m.Teaayi ffu elpisyielded pairs cell four of analysis The mV. 80 and mV 40 of pulses A 2 el eeegnee oepesete x3GPo R76H- or Cx43-GFP either express to engineered were cells N2a ) c j f4 pS. 41 of j .R6 uatepesn elpairs cell mutant-expressing R76H ). P , .1.( 0.01). B h mutant The ) Journal of Cell Science tiig(le.Wsenbosrvae htC2 ( Cx26 that revealed blots Western ( (blue). Cx26 staining for immunolabeled Cx43. and engine or R33X-GFP were Cx26 Cx32 with or mutants Cx26 Cx43 expressing of Co-expression 5. Fig. o F BD.GPHwsue salaigcnrl(,) cl as 20 bars: Scale (B,D). control loading a as immunoblotting by used denoted was as GAPDH mutants (B,D). the GFP co-expressed for that cells in levels high at rdt oepesGP 7HGPor R76H-GFP GFP, co-express to ered A )orCx32( B n x2( Cx32 and ) C olwdb Hoechst by followed ) D eacells HeLa )wereexpressed m m. ayptet xii te eomte htinclude disorders scope that broad CNS The 2009). deformities al., and et Paznekas other 2003; incontinence al., et abnormalities, exhibit (Paznekas urinary developmental these patients hyperkeratosis, to teeth, 2009). eyes, al., addition seminal many et the Paznekas in This 2003; affects tissue al., However, et typically 2003). in (Paznekas skeleton that abnormalities and al., digits development to et organ GJIC and Cx43-based (Paznekas until linked a Cx43 not discovery the was as in encodes mutations it to linked that but classified definitively was ago it was that decades 2003 many (ODDD) disease developmental dysplasia Oculodentodigital shown). Discussion not (data failed mutant Cx37 R33X or Cx40 co- the with However, if interacted mutant GJIC. physically determine R33X the of to the inhibitor attempts that trans-dominant reduced immunoprecipitation suggest a alone. Cx40 as findings either acting These significantly 8C,D) with was 8). (Fig. co-expressed (Fig. mutant when Cx37 Cx37 coupling or dye R33X increased cells or of HeLa Cx43 incidents in the 8A,B) achieved wild-type (Fig. levels Conversely, and the Cx40 over mutant cells expressing R76H HeLa revealed in studies the coupling transfer Dye both mutants. R33X that or R76H the either ufcetC4-ae JCadalwptet omaintain to patients allow provide and to suffice GJIC autosomal could that Cx43-based gene to possibility wild-type the linked sufficient the for disease harboring allow allele a would the in exclusively which expressed mutations be is ODDD dominant Originally, Cx43 to 2006). that thought (Laird, know tissues was we human as distinct may surprise, 35 patients ODDD over great all, of not but be some, not in found tissues disease of eesv x3mtnsaddsae2861 disease and mutants Cx43 Recessive uniid S o infcn.Saebr:20 bars: Scale was significant. cells not expressing NS, cells connexin expressing quantified. surrounding mutant to or dye GFP passing of incidences phase-contrast the under and dye and microscopy to injection prior dye imaged after were microinjection, Microinjected asterisks) 350. by Alexa (denoted from cells fluorescence post-injected the of pre- represent Images the cells fluorescence. of GFP Images represent (B,D). cells cells injected represent microinjected parentheses of dye in number of Numbers the incidences (B,D). the quantified and were 350, transfer Alexa with microinjected were ( i.6 x3mtnshv otasdmnn fet nCx26 on effects trans-dominant function. no Cx32 have and mutants Cx43 6. Fig. C , D n oepesn F,R6-F rR3-F mutants R33X-GFP or R76H-GFP GFP, co-expressing and ) eaclsepesn x6( Cx26 expressing cells HeLa m m. A , GJA1 B rCx32 or ) gene Journal of Cell Science diin ocasclOD n fteeptet exhibited patients these of In one 2006). ODDD al., et classical (Richardson to ODDD addition, with defects developmental associated severe typically exhibit and mutant the R33X recessive the examined severity. how variable we and with study, disease mutants to present disease-linked translate the these mutations of In loop extracellular properties 2004). domain 1st unique the the al., of within region et extreme substitution (Pizzuti the at (R76H) be to histidine an predicted and a 2006) to al., et arginine the (Richardson (R33X) a of encoded Cx43 that truncated understanding identified severely when were mutations our complicated al., recessive more autosomal However, et became two al., Stewart mutants 2010). 2005; Cx43 et al., of al., Musa characteristics et et 2008; Shibayama 2008; Toth al., 2012; al., 2013; al., et 2009; et et McLachlan Manias Shao 2012; 2005; al., 2006; 2009; al., al., al., et et Lai et et Lorentz 2006; McLachlan al., 2007; Flenniken et Dobrowolski al., 2007; Gong 2007; et al., al., Langlois 2011b; et et Gong Dobrowolski 2005; al., al., al., 2008; et et Churko al., et 2012; et al., Dobrowolski et Churko Churko 2011a; 2010; al., et wild-type (Churko co-expressed Cx43 and these on mutants us of dominant-negative function by or gain-of-hemichannel Many mutants categorized mutants, 1996). loss-of-function been as Jongsma, have others mutants and where dominant (Gros heart the autosomal abundant in case is the be to Cx43 seems as function organ essential Cx40. or Cx37 with mutants Cx43 of Co-expression 7. Fig. 2862 F rteR3-F uat ** mutant. R33X-GFP the or GFP pl (GJ) junction gap Cx40 immunolabeled ( Cx40. for immunolabeled co and to engineered were Cx40 expressing muoltigfrGP AD a sda odn oto.Saebr 20 bar: Scale control. loading by a denoted as used as co- was that Cx43-GFP GAPDH cells GFP. or in for GFP levels immunoblotting high mutants, at GFP-tagged expressed were the (D) expressed Cx37 and (C) Cx40 that revealed w on ainshv endcmne ohro the harbor to documented been have patients young Two ora fCl cec 2 (13) 126 Science Cell of Journal B h ecnaeo el containing cells of percentage The ) P , .1 ( 0.01. epesGP 7HGPo R33X-GFP or R76H-GFP GFP, -express qe a uniidi el expressing cells in quantified was aques C , D etr lt o connexins for blots Western ) ( A eacells HeLa ) m m. hta5%cmlmn fwl-yeC4 xrsinwas is what expression unlike Cx43 not Cx43 the wild-type development, in normal of observed preserve complement suggesting to 2006), 50% sufficient al., et a (Richardson that unaffected appeared mutation clinically R33X be the to for these heterozygous of parents were the who Interestingly patients substantial 2006). any motor al., for the in et followed (Richardson delay of not period no was sister she exhibited albeit but affected development, ODDD skill The severe threatening 2006). had life also al., a proband have et to (Richardson of considered indicative was condition and defects, defects, speech CNS and stark motor severe of characteristics n te et n kltldfriis(izt ta. 2004). al., et (Pizzuti deformities skeletal and nose teeth pointed cataracts, other congenital and hypotrichosis, stature, having shared small by characterized known a patient is disorder which ODDD developmental syndrome the Hallermann-Streiff related this as a again Interestingly with where symptoms 2004). clinical al., normal mutant clinically et be R76H to (Pizzuti found of a were inheritance parents harbored homozygous carrying heterozygous patient of instance a another ODDD, In 1995). al., et ubro irijce el BD.** (B,D). cells mutant microinjected R33X of 20 the number bars: whereas Scale transfer, transfer. dye dye increased inhibited mutant R76H the or Cx43 wa m cells or expressing GFP connexin surrounding of incidences the d and after microscopy microinjection, dye dye of to incidences prior the imaged Microin and (B,D). 350 Alexa quantified with were microinjected transfer Cx37. were and mutants Cx40 the on or effect GFP ( trans-dominant Cx40 a expressing had cells mutant HeLa R33X The 8. Fig. +/ 2 A , B eeoyoskoku ie(Reaume mice knockout heterozygous )orCx37( m .Nmesi aetee ersn the represent parentheses in Numbers m. etdcls(eoe yatrss were asterisks) by (denoted cells jected P uniid oeta h xrsinof expression the that Note quantified. s , eijcinadudrphase-contrast under and injection ye tn xrsigclspsigdeto dye passing cells expressing utant .1 *** 0.01; C , D n oepesn,Cx43-GFP, co-expressing, and ) P , 0.001. Journal of Cell Science as ern osadplolna eaoem hnmutated to when known keratoderma recessive and palmoplantar highly and members is or loss family hearing residue connexin dominant cause R76 of the The is to among disease. substitution conserved linking Cx43-linked the of of when inheritance nature importance the that critical a indicates with al., which substituted et (Pizzuti was 2004) ODDD 76 dominant autosomal position acquire at patients serine, residue arginine the When ai fmtn oedgnu x3i tartoo : which 1:1 of ratio a molecular is at the is it that Cx43 evidence endogenous However, no to completed. have mutant we of causing surface is that ratio cell note assembly to possibly the to mutant to junction important Cx43 the Cx43 gap of of wild-type trafficking where ability reduced the with and in misfolding interact rooted be co- physically R33X to that the of appeared cells effect NRK mutant dominant-negative The Cx43-positive mutant. in the of seen expressed reduction plaques similar junction a gap and of Cx43 incidence by express dominant- the Cx43-based modest reducing coupling Cx43 a any dye co-expressed had of on mutant absence effect R33X negative the the in suggesting Interestingly, survive strongly GJIC. patients channels, R33X junction cell to gap that failed the functional mutant throughout this any found surprisingly, form Not was nucleus. with and the tagged (FLAG), and when cytoplasm even tag surface, epitope cell small the to the a traffic to of failed it assessment cells, accurate mutant. more the of electrical a adjoining capacity junction was gap to probably functional of dye cell level is any normal expressing 15% whether conductance mutant the to the suggest we as from are ‘‘no’’ cells, pass here or ‘‘yes’’ performed to is simple studies observed assessment) a transfer from on dye transfer suffer the based they dye that although survive, Given yellow to to ODDD. patients that Lucifer allow assessment) to mutant and sufficient R76H (electrophysiology 350 the 15% of (Alexa ability from forming Remarkably, ranges GJIC. channel trans- non-Cx43-based patients residual of of ODDD the levels these that evidence normal suggests have further no connexins may Cx40 or co-expressed exhibited Cx37 other as mutant such on the R76H properties addition, In the dominant-negative normal. clinically that mutant be to R76H fact parents the the the by for sufficient provided this of activity utilizing Collectively, well channel as be itself. allele parents residual normal to the the on expected heterozygous encoded as Cx43 be of complement would the full plaque the patient mutant and Thus, R76H between surface transfer homozygous dye cells. cell R76H of level the the overall expressing to the on that Cx43 the or either formation found wild-type on of properties we dominant-negative trafficking Cx43, no of exhibited wild-type capable mutant dominant also co-expressed are were mutants linked to mutant ODDD many Consistently, R76H for Although 1999). al., dye. functionally the al., passing reported from et et Elenes pS formed pS (Bukauskas be 2000; 110–120 41 channels al., Cx43 expected et of to wild-type Bukauskas the 2001; GFP-tagged conduction below found and gap unitary well untagged into a were is assembled exhibited and which that surface and of cell compromised channels the subset to a junction trafficked least it at cells of function the of likely for connexins. is residue importance this suggesting 2009) critical al., et (Paznekas Cx26 in hnteR6 uatwsepesdi JCdfcetHeLa GJIC-deficient in expressed was mutant R76H the When hnteR3 uatwsepesdi JCdfcetHeLa GJIC-deficient in expressed was mutant R33X the When , 0 nHL el tbyegnee to engineered stably cells HeLa in 40% . 0 ae x3GI per obe to appears Cx43-GJIC based 60% , 0 nCx43-based in 50% . 40% ti aeyc-xrse ntesm el sC2 rC3 (So Cx32 or Cx26 as cells 2003), same al., the (Gemel et in Laux-Fenton tissues co-expressed 1996; rarely other Jongsma, is skin, and it and heart, Gros cornea 2012; the the al., vessels, of et cells that blood in testis, suggesting Cx40 ovaries, and/or frequently Cx32, is Cx37 Cx43 and with Although co-expressed non-specific. Cx26 not is expressed effects effect GJIC inhibitory that no exhibited cells it whereas on to Cx40, member mutant and engineered 21 R33X Cx37 cells the co-express the on notion, of effects this trans-dominant-negative members with exhibited related Consistent other family. of interfere in connexin possible function translated and Cx43 interact the to of with sufficient fragment be acid may we patients amino 2003), R33X 32 al., the et that (Lagree reasoned process oligomerization co-expressed the of governing status functional there the expressed, in highly reduction is Cx43. mutant clear R33X a the was data when clinically our Conversely, that exhibit 2006). showed not al., et do R33X (Richardson patients) ODDD (parents the detectable recessive carriers Cx43 autosomal of mutant endogenous R33X R33X of levels of heterozygous status low why functional explain the expressing might alter cells to patients. NRK failed ODDD mutant harboring that R33X of fact parents The in case the be would u otersda hne ciiyrtie ytemtn n the and mutant patients the R33X by retained than activity strictly channel R76H morbidities residual that fewer the causes to propose from due We Hallermann-Streiff mutant suffer 2004). additional will al., R76S patients et with exhibiting related (Pizzuti the symptoms in into the ODDD associated as functionally whereas transcends severe syndrome, commonly as are it not but channels is make symptoms ODDD patients to the case, ability R33X this the albeit In retains compromised. we channels, mutant hand, R76H other functional the the On that function. for discovered and ODDD available development of be remains organ symptoms Cx43 proper clinical functional also no sufficient that have suggesting may patients of heterozygous R33X form the of severe Interestingly members parents date. and to extreme ODDD family most reported clinically the in connexin other resulting that compromised predict related to we deemed and are GJIC, closely Cx43-based mutant no R33X with the surviving harbor be that From patients ODDD. studies, to linked our mutants Cx43 recessive of autosomal known number junctions assembly. total the junction gap altering gap least for in at available support is molecules would reduction mutant Cx40 mutant the the observed connexin, that expressing notion an cells the the in The passage was Cx40 assembly. of of than junction there composed gap rather that misfolding and degradation pathway fact a secretory for the in it through targeting resulting transient is prematurely Cx40 unstable or but Cx37 case either with and the of mutant be mechanism the still the of could interaction is mechanism the this this that whether suspect Cx37 to We with as action. interact concerns could raising mutant Cx40 affected R33X could or the we of However, that disease. show scope the directly Cx40 patients of not the severity and overall broadening in Cx37 the and and with generated tissues function interact Cx43 may their mutant of inhibiting R33X propose fragment We the 1999). N-terminal harboring al., et both (He the with Cx40 and channels that 1997) heteromeric be al., form et to (Brink to Cx37 documented thought whereas been Cx32, not or has Cx26 is it either with Cx43 co-oligomerization Furthermore, of capable 2004). Willecke, and ic h -emnso x3i huh ob epnil for responsible be to thought is Cx43 of N-terminus the Since nsmay hsi h is hrceiaino h nytwo only the of characterization first the is this summary, In eesv x3mtnsaddsae2863 disease and mutants Cx43 Recessive ¨hl Journal of Cell Science oepesC4.HL el tbyepesn x2wr eeosypoie by have provided GA). generously we University, engineered were as (Emory transiently Cx32 or expressing Koval infection 2002) stably Michael retroviral al., cells Dr et HeLa by Qin Cx40. Cx26 2000; express al., to or et Cx37 (Mao 100 previously Cx43, described serum, express bovine to engineered fetal 10% 100 with supplemented D-glucose prxmtl,12mnatrmconeto,dgtliae eecletdwt a with collected were microscope. pressure images epifluorescence digital automated microinjection, inverted after IRE2 FemtoJet pressure min DM 1–2 Eppendorf Leica were 350 Approximately, a Alexa an mutants mM to using 10 attached or or microinjector Canada) yellow Cx43 Lucifer Probes, 5% GFP-tagged contain (Molecular pipette expressing glass a cells with microinjected or cells Control studies transfer Dye fixed and coverslips glass 4 on at grown al., acetone were et methanol/20% cells Gong 80% Briefly, 2007; with 2008). al., al., et et (Gong Manias described 2006; previously as immunolabeled were Cells Immunocytochemistry forward the using the by introduce engineered (5 (London, to was Cx43 Industries construct human GFP- Biotech of the encoding primer Norclone encoding R33X-GFP construct from (McLachlan cDNA The purchased human described ON). was The mutant previously 2005). al., R76H was et tagged Roscoe Cx43-GFP 2008; human al., et encoding vector cDNAs The Cx43 mutant of Engineering cultured were Cells Collection. carcinoma) Culture cervical 37 Type (mouse (human at American N2a HeLa from Invitrogen. purchased and were from (NRK) cells obtained kidney were rat reagents normal culture neuroblastoma), and sera, media, All reagents and culture Cell burden. Methods disease and less Material or and greater as have disease, distinct understanding may to better patients clearly a ODDD linked provide why are we two to discoveries, mutants these elucidated on Cx43 based how have family of we connexin mechanisms the co-expressed Finally, inhibits other and of members. cross-talks ability mutant the forming that channel evidence any of lack 2864 A G A T T oitouethe introduce to with CT) digested GTA AAA TGA AAA oiiefrC4 rC4 a ucinpauswsplotted was plaques also junction were gap cells that Cx40 cells or plaques or GFP-positive junction (two HeLa Cx43 of gap evidence percent for The Cx40 clear positive interfaces. or or exhibited cell–cell Cx43 of cells locations immunolabeled cells same at of these plaques) whether images NRK disguisable and selected more field randomly imaged each in Ten cells we respectively. Cx40, R33X-GFP, or (60 Cx43 or for 2003; immunolabeled Cx43-GFP al., et GFP, (Qin described previously 2004). as Zeiss al., microscope on 10 et analyzed confocal Veitch at and Meta mounted, 33342 water, 510 555 distilled Hoechst Alexa LSM in detected rinsed to with were was conjugated stained Coverslips binding antibodies Probes). were antibody anti-rabbit Nuclei Primary from donkey dilution. purchased (Invitrogen). or (Sigma- 1:100 were anti-mouse antibody a antibodies goat anti-Cx43 at Cx40 using of and used dilution Cx37 and 1:500 Cx32, Invitrogen a anti-Cx26, with All labeled Aldrich). were Cx43 mutant oteicuino mn cdcmoet ftemlil lnn ieand site cloning multiple the of SDS. components with interaction acid upon amino characteristics due folding slightly presumably of in a shown) differences inclusion at not (data resolved the human was GFP GFP vector of recombinant Expressed to encoding than acids GFP. GFP weight a amino free control, molecular 32 expressing a higher cells first As sequence. engineer the linker to that of acid used confirmed C-terminus amino 7 sequencing the a DNA to via Cx43 in-frame R33X-RFP. fused encoding was vector with RFP a Moscow, digested generate was was to 16/10, vector RFP vector pTaqRFP Miklukho-Maklaya R33X The GFP-tagged JSC, linker. Hin acid The (Evrogen amino Russia). 4 Evrogen 117420, a p3 via from the epitope in- with purchased fused FLAG GFP-tagged was the replaced the mutant R33X to engineer was the frame to that vector confirmed used analysis GFP as Sequence (Sigma-Aldrich). approach the same where the mutant follow primer to reverse a proceeded amino used and seven we a mutant, via R33X GFP FLAG-tagged of (5 the N-terminus acids engineer the amino To to 32 linker. in-frame first acid fused the was that Cx43 confirmed human analysis from sequence DNA vector. (Clontech) 9 oqatf a ucinpausi el oepesn onxn oehrwith together connexins co-expressing cells in plaques junction gap quantify To 6 GGCGACCAAATAAAGACG oitoueaBm1site BamH1 a introduce to CTG) GTA AAA TGA AAA CGA ATC CGG -GCG IIrsrcinezmst xieteR3 oigrgo ro olgtn to ligating to prior region coding R33X the excise to enzymes restriction dIII m 60 ˚ n %CO 5% and C /lsrpoyi n MLguaie eaclswr stably were cells HeLa L-glutamine. mM 2 and streptomycin g/ml m )frec odto eesoe o h oa ubro GFP-positive of number total the for scored were condition each for m) ora fCl cec 2 (13) 126 Science Cell of Journal Hin Hin dIII/ 2 IIst n h ees rmr(5 primer reverse the and site dIII nDlec’ oiidEsnilMdu DE) . g/l 4.5 (DMEM), Medium Essential Modified Dulbecco’s in Bam 9 GCGCAGCTAGGTGCTGACG) AGC TGG GAC GGT ATG CTT AAG GAC -GAC Iadcoe nothe into cloned and HI ˚ o 5mnts el xrsigC4 or Cx43 expressing Cells minutes. 15 for C Bam Ist.TePRpout were products PCR The site. HI Hin dIII/ 9 TGGTCGCGA CCG GAT -TCG 6 Bam 6 lgCV1 vector flag-CMV-14 ...(** s.e.m. IctpEGFP-N1 HI-cut m m /l(Molecular g/ml /lpenicillin, g/ml Bam P , Iand HI 0.01). otcigclswsqatfe samaueo y rnfra previously as transfer dye of 2006). measure more al., et or a McLachlan one GFP, 1999; as to al., microinjected transfer et quantified of (Jordan dye percentage was described exhibited The cells that 350. and cells contacting Alexa presence mutant-expressing or or the yellow connexin reflected Lucifer images of (pre- ‘‘post-injection’’ OpenLab GFP spread for whereas using initially imaged images), Japan) were cells injection Photonics, analysis, data For (Hamamatsu (Improvision). software camera device charge-coupled nirgn aaa rIDe80cnuae eodr niois(green; dilution; antibodies NE). Odyssey 1:10,000 Lincoln, the secondary Biosciences, using (red; (Li-Cor visualized conjugated System and 680 Imaging 800 PA) Chemicon Infrared Gilbertsville, Fluor IRDye Rockland, (1:10,000; dilution; Alexa or revealed 1:10,000 The antibody were with Canada) binding. bands GAPDH Immunoreactive incubation antibody Invitrogen, loading. anti-GFP for 1-hour protein for for following probed control control a as also a cases, and MAB374) some as were 36-4900) (1:500; In used antibody. membranes Invitrogen was anti-Cx37 MAB3580) GFP (1:200; polyclonal Chemicon recombinant (1:1000; anti-Cx40 C3470), anti-GFP polyclonal monoclonal Sigma 40-4300), 71-0500), (1:500; Invitrogen Invitrogen (1:500; anti-Cx32 anti-Cx26 polyclonal polyclonal C6219), Sigma (1:5000; Cx43 Co-immunoprecipitation Na 0.5% mM 1 EGTA, mM NaF, 1 mM buffer EDTA, 1 mM detergent-based X-100, 1 in 7.4, Triton pH lysed 1% Tris-HCl, were P-40, mM 10 cells Nonidet NaCl, expressing mM mutant (150 and Connexin blotting Western sdfriaeaqiiin aawspoesduigLMsfwr n lte as scanning plotted and laser software overall repeated LSM the using to in processed due was decrease fields Data slight acquisition. image a image fluorescent for was used calcein into there calcein the cases green-fluorescent after of some of seconds brightness In recovery 6 and cells. the every assess cell photobleached to and the the seconds RFP bleaching of 198 by before saturated for area collected not bleaching were entire were that the Images areas the cell fluorescence. throughout and exhibited saturation cells selected expressing pixel were mutant expressing ‘‘low’’ for RFP mutant mutant ‘‘High’’ laser. R33X-RFP exhibited equipped argon the an cells microscope cells with of or bleached confocal levels cells cells calcein META low green-fluorescent Untransfected Live fluorescence. and PBS. 510 (calcein) in high LSM green twice expressing and washing Zeiss (RFP) 10 to a red prior detecting with on min 10 imaged incubated for were medium were culture cells in R33X-RFP. Probes) with hours, transfected 24 were cells After NRK Cx43-positive of cultures (FRAP) Monolayer photobleaching after recovery Fluorescence junctional macroscopic The (g CA). City, Cx43-GFP. conductance Axopatch Union control, two Instruments, its using (Axon or by amplifiers performed mutant 200B were R76H-GFP recordings the patch-clamp whole-cell with Double transfected were cells N2a electrophysiology clamp Patch NP-40, 0.5% EGTA, mM 1 EDTA, mM 1 Na mM NaCl, 1 mM and X- 150 NaF Triton mM (1% Tris, buffer 1 RIPA mM in lysed 10 were R33X-GFP 100, expressing cells or cells NRK t1 H,adtercre ntr urnswr iial itrdb low-pass a by filtered digitally presentation. were for digitized channel currents Hz) and open unitary (200 kHz recorded and filter 1 the at baseline Gaussian Gaussian and filtered the kHz, low-pass with of 10 was histograms variance signal at current and these junctional mean The fitting the current. then determine to and functions histograms amplitude current sesda rvosydsrbd(i ta. 00.T nueacrc fteg the of accuracy ensure To 2010). al., et (Xin described previously as assessed n muorcpttdb sn niC4 niois(2 antibodies anti-Cx43 4 using at C6219) by immunoprecipitated and lae rti –ehrs ed i B)fr2huso h okra 4 at rocker the on hours 2 for PBS) (in beads A–Sepharose Protein cleaned esrdapiue fuiaycret(I current unitary of amplitudes measured I nioyba ope a etiue t1940 at centrifuged was complex bead antibody rmtesre eitneo ahptheetoei h g the in electrode patch each of resistance series the from eahdfo h ed ybiigfr5mntsi 30 in minutes 5 for boiling by beads the 500 from with detached times proteins bound three from washing separated were by proteins Unbound aspirated. were supernatants 04.Rcrig rmcl ar ipaigoeo w hneswr sdto used were channels two The or amplitudes. one current displaying unitary pairs the cell determine from (V Recordings voltage 2004). junctional correct to used was compensation off-line %ml tro eprtr n nuae ihpiayatbde vrih at overnight antibodies primary with in incubated blocked and membranes, temperature 4 nitrocellulose room onto at milk transferred 5% were proteins The PAGE. 40 Approximately inhibitors). n rnfre ontoells ebae hc eepoe ihat-F and anti-GFP with probed were antibodies. which anti-Cx43 membranes nitrocellulose to transferred and containing buffer j ˚ eeete eemndb ietmaueet rb is ltigall-points plotting first by or measurements direct by determined either were s .C4,C2,C3,C3,C4 n F eedtce sn oylnlanti- polyclonal using detected were GFP and Cx40 Cx37, Cx32, Cx26, Cx43, C. ˚ vrih.Imn opee eepeiiae ih30 with precipitated were complexes Immune overnight. C j fC4-F n 7HGPgpjnto hneswere channels junction gap R76H-GFP and Cx43-GFP of ) b mratehnl ape eersle y1%SDS-PAGE 10% by resolved were Samples -mercaptoethanol. 3 VO m 4 .Teclua yae eehmgnzd cleared, homogenized, were lysates cellular The ). fpoenprln a eovdo n1%SDS- 10% in on resolved was lane per protein of g m fRP ufr n h on ope was complex bound the and buffer, RIPA of l j yteV the by ) c j m a acltdb iiigthe dividing by calculated was /lclenA (Molecular calcein-AM g/ml g j ; ,at4 c j m 5 j lof2 acltos(uae al., et (Musa calculations I j /V ˚ o iue and minutes 2 for C 3 VO j 6 m h mltdsof amplitudes The . /ecin Sigma g/reaction; j aml loading Laemmli rosresulting errors ) 4 n proteases and m fpre- of l ˚ .The C. j , Journal of Cell Science ee,J,Nlo,T . ut .M n ee,E C. E. Beyer, and M. J. Burt, K., T. Nelson, J., Gemel, lnie,A . son,L . nesn . iiet,N,Feig . Gittens, C., Fleming, N., Ciliberti, N., Anderson, R., L. Osborne, M., A. Flenniken, lns . uat .adMrn,A P. A. Moreno, and M. Rubart, S., Elenes, orwlk,R,Hri,G,Lcnr . Wo H., Lechner, G., Hertig, R., Dobrowolski, easaitclysgiiatdifference. software. significant statistical statistically SigmaStat a sample the be independent using by two-tailed done Student’s were the using analyzed orwlk,R,Sse . crce,J . akn,M,Km .S,Rcasa,M., Rackauskas, S., J. Kim, M., Watkins, W., J. Schrickel, P., Sasse, R., Dobrowolski, K. Willecke, and A. Laird, Sommershof, and R., D. Dobrowolski, Bai, J., Sampson, J., Lee, A., Macdonald, J., J. Kelly, M., J. Churko, l ausaepeetda mean as presented are values All Statistics ( Prism4. time GraphPad over FRAP percent the hro .M,Sa,Q,Gn,X . wbd,K . a,D,Smsn .and J. Sampson, D., Bai, J., K. Swoboda, Q., X. Gong, Q., Shao, M., J. Churko, W. D. Laird, and Q. Shao, X., Pan, S., Langlois, M., J. Churko, S. G. Goldberg, and B. D. Alexander, of Institute References Canadian D.B.). the and from D.W.L. grants (to by Research supported Health was work This Funding the performed manuscript. D.B. the and of and writing L.X. project pilot the the A.M., supervised performed mutants. D.W.L. R.L. experiments. Cx43 electrophysiological experiments. the designed FRAP on constructs, the the studies of did many and prepared of experiments most directly prepared Q.S. and experiments figures. the the of majority the performed T.H. of conflicts contributions Author no declare authors confocal The the with analysis. assistance interest. FRAP his for and Simek imaging Jamie thank authors The Acknowledgements hro .M,Ca,J,Sa,Q n ar,D W. D. Laird, and Q. Shao, J., Chan, M., J. Churko, K. V. Verselis, and V. M. Bennett, A., Bukauskiene, F., F. Bukauskas, Laird, D., P. Lampe, V., M. Bennett, A., Bukauskiene, K., Jordan, F., F. H., Bukauskas, K. Seul, M., E. Westphale, E., Peterson, K., Banach, K., Cronin, R., P. Brink, fcnei3 rcnei4 ihcnei4 eetvl fet intercellular affects selectively connexin43 with connexin40 transfer. or molecular connexin37 of dysplasia. oculodentodigital of al. et model L. mouse Moreno, C., a 132 Lounsbury, in B., L. mutation Kelsey, missense Q., X. Gong, E., J. sltdpiso aieara el smdae yhmgnosadheterogeneous and homogeneous by mediated is channels. cells junction atrial gap canine of pairs isolated eitdgpjntoa opigi h eecyeo ibbd ed oaltered to leads buds limb of mice. mesenchyme in the morphogens K. of in Willecke, expression coupling and junctional H. gap Schorle, mediated R., Bauer, D., Eckert, raz . hnm . imn,K,Dgn .e al. et J. humans. Degen, in oculodentodigitaldysplasia hereditary K., of model new Tiemann, a represents mutant A., mouse connexin43G138R Ghanem, C., Troatz, differentiation. W. D. odfcetgpjnto channels. junction addition gap in activity deficient hemichannel to increased cause mutations Cx43 dysplasia-associated defects. wound-healing have may 32 patients that suggest patients dysplasia W. D. Laird, uatrgltshi rwhadhi ie opooyi os oe fhuman of model mouse a in morphology fiber hair dysplasia. and oculodentodigital growth hair regulates mutant from distinct is differentiation mutants. keratinocyte connexin43 impair disease-linked to other mutant connexin43 fs260 the of fused protein. connexin43 and fluorescent connexin43 green from assembled with cells. channels living junction gap of in properties coupling functional USA and Sci. channels Acad. K. Natl. junction Proc. V. gap protein Verselis, fluorescent and W. D. connexin37. human and connexin43 C. C1386-C1396. rat E. from Beyer, formed and channels V. S. 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Biochem. 81 137-152. , ¨rsdo 131 re,P,Wl,V,Dce N., Dicke, V., Wulf, P., ¨rfer, 219 20) oeoculodentodigital Some (2007). 2197-2204. , 21a.TeG0 connexin43 G60S The (2011a). 21) nuil coexpression Inducible (2012). 10 429 18 9-17. , 20) oso connexin43- of Loss (2009). P ur e.Chem. Med. Curr. 990-1004. , 2899-2911. , , 473-483. , .5wscniee to considered was 0.05 20) h conditional The (2008). m .Physiol. J. Am. t 21) h potency The (2010). ts.Alanalyses All -test. n 20) Gja1 A (2005). 5 20) Gating (2001). Development –0 using 7–10) u.Mutat. Hum. 10 2045- , 273 , og .Q,Sa,Q,Lnli,S,Bi .adLid .W. D. Laird, and D. Bai, S., Langlois, Q., Shao, Q., X. Gong, caha,E,Mna,J . og .Q,Lusuy .S,Sa,Q,Bernier, Q., Shao, S., C. Lounsbury, Q., X. Gong, L., J. Manias, E., McLachlan, og .Q,Sa,Q,Lusuy .S,Bi .adLid .W. D. Laird, and D. Bai, S., C. Lounsbury, Q., Shao, Q., X. Gong, a,A . ehegr . iigo,D,Glpa,J,Lid .W n as .C. C. Naus, and W. D. Laird, J., Galipeau, D., Lidington, J., Bechberger, J., A. 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Bai, M., S. hrceiaino J1faehf uaincuigouoetdgtldysplasia oculodentodigital causing mutation keratoderma. frameshift palmoplantar and GJA1 a of characterization 20) ernldfeetainadgot oto fnuo2 el fe retroviral after cells neuro-2a connexin43. of of control delivery growth gene and differentiation Neuronal (2000). 20) aeo onxn3i ada isehrorn ies-ikdconnexin43 disease-linked a harbouring tissue cardiac in mutant. connexin43 of Fate (2008). epidermis. the in differentiation Chem. keratinocyte regulate levels Connexin (2007). ragmn fcnei4 nrthatgpjnto membranes. junction gap heart rat in connexin43 of arrangement hrceiaino a ucinpoen ntebadro x3mtn mouse mutant Cx43 of bladder the communication in dysplasia. proteins for oculodentodigital junction of evidence models gap molecular of Characterization cornea: rat the in compartments. patterns expression ua rattmrclsihbt nvv uo rwhb ehns htis that mechanism a by growth tumor vivo communication. intercellular in 277 junctional gap inhibits significant cells of independent W. tumor D. breast Laird, human and A. 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