Turk J Biochem 2017; aop
Research Article
Hande Aypek and Gülistan Meşe*
Alteration of protein localization and 
intracellular calcium content due to 
connexin26 D50A and A88V mutations
D50A ve A88V mutasyonlarına bağlı connexin26 
protein lokalizasyonunun ve hücre içi kalsiyum 
miktarının değişimi
DOI 10.1515/tjb-2016-0292 Conclusion: Retention of Cx26 in the Golgi apparatus and 
Received November 30, 2015; accepted July 13, 2016 alteration in the intracellular calcium content due to KID 
Abstract syndrome mutations may influence various cellular pro-
cesses that might contribute to development of epidermal 
Introduction: Connexins (Cx) play essential roles in cellular phenotypes.
homeostasis by forming gap junctions and non-junctional Keywords: Connexin26; Mutations; Keratitis-ichthyosis-
hemichannels. In vitro characterization of Cx26 mutations deafness (KID) syndrome; Intracellular calcium; Golgi 
causing keratitis-ichthyosis-deafness (KID) syndrome, apparatus.
were shown to form leaky hemichannels. The molecular/
cellular mechanisms affected by aberrant hemichannels 
have recently been elucidated. Here, we further wanted Özet
to characterize Cx26 KID syndrome mutations, D50A and 
A88V, which were shown to form aberrant hemichannels Giriş: Connexin molekülleri (Cx) kurdukları hücrelerarası 
and remained unaddressed in the literature. kanallar ve hücrelerarası olmayan yarı kanallar sayesinde 
Methods: Neurobiotin uptake assay in HeLa and N2A cells hücre yaşamını dengelemede önemli roller üstlenirler. İn 
transfected with Cx26-WT, D50A or A88V verified the pres- vitro analizlerle keratitis-ichthyosis-deafness (KID) send-
ence of aberrant hemichannels and immunofluorescent romuyla ilişkili Cx26 mutasyonlarının düzgün kapanama-
staining with fluorescent microscopy determined cellular yan yarı kanalların oluşmasına sebep oldukları gösteril-
localization of Cx26. Finally, intracellular calcium content miştir. Bu tip anormal çalışan yarı kanalların etkilediği 
was examined by using calcium indicator, Fluo-3AM, and moleküler / hücresel mekanizmalar son zamanlarda araş-
flow cytometer. tırılmaya başlanmıştır. Bu çalışmada amacımız, daha 
Results: Cx26-D50A and A88V mutations prevented the önce yarı kanallar yaptıkları gösterilen Cx26’da oluşan 
formation of gap junction plaques at cell-cell appositions KID sendromu mutasyonlarından D50A ve A88V’nin 
and mutant proteins were observed to localize to the Golgi hücre biyolojisinde yaptığı değişiklikleri incelemektir.
apparatus. Further, comparison of intracellular calcium Yöntemler: Cx26-WT, D50Y ve A88V ile transfekte edilen 
content showed an increase in calcium amount in cells HeLa ve N2A hücrelerinde neurobiotin boya alım deney-
containing Cx26-D50A and A88V relative to Cx26-WT. leriyle anormal yarı kanalların varlığı teyit edilmiş ve flo-
resan mikroskop ve immunoboyamasıyla Cx26’nın hücre 
içi lokalizayonu belirlenmiştir. Son olarak, kalsiyum belir-
*Corresponding author: Gülistan Meşe, PhD, Department of teci, Fluo-3AM ve akış sitometresi kullanarak hücre içi kal-
Molecular Biology and Genetics Rm: D206, Izmir Institute of siyum içeriği belirlenmiştir.
Technology, Urla 35430, Izmir, Turkey, Phone: + 90 232 750 7312, 
Fax: + 90 232 750 7303, e-mail: gulistanmese@iyte.edu.tr Bulgular: Cx26-D50A ve A88V mutasyonları hücreler arası 
Hande Aypek: Department of Molecular Biology and Genetics, Izmir kanalların oluşumunu engellemiş, aynı zamanda mutant 
Institute of Technology, Urla, Izmir, Turkey proteinler hücrede Golgi aygıtında lokalize olmuşlardır. 
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2      Hande Aypek and Gülistan Meşe: Cx26 KID syndrome mutations
Ek olarak normal Cx26 ile kıyaslandığında Cx26-D50A ve of the characterized Cx26  mutations associated with 
A88V bulunduran hücrelerde hücre içerisindeki kalsiyum  keratitis-ichthyosis-deafness (KID) syndrome were shown 
miktarının arttığı tespit edilmiştir. to lead to the formation of aberrant hemichannels [13–15, 
Sonuç: KID hastalarında görülen epidermal bozukluk- 21]. Therefore, the formation of abnormal hemichannels 
ların oluşmasında, mutant Cx26 proteinlerinin Golgi due to Cx26 mutations was suggested to be the underlying 
aparatında tutularak ve anormal yarı kanal oluşturup cause of epidermal anomalies observed in KID syndrome 
hücresel kalsiyum dengesini bozarak çeşitli hücre fonksi- patients. In this respect, mutant Cx26 hemichannels are 
yonlarında neden olduğu değişimlerin rol oynayabileceği thought to interfere with keratinocyte homeostasis by 
düşünülmektedir. releasing signaling molecules such as Ca2 + and ATP into 
Anahtar kelimeler: Connexin26; Mutasyon; Keratitis- the extracellular environment as a paracrine element and 
ichthyosis-deafness (KID) sendromu; Hücresel kalsiyum; alter the epidermal physiology [22, 23]. However, the exact 
Golgi aygıtı. mechanisms of how aberrant hemichannels influence keratinocyte homeostasis and generate skin phenotypes 
of KID syndrome are not completely known. One of the 
components important for the epidermis is the mainte-
Introduction nance of calcium homeostasis as abnormal calcium sign-
aling was implicated with several human disorders [24, 
Connexins (Cx) are one of the building blocks of gap 25]. In that respect, here we aimed to determine altera-
junctions in vertebrates. At least 21 connexin isoforms tions in the Cx26 biogenesis and the intracellular calcium 
have been identified in the human genome which are content caused by KID syndrome associated Cx26-D50A 
expressed throughout the body in a cell/tissue type and and A88V mutations, which were previously shown to 
developmental stage-specific manner [1, 2]. Gap junc- cause elevated membrane currents in Xenopus oocytes 
tions mediate various cellular processes including both and HeLa cells [26].
the delivery of nutrients and removal of waste from avas-
cular organs, electrical and metabolic coupling of cells, 
cell growth, proliferation, and differentiation by allowing 
the passage of molecules between adjacent cells [3–5]. In Materials and methods
addition to the formation of intercellular channels that 
couple adjacent cells, connexins also function as non- Site-directed mutagenesis to generate 
junctional hemichannels that can facilitate the exchange Cx26 mutant clones
of molecules across the plasma membrane and regulate 
cellular/tissue homeostasis [6]. Under physiological con- Human Cx26 WT (wild-type) cDNA was used to generate 
ditions, hemichannels are closed to prevent the leakage Cx26-D50A and A88V missense point mutations by using 
and uncontrolled uptake of molecules across the plasma site-directed mutagenesis. After cloning of PCR products 
membrane [7–9]. On the other hand, external insults, into pBlueScript cloning vector, location and insertion of 
stress or mutations in connexin genes can disrupt proper mutations were verified by sequencing (Macrogen Europe, 
sealing of hemichannels and negatively affect cellular The Netherlands). Then, Cx26-WT, D50A and A88V cDNAs 
physiology [10–15]. were subcloned into pIRES2EGFP2 and pCS2+ mammalian 
The importance of connexin mediated cellular com- expression vectors.
munication for human physiology is better understood 
with characterization of various human disorders and 
their associations to mutations in several connexin iso- Cell culture
forms, such as cataract (Cx46, Cx50), hearing loss, skin 
disorders (Cx26, Cx30) and hereditary peripheral neu- HeLa and neuro-2A (N2A) (ATCC, USA) were maintained 
ropathy (Cx32) [3, 16–19]. In vitro characterization of in Dulbecco’s Modified Eagle Medium (DMEM, Thermo 
disease-associated connexin mutations showed that Scientific HyClone, USA) supplemented with 10% fetal 
mutations affect various processes in connexin biogen- bovine serum (FBS) (Biological Industries, Israel) and 
esis and function including but not limited to the genera- 1% penicillin/streptomycin (GIBCO, USA) in a humidified 
tion of truncated proteins, altered trafficking, prevention chamber with 5% CO2 and 37°C. Cells were plated in 6 well 
of gap junction channel formation and function as well plates for transfection with Lipofectamine 2000 reagent 
as altered hemichannel activities [20]. For example, all (Invitrogen, USA) on the following day with 1 : 2 DNA to 
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Hande Aypek and Gülistan Meşe: Cx26 KID syndrome mutations      3
Lipofectamine 2000 ratio following the manufacturer’s antibodies [rabbit anti-Cx26 antibody (1 : 500) and mouse 
protocol. 3.2 mM CaCI2 was added in medium to close pro- anti-golgin-97 (1 : 1000) (Invitrogen, USA)] was followed 
spective hemichannels and cells were used within 24–36 h by the application of secondary antibodies [Alexa555-con-
after transfection. jugated goat anti-rabbit and Alexa488-conjugated goat 
anti-mouse secondary antibody (1 : 200, Invitrogen, USA)] 
and 1 μM DAPI for 45  min at RT in dark. After washing 
Neurobiotin dye uptake assay with PBS, coverslips were dipped in distilled water, dried 
and mounted on glass slides. Staining was verified under 
N2A cells transfected with pIRES2EGFP2 clones were used fluorescence microscope (IX83, Olympus, Japan) with 
for fluorescent dye uptake assays with neurobiotin (NB, × 40 objective and images were taken with a CCD digital 
287 Da, + 1 charge, Vector Labs, USA). Twenty four hours camera.
after transfection, cells were washed with PBS and incu-
bated with Ca2 + free medium for 20 min at 37°C. Cells were 
then incubated with 0.5 mg/mL NB for 20 min and were Measurement of intracellular calcium levels
washed with PBS containing 3.2 mM CaCl2 three times for 
10 min. After fixation of cells with 4% paraformaldehyde Intracellular calcium content in N2A cells (5 × 105) trans-
(PFA) for 20 min at room temperature (RT), cells were per- fected with pCS2+ clones was determined by using Fluo-
meabilized with 0.1% Triton-X 100 for 10  min, blocked 3AM Ca2 + indicator (Invitrogen, USA) and flow cytometry 
with 3% BSA-0.1% TritonX-100 for 15 min and then were [23]. After washing cells with Ca2 + free PBS twice for 5 min, 
incubated with tetra-methyl rhodamine isothiocyanate cells were incubated with 5 μM Fluo-3AM at 37°C for 
(TRIT-C) conjugated streptavidin (1 : 1000 dilution, Pierce, 30  min. Then, cells were washed with PBS, trypsinized, 
USA) for 30 min at RT in dark. Images were acquired with pelleted and resuspended in 300 μL Hank’s Balanced Salt 
a fluorescence microscope (IX71, Olympus, Japan) using Solution (HBSS) [28] and analyzed with FACSCANTO (BD 
the same exposure times for all conditions. Image analy- Biosciences, USA).
sis for signal intensity was performed with ImageJ (NIH, 
USA) program. Background subtraction was applied to all 
images before thresholding and signal intensities of fluo- Statistical analysis
rescence within cells were determined.
All results were expressed as mean ( ± standard devia-
tion). Groups were compared using ANOVA followed by 
Immunofluorescent staining Student-Newman-Keuls post-hoc comparison analysis. 
Statistical significance was considered for p < 0.05.
HeLa cells (2.5 × 105) were cultured on glass coverslips in 
6  well plates and transfected with pCS2+ clones. After 
washing with PBS, cells were fixed with 4% PFA for 
20 min, permeabilized with 0.1% Triton-X 100 for 15 min Results
and blocked with 3% bovine serum albumin (BSA) for 1 h 
at room temperature (RT). A 1 : 500 dilution of a polyclonal Verification of the ability of Cx26-D50A 
rabbit anti-Cx26 antibody (Invitrogen, USA) was applied and A88V mutations to form aberrant 
for 1 h at RT followed by an incubation with 1 : 200 dilution hemichannels
of Alexa555-conjugated goat anti-rabbit antibody (Invitro-
gen, USA), 1 : 200 dilution of Alexa Fluor 488 conjugated Cx26-D50A and A88V mutations were previously shown 
Phalloidin (Invitrogen, USA) and 1 μM DAPI for 45  min to cause elevated membrane currents in cRNA injected 
at RT in dark. Antibodies and DAPI was removed by PBS Xenopus oocytes and in HeLa cells, suggesting the pres-
wash and coverslips were dipped in distilled water, dried ence of active hemichannels [26]. We wanted to confirm 
and mounted on glass slides. Staining was verified under if these mutations were also able to facilitate the uptake 
fluorescence microscope (IX83, Olympus, Japan) with of fluorescent dyes in transfected N2A cells (Figure 1). In 
× 40 objective and images were taken with a CCD digital sample images, neurobiotin fluorescent intensities were 
camera [27]. observed to increase in cells with Cx26-WT relative to the 
For co-immunofluorescent staining of golgin-97 and negative control, pIRES2EGFP2 transfected cells. Further, 
Cx26, 1  h incubation at room temperature with primary cells with Cx26-D50A and A88V had higher neurobiotin 
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4      Hande Aypek and Gülistan Meşe: Cx26 KID syndrome mutations
A hemichannels and also showed that these hemichannels 
allow the passage of larger molecules than ions across the 
plasma membrane [26].
Effect of Cx26-D50A and A88V mutations 
on the Cx26 protein biosynthesis and 
trafficking
To examine the effects of KID syndrome associated Cx26-
D50A and A88V mutations on protein biosynthesis and 
trafficking, HeLa cells transiently transfected with pIRE-
S2EGFP2 Cx26-WT, D50A and A88V constructs were used 
for immunofluorescent staining (Figure 2). Cells express-
ing Cx26-WT, D50A and A88V constructs were able to 
synthesize Cx26 proteins as observed by Cx26 signals in 
representative images in Figure 2 (red signals). Further-
more, Cx26-WT expressing cells had gap junction plaques 
B at the cell-to-cell contact sites between adjacent cells 
(Figure 2A, white arrows). On the other hand, no gap 
junctional plaques were observed between neighboring 
cells transfected with either Cx26-D50A or A88V mutant 
constructs (Figure 2A, white arrows), suggesting that they 
impaired the formation of gap junction channels between 
adjacent cells as also shown by Mhaske et al. [26].
To examine whether these mutations caused defects 
in Cx26 protein trafficking, co-immunostaining of Cx26 
and golgin-97, a Golgi apparatus marker, was performed 
(Figure 2B). There was no co-localization between Cx26 
(red signal) and golgin-97 (green) in Cx26-WT expressing 
cells. In contrast, Cx26-D50A and A88V mutant proteins 
Figure 1: Aberrant hemichannel activities of Cx26-D50A and A88V. were detected to extensively overlap with golgin-97 in 
(A) Representative images from neurobiotin uptake assays. pIRE- transfected cells as observed by yellow signals in trans-
S2EGFP2 transfected cells were used as a negative control. Green 
is for GFP expression and red is for neurobiotin signal. Scale bar fected cells (Figure 2B). These suggested that a fraction 
90 μm. (B) Comparison of fluorescent dye intensities in transfected of proteins retained in the Golgi apparatus while others 
cells. Cx26-D50A and A88V were compared with both negative formed hemichannels on the plasma membrane.
control pIRES2EGFP2 and Cx26-WT (p-value * < 0.001 comparison 
between pIRES2EGFP2 and Cx26-WT, D50Y, A88V; p** < 0.001 com-
parison between Cx26-WT and mutants D50Y and A88V). Effect of Cx26-D50A and A88V mutations 
on intracellular calcium signals
signals than cells with Cx26-WT (Figure 1A). Fluorescent 
signal intensity analysis from at least 15 images from Calcium signals are indispensable for proper functioning 
three different experiments suggested that the fluorescent of keratinocytes and abnormalities in calcium homeosta-
intensity in cells transfected with Cx26-WT increased by sis have been shown to lead skin disorders [25]. In order to 
2-fold (p < 0.001) compared to cells with pIRES2EGFP2 investigate whether Cx26-D50A and A88V mutations cause 
(Figure 1B). Further, Cx26-D50A and Cx26-A88V resulted any change in the internal calcium levels, we determined 
in 3.2 and 3.6-fold (p < 0.001) increase in neurobiotin cellular calcium content by a calcium indicator Fluo-3AM 
uptake relative to pIRES2EGFP2 cells and 1.6 and 1.8- using flow cytometry (Figure 3). Comparison of Fluo-3AM 
fold (p < 0.001) increase compared to cells with Cx26-WT, signals among groups suggested that calcium amount in 
respectively. Neurobiotin uptake assay confirmed the cells with Cx26-WT and cells transfected with the negative 
ability of Cx26-D50A and A88V mutations to form aberrant control, pCS2+ were similar (p = 0.12). However, cells with 
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Hande Aypek and Gülistan Meşe: Cx26 KID syndrome mutations      5
Figure 2: Comparison of Cx26-WT, D50A and A88V localization and trafficking in gap junctional communication deficient HeLa cells.
(A) Cx26-WT, D50A and A88V transfected HeLa cells were co-stained with phalloidin for actin (green) and Cx26 antibody (red). Blue is DAPI 
staining of the nucleus. White arrows point out the gap junction plaques between Cx26-WT cells and red arrows show cell-cell contact sites 
of cells transfected with Cx26-D50A and A88V. Cells with only green and blue signals indicate untransfected cells. Scale bar 20 μm. (B) 
Cx26-WT, D50A and A88V transfected cells were co-stained with Cx26 (red) and the Golgi apparatus marker, golgin-97 (green) antibodies. 
Blue shows DAPI staining of the nucleus. Yellow signals in Cx26-D50A and Cx26-A88V point out the co-localization of Cx26 and golgin-97. 
Scale bar 20 μm.
Cx26-D50A and A88V had 1.6-fold increase in the intra-
cellular calcium amount with respect to Cx26-WT cells 
(p = 0.04) (Figure 3). These changes in calcium content 
suggested that Cx26-D50A and A88V mutations interfered 
with calcium mechanisms in these cells.
Discussion
KID syndrome is a rare congenital disorder associated 
with dominant Cx26  mutations. The molecular mecha-
nisms leading to KID syndrome skin phenotypes is not 
exactly known and in vitro characterization of Cx26 muta-
Figure 3: Effect of Cx26-D50A and A88V hemichannels on intracel-
lular calcium levels. tions has helped to understand the effect of mutations 
Intracellular calcium content was compared among mutants, on cell homeostasis and hence the generation of skin 
Cx26-WT and pCS2+ containing cells (p-value * < 0.05). anomalies in affected individuals. Here, we characterized 
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6      Hande Aypek and Gülistan Meşe: Cx26 KID syndrome mutations
the properties of KID syndrome associated Cx26-D50A membrane where they formed non-junctional hemichan-
and A88V mutations, which were shown to form aber- nels. However, these hemichannels were not able to dock 
rant hemichannels by electrophysiological analysis [26]. with each other to complete the formation of gap junc-
D50A and A88V mutations resulted in an increase in the tion channels on the plasma membrane. Furthermore, a 
uptake of neurobiotin into cells from the extracellular fraction of Cx26-D50A and A88V mutant proteins were 
environment, implicating the ability of mutant hemichan- observed to accumulate in the Golgi apparatus, suggest-
nels to transfer larger molecules than small ions into cells. ing defects in Cx26 trafficking. Cx26 proteins were shown 
Moreover, these mutations prevented the formation of gap to be transported to the plasma membrane either passing 
junction channels at the cell-to-cell junctions and a frac- through the Golgi apparatus or by bypassing the Golgi 
tion of mutant proteins were observed to be retained in the apparatus depending on the cell type, suggesting dif-
Golgi apparatus compared to Cx26-WT. Finally, mutations ferent membrane transport pathways for Cx26 [36, 37]. 
caused an elevation in the intracellular calcium levels in Further, Cx26-D66H mutation associated with Vohwin-
cells containing D50A and A88V compared to cells with kel syndrome and R184Q mutation linked to dominant 
Cx26-WT. These results confirmed the activities of leaky hearing loss were also shown to be retained in the Golgi 
hemichannels due to Cx26-D50A and A88V mutations and apparatus [38, 39]. These mutations might impair the 
may also implicate the involvement of altered protein traf- folding of the proteins, resulting in their accumulation in 
ficking and elevated intracellular calcium content due to the Golgi apparatus that may play role in the generation 
aberrant hemichannels in KID syndrome. of stress responses to influence cellular processes [40]. 
Cx26 mutations cause both non-syndromic and syn- Thus, both the leaky hemichannel activity and retaining 
dromic deafness. In vitro characterization of mutations of mutant proteins in the Golgi apparatus can alter cellu-
leading to non-syndromic deafness suggested these lar physiology.
mutations are loss-of-function mutations and they only In conclusion, characterization of Cx26  mutations 
affect the cochlear function but have no effect on other provide an invaluable tool in the elucidation of molecular 
Cx26 expressing tissues such as the skin and the liver mechanisms underlying the KID syndrome. It will help 
[15, 29–31]. On the other hand, Cx26  mutations associ- us to expand our understanding of the disorders, the role 
ated with syndromic deafness were shown to result in of Cx26 in epidermal homeostasis and also our ability to 
the acquisition of novel functions which influence the develop new therapeutic approaches for patients.
homeostasis of both the inner ear and the skin and are 
considered as gain of function mutations [14, 32, 33]. For Acknowledgments: We thank Thomas W. White from 
example, Cx26 mutations linked to KID syndrome were Stony Brook University, NY, USA for kindly providing 
shown to form aberrant hemichannels and activities of pIRES2EGFP2, pCS2+ and pBSBK-Cx26WT vectors. Expert 
abnormal hemichannels are suggested to be the main technical help from Drs. Ozden Yalcin-Ozuysal, Engin 
mechanisms underlying skin pathologies [22, 26, 33, 34]. Ozcivici and Izmir Institute of Technology, Biotechnol-
The molecular mechanisms related to this phenotype ogy and Bioengineering Research and Application Center 
might be the uncontrolled release of signaling molecules is well appreciated. This work was supported by The Sci-
including ATP into the extracellular environment through entific and Technological Research Council of Turkey 
aberrant Cx26  hemichannels and therefore the disrup- Grant (210T035) and FP7 Marie Curie Re-Integration Grant 
tion of calcium signaling. Changes in calcium homeo- (PIRG08-GA-2010-277101).
stasis can affect different cellular processes including 
proliferation, differentiation and survival that can con- Conflict of interest: Authors have no conflicts of interest.
tribute to the development of epidermal abnormalities 
[25, 35]. Here, we showed that expression of Cx26-D50A 
and A88V mutations in mammalian cells resulted in an References
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Hande Aypek and Gülistan Meşe: Cx26 KID syndrome mutations      7
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