Functional Characterization of a GJA1 Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma

Abstract

A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms. A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms. Gap junctions are unique intercellular channels that directly connect the cytoplasm of neighboring cells, which are formed by integral membrane proteins called connexins (Cxs) 2The abbreviations used are: Cxs, connexins; ANOVA, analysis of variance; ER, endoplasmic reticulum; GFP, green fluorescent protein; PDI, protein-disulfide isomerase; T259, Cx43 mutant truncated at residue 259. 2The abbreviations used are: Cxs, connexins; ANOVA, analysis of variance; ER, endoplasmic reticulum; GFP, green fluorescent protein; PDI, protein-disulfide isomerase; T259, Cx43 mutant truncated at residue 259. (1Willecke K. Eiberger J. Degen J. Eckardt D. Romualdi A. Guldenagel M. Deutsch U. Sohl G. Biol. Chem. 2002; 383: 725-737Crossref PubMed Scopus (998) Google Scholar). Each apposing cell contributes a hexameric arrangement of connexins, also known as a connexon, which dock to form a gap junction channel. A connexin polypeptide chain spans the plasma membrane four times, consisting of four transmembrane domains (M1 to M4), two extracellular loops (EL-1 and EL-2) and one intracellular loop (IL), with both the N and C termini (AT and CT, respectively) located in the cytoplasm (2Saez J.C. Berthoud V.M. Branes M.C. Martinez A.D. Beyer E.C. Physiol. Rev. 2003; 83: 1359-1400Crossref PubMed Scopus (980) Google Scholar, 3Laird D.W. Revel J.P. J. Cell Sci. 1990; 97: 109-117Crossref PubMed Google Scholar, 4Falk M.M. Buehler L.K. Kumar N.M. Gilula N.B. EMBO J. 1997; 16: 2703-2716Crossref PubMed Scopus (135) Google Scholar) (Fig. 1). Whereas connexin transmembrane domains and extracellular loops are highly conserved, the intracellular loop and C terminus are known to be the most variable domains across different connexin family members, suggesting that these domains play important roles in the differential functions of connexins. Connexins exhibit complex tissue distribution patterns that are temporally and spatially regulated during development and differentiation. Among connexin family members (21 in human and 20 in mouse), the GJA1-encoded protein (Cx43) exhibits the most ubiquitous distribution, playing important roles in many tissue types and physiological processes (5Kretz M. Maass K. Willecke K. Eur. J. Cell Biol. 2004; 83: 647-654Crossref PubMed Scopus (66) Google Scholar). Cx43 channels can be regulated by a pH-sensitive “ball-and-chain” mechanism where the C terminus acts as a gating particle and a region within the intracellular loop serves as a receptor (6Morley G.E. Taffet S.M. Delmar M. Biophys. J. 1996; 70: 1294-1302Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 7Duffy H.S. Sorgen P.L. Girvin M.E. O'Donnell P. Coombs W. Taffet S.M. Delmar M. Spray D.C. J. Biol. Chem. 2002; 277: 36706-36714Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The cytoplasmic acidification renders the “particle” able to bind to the “receptor”, leading to the closure of the Cx43 gap junction channel. This pH gating mechanism was supported by the findings that a truncated mutant of Cx43, where the last 125 amino acid residues were deleted from the CT, became insensitive to intracellular pH change. Furthermore, if the deleted tail segment was co-expressed with the truncated Cx43 the impaired pH gating could be partially restored (6Morley G.E. Taffet S.M. Delmar M. Biophys. J. 1996; 70: 1294-1302Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Channel gating, connexin assembly, and degradation events are also thought to be regulated in part by phosphorylation of one or more of the 14 serine/tyrosine phosphorylation sites encoded-within the C-terminal region of Cx43 (8Lampe P.D. Lau A.F. Int. J. Biochem. Cell Biol. 2004; 36: 1171-1186Crossref PubMed Scopus (498) Google Scholar, 9Kim D.Y. Kam Y. Koo S.K. Joe C.O. J. Biol. Chem. 1999; 274: 5581-5587Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 10Lampe P.D. TenBroek E.M. Burt J.M. Kurata W.E. Johnson R.G. Lau A.F. J. Cell Biol. 2000; 149: 1503-1512Crossref PubMed Scopus (462) Google Scholar, 11Laird D.W. Biochim. Biophys. Acta. 2005; 1711: 172-182Crossref PubMed Scopus (240) Google Scholar, 12Solan J.L. Lampe P.D. Biochim. Biophys. Acta. 2005; 1711: 154-163Crossref PubMed Scopus (253) Google Scholar, 13Moreno A.P. Biochim. Biophys. Acta. 2005; 1711: 164-171Crossref PubMed Scopus (60) Google Scholar). The Cx43 C terminus is also able to directly or indirectly interact with a variety of proteins, such as ZO-1, ZO-2, α- and β-tubulin, β-catenin, CIP85, and drebrin (14Duffy H.S. Delmar M. Spray D.C. J. Physiol. Paris. 2002; 96: 243-249Crossref PubMed Scopus (113) Google Scholar, 15Herve J.C. Bourmeyster N. Sarrouilhe D. Biochim. Biophys. Acta. 2004; 1662: 22-41Crossref PubMed Scopus (123) Google Scholar, 16Wei C.J. Xu X. Lo C.W. Annu. Rev. Cell Dev. Biol. 2004; 20: 811-838Crossref PubMed Scopus (331) Google Scholar, 17Lan Z. Kurata W.E. Martyn K.D. Jin C. Lau A.F. Biochemistry. 2005; 44: 2385-2396Crossref PubMed Scopus (51) Google Scholar, 18Laird D.W. Biochem. J. 2006; 394: 527-543Crossref PubMed Scopus (639) Google Scholar) which are expected to regulate the assembly and functional status of Cx43 gap junction channels. The mounting number of clinical studies has revealed a role for connexin mutations in a wide spectrum of human diseases (16Wei C.J. Xu X. Lo C.W. Annu. Rev. Cell Dev. Biol. 2004; 20: 811-838Crossref PubMed Scopus (331) Google Scholar, 19Kelsell D.P. Di W.L. Houseman M.J. Am. J. Hum. Genet. 2001; 68: 559-568Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 20Richard G. Clin. Exp. Dermatol. 2003; 28: 397-409Crossref PubMed Scopus (57) Google Scholar). Oculodentodigital dysplasia (ODDD), a rare inherited autosomal dominant disorder characterized by developmental abnormalities of the face, eyes, limbs, and dentition, has been linked to mutations in the GJA1 gene (21Paznekas W.A. Boyadjiev S.A. Shapiro R.E. Daniels O. Wollnik B. Keegan C.E. Innis J.W. Dinulos M.B. Christian C. Hannibal M.C. Jabs E.W. Am. J. Hum. Genet. 2003; 72: 408-418Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Although the Cx43 mutations associated with ODDD are broadly distributed, the majority of the known mutations are located in the N-terminal half of the gene sequence encoding Cx43. Interestingly, the bulk of these mutations are familial with the remaining cases representing sporadic mutations. Also, in all but one case, ODDD-linked GJA1 gene mutations alter a single amino acid, leaving the remainder of the 382 amino acid residues of the Cx43 sequence unaltered. The only mutation localized to the C-terminal region of Cx43 is a frameshift mutation arising from the deletion of two nucleotides (780-781del) (22van Steensel M.A. Spruijt L. van der Burgt I. Bladergroen R.S. Vermeer M. Steijlen P.M. van Geel M. Am. J. Med. Genet. A. 2005; 132: 171-174Crossref Scopus (74) Google Scholar). The altered sequence translates into 46 aberrant amino acid residues being added after residue 259 until it reaches a premature stop codon after encoding an amino acid residue at the 305 position. The incorporated sequence of 46 aberrant amino acids does not correspond to any known functional polypeptide domain as determined by a BLAST search (22van Steensel M.A. Spruijt L. van der Burgt I. Bladergroen R.S. Vermeer M. Steijlen P.M. van Geel M. Am. J. Med. Genet. A. 2005; 132: 171-174Crossref Scopus (74) Google Scholar). Thus far, this is the only familial Cx43 ODDD-linked mutation where patients suffer from not only the wide range of syndromes associated with ODDD but also palmoplantar keratoderma. Given that the last native 123 amino acid residues are lost from this frameshift Cx43 mutant it is postulated that the reason for patients suffering from increased disease burden may be related to a critical role played by the Cx43 C terminus in the skin. In fact, mice expressing a Cx43 mutant where the last 125 amino acids of Cx43 are missing die within 1 week after birth because of the loss of skin barrier function (23Maass K. Ghanem A. Kim J.S. Saathoff M. Urschel S. Kirfel G. Grummer R. Kretz M. Lewalter T. Tiemann K. Winterhager E. Herzog V. Willecke K. Mol. Biol. Cell. 2004; 15: 4597-4608Crossref PubMed Scopus (115) Google Scholar). In the present study, we fully characterized the functional status of the fs260 mutant as it is distinct from other Cx43 ODDD-linked single residue mutations. Importantly, the mechanism responsible for the trafficking defect is encoded within the 46 aberrant amino acids attached to the Cx43 sequence after residue 259. Through the collective use of well understood cell culture models and keratinocytes, our studies shed new insights into why patients harboring this mutant exhibit both ODDD and skin disease. Cell Culture and Reagents—All cell culture reagents were obtained from Invitrogen or Sigma. The mouse neuroblastoma (N2A) cells, the human cervical carcinoma (HeLa) cells and the normal rat kidney (NRK) cells were purchased from ATCC (American Type Culture Collection). Rat epidermal keratinocytes (REKs) were generously provided by Dr. Vincent C. Hascall (Cleveland Clinic Foundation, Cleveland, OH). Cells were cultured in regular high glucose DMEM supplemented with 10% fetal bovine serum, 2 mm l-glutamine (except N2A cells), 100 units/ml penicillin, and 100 μg/ml streptomycin. All the cell lines were maintained at 37 °C in a moist environment of 95% air and 5% CO2 and subcultured directly into 35 mm tissue culture dishes or plated on glass coverslips as required. Engineering of Mutant Cx43 cDNAs—The engineering of human Cx43-GFP construct was described previously (24Roscoe W. Veitch G.I. Gong X.Q. Pellegrino E. Bai D. McLachlan E. Shao Q. Kidder G.M. Laird D.W. J. Biol. Chem. 2005; 280: 11458-11466Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The human Cx43 mutation-fs260 was constructed with the QuikChange™ Site-directed mutagenesis kit (Stratagene, La Jolla, CA) by using forward 5′-GCTGAGCCCTGCCAAAGACTGGGTCTCAAAAATATGC-3′ and reverse 5′-GCATATTTTTGAGACCCAGTCTTTGGCAGGGCTCAGC-3′ primers and cloned into pcDNA3.1 expression vectors. Sequence analysis confirmed that no other mutation was present. Two nucleotides (780-781TG) were deleted resulting in a frameshift beginning after residue 259 until reaching a premature stop codon after encoding residue 305. The Cx43 mutant, designated fs260 (representing frameshift after encoding residue 259), encoded an irrelevant sequence of 46 aberrant amino acid residues at the C terminus. The fs260-GFP cDNA was generated by PCR using the forward 5′-CGGGGTACCAACATGGGTGACTGGAGC-3′ and reverse 5′-CGGTGGATCCTTGCTTGCTTG-3′ primer by adding KpnI and BamHI restriction sites, then cloning into the pEGFP-N1 vector (BD BioSciences). The resulting construct (fs260-GFP) encoded 6 amino acids linking fs260 to GFP in-frame was sequenced for verification. The T259 mutant was derived from the fs260 construct by deleting the sequence encoding the 46 aberrant amino acids. The primers used to generate the T259 mutant were forward 5′-CGGGGTACCAACATGGGTGACTGGAGC-3′ and reverse 5′-CGGTGGATCCCAGTCTTTGGCAGGGCTC3′. The T259 mutant was then incorporated into the pEGFP-N1 vector by using the KpnI and BamHI restriction sites as described above. Sequencing indicated that truncated Cx43 at residue 259 was linked in-frame by 6 amino acids to GFP. The fs260-GFP construct was further mutated to substitute the putative endoplasmic reticulum FF motif, encoded within the aberrant 46 amino acids, to an AA motif (fs260-FF/AA) by using the forward primer 5′-CGACAGAAACAAGCCGCCTTGCCGCAATTACAACAAGCAAGCAAG, and reverse primer 5′-CTTGCTTGCTTGTTGTAATTGCGGCAAGGCGGCTTGTTTCTGTCG. All GFP-tagged variants were further cloned into the AP2 retroviral vector as described previously (25Qin H. Shao Q. Curtis H. Galipeau J. Belliveau D.J. Wang T. AlaouiJamali M.A. Laird D.W. J. Biol. Chem. 2002; 277: 29132-29138Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Transient Transfection and Retroviral Infection—Cells were transfected with human Cx43-GFP, fs260-GFP, T259-GFP, or fs260-FF/AA-GFP by Lipofectamine 2000 (Invitrogen) as described previously (24Roscoe W. Veitch G.I. Gong X.Q. Pellegrino E. Bai D. McLachlan E. Shao Q. Kidder G.M. Laird D.W. J. Biol. Chem. 2005; 280: 11458-11466Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In brief, cells were allowed to reach 50% confluence in 35-mm culture dishes, the plasmids were preincubated with Lipofectamine 2000 reagent for 15 min at room temperature, the complexes were then incubated with the cells for 4-6 h. Cells were washed and incubated in regular medium for 24-48 h before being used for patch-clamp recording, or fixed for immunocytochemistry. For fs260 and free GFP co-transfection experiments, the vectors encoding GFP and fs260 were used at a 1:5 ratio, respectively, to ensure that cells that expressed GFP also expressed fs260. In co-transfection experiments, the cDNAs of fs260-GFP and Cx43-mRFP (Cx43 tagged to monomeric dsRed) were pre-mixed at various ratios prior to being used in transfections. For cell infection, REKs at 30-40% confluence were subjected to two rounds of retroviral infection with retroviral particles encoding GFP-tagged fs260, T259, G138R or GFP as described previously (26Thomas T. Telford D. Laird D.W. J. Biol. Chem. 2004; 279: 19157-19168Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Infected cells were passed at least twice, evaluated to ensure at least 90% expression efficiency, and subjected to Western blot analysis. Western Blot Analysis—REKs expressing GFP-tagged fs260, T259, G138R or GFP, were subjected to lysis buffer containing 50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 0.02% sodium azide, 100 μg/ml phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.1% SDS, 50 mm NaF, 2 mm EDTA, and a protease inhibitor mixture from Roche Applied Science (Mississauga, ON, Ref. 27Laird D.W. Castillo M. Kasprzak L. J. Cell Biol. 1995; 131: 1193-1203Crossref PubMed Scopus (222) Google Scholar). The BCA assay reagent kit (Pierce) was used to determine protein concentrations and 20 μg of protein per lane were separated by 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and subsequently immunoblotted for Cx43 (antibody to the C-terminal of Cx43; 1:5,000 dilution, Sigma; antibody to the N-terminal of Cx43 1:100 dilution, Fred Hutchinson Cancer Research Center Antibody Development Group, Seattle, WA). The membranes were stripped and re-probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to assess loading levels. Antibody binding was detected using the enhanced chemiluminescence system (Pierce). Immunocytochemistry—Cell immunofluorescent labeling and imaging were performed as previously described (24Roscoe W. Veitch G.I. Gong X.Q. Pellegrino E. Bai D. McLachlan E. Shao Q. Kidder G.M. Laird D.W. J. Biol. Chem. 2005; 280: 11458-11466Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Briefly, cells grown on 12-mm glass coverslips were fixed with 80% methanol/20% acetone (or a 3.7% formaldehyde solution for GFP-expressing cells) at 4 °C for 15 min. HeLa and NRK cells transfected with fs260-GFP or T259-GFP were labeled with a 1:500 dilution of an anti-protein disulfide isomerase (PDI) antibody (Stressgen, Biotechnologies, Victoria, BC), a 1:1000 dilution of an anti-giantin antibody (Babco, Berkeley Antibody Company, Richmond, CA) or a 1:200 dilution of an anti-TGN38 antibody (BD Transduction Laboratories, Lexington, KY). NRK cells expressing fs260-GFP, T259-GFP, or fs260-FF/AA-GFP were also labeled with a 1:50 dilution of anti-Cx43 monoclonal antibody (Fred Hutchinson Cancer Research Center Antibody Development Group). Washed cells were incubated for 1 h in goat anti-mouse or donkey anti-rabbit antibodies conjugated to Texas red or FITC (Jackson ImmunoResearch Laboratories). In some cases, cell nuclei were labeled with Hoechst 33342 (1:1000 dilution; Molecular Probes). As described previously (24Roscoe W. Veitch G.I. Gong X.Q. Pellegrino E. Bai D. McLachlan E. Shao Q. Kidder G.M. Laird D.W. J. Biol. Chem. 2005; 280: 11458-11466Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 27Laird D.W. Castillo M. Kasprzak L. J. Cell Biol. 1995; 131: 1193-1203Crossref PubMed Scopus (222) Google Scholar), the labeled cells were analyzed and imaged on a Zeiss (Thornwood, NY) LSM 510 META confocal microscope. Digital images were prepared using Zeiss LSM, Adobe Photoshop 7.0 and CorelDraw 12 software. To quantify the incidences of gap junction plaque formation, at least 8 fields (0.09 mm2 were and over were for condition NRK cells and over were The are as The was used to junctional intercellular coupling N2A cell expressing mutant or wild-type Cx43 was using whole cell patch as described previously (24Roscoe W. Veitch G.I. Gong X.Q. Pellegrino E. Bai D. McLachlan E. Shao Q. Kidder G.M. Laird D.W. J. Biol. Chem. 2005; 280: 11458-11466Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). was by In labeling only N2A cell expressing both fs260-GFP and Cx43-mRFP were for NRK cells or REKs or cells transfected with Cx43 were with single whole cell patch The cell membrane capacitance was determined and the gap junctional conductance was then This single cell capacitance recording was by and A.D. van A.P. 1996; PubMed Scopus Google Scholar, A.D. A. van A.P. J. Exp. 2001; Google Scholar), which is for the gap junctional coupling cells within or Briefly, cell cells) were for capacitance recording, and the cell in the of a was at A was then to a The was at and at 100 The and the were and analysis were performed a and The conductance the cell and cells, was by the where is the conductance the patch and the is the of the is that the conductance of conductance A.D. A. van A.P. J. Exp. 2001; Google Scholar). The are as The and were used to The Cx43 fs260 Mutant to the in the or of in the GJA1 gene encoding Cx43 (Fig. the autosomal dominant disorder known as oculodentodigital In the of the frameshift mutation by the deletion of nucleotides and patients be expected to a truncated of Cx43 where 46 aberrant amino acid residues are added after residue 259 (Fig. a Cx43 mutant the majority of putative or known phosphorylation sites (Fig. and a new motif be into the encoded aberrant sequence (Fig. In all the provided by proteins that bind the last 123 amino acid domain of Cx43 be To to the of this frameshift the fs260-GFP mutant was expressed in a variety of cells to assess revealed that in HeLa cells, fs260-GFP formed gap junctional at the but a and intracellular analysis revealed that fs260-GFP in part with suggesting a of fs260-GFP was localized in the endoplasmic reticulum (Fig. fs260-GFP was to with the (Fig. The was more in NRK cells where the the mutant and was (Fig. the with the was also (Fig. When expressed in NRK cells or REKs the intracellular distribution of fs260-GFP suggesting that endogenous wild-type Cx43 could not distribution to the cell (Fig. C and for Cx43 in NRK cells revealed the of gap junction plaques at (Fig. fs260 and reduced the of endogenous Cx43 gap junction plaques at in NRK NRK cells expressing fs260-GFP T259-GFP or fs260-FF/AA-GFP were for endogenous Cx43 using an antibody that only endogenous Cx43 and not the co-expressed cells expressing the fs260-GFP or fs260-FF/AA-GFP was a loss of gap junction plaques plaques were in cells the mutant Hoechst 33342 was used to cell nuclei in C. In cells expressing T259-GFP, Cx43 gap junctions were both mutant expressing and cells of these results revealed that gap junction plaques were in of the fs260-GFP or cells gap junctions were in over 80% of the cells expressing T259-GFP A of 8 fields (0.09 were for the or of gap junction plaques at were for fs260 mutant the assembly of Cx43 gap junctions in REKs and to the of endogenous Cx43. REKs were with retroviral particles encoding GFP, fs260-GFP, T259-GFP, or transfected with a cDNA encoding fs260-FF/AA-GFP prior to with an anti-Cx43 to the C-terminal domain that gap junction plaques were fs260 or cells but or GFP-expressing Hoechst 33342 was used to cell Western blot revealed that the expression of the fs260 mutant reduced the of endogenous Cx43 Western blot is of The T259 Mutant to the Cell assess the role of the 46 amino acid residues in the aberrant of the fs260 mutant, a truncated Cx43 mutant was at 259 1). the fs260 mutant, T259-GFP was expressed in HeLa or NRK cells this truncated mutant was to the plasma membrane and formed gap junctional plaques at (Fig. In HeLa cells, the also that this truncated mutant was also partially in intracellular (Fig. A and a that was in NRK cells (Fig. C and The fs260 Mutant the of Gap from if the expression of the fs260-GFP mutant reduced the of gap junctions formed from endogenous Cx43, transfected NRK cells were for endogenous Cx43 the antibody used is for the C-terminal domain and only endogenous As fs260-GFP localized to intracellular (Fig. gap junction plaques were as neighboring cells (Fig. in cells expressing the mutant, endogenous Cx43 was to intracellular of the and was a in gap junction plaques at the (Fig. Furthermore, NRK cells expressed T259-GFP, endogenous Cx43 formed gap junctions at where it with T259-GFP (Fig. To assess the putative FF motif encoded in the aberrant 46 amino acid domain was responsible for the retention of the fs260 mutant within intracellular the FF motif was mutated to AA and expressed in NRK cells (Fig. the fs260-FF/AA-GFP mutant was typically in intracellular with for assembly into gap junction plaques (Fig. the fs260-FF/AA-GFP mutant, the fs260 mutant, reduced the number of gap junction plaques formed from endogenous Cx43