Alteration of protein localization and intracellular calcium content due to connexin26 D50A and A88V mutations

Site-directed mutagenesis to generate Cx26 mutant clones

Human Cx26 WT (wild-type) cDNA was used to generate Cx26-D50A and A88V missense point mutations by using site-directed mutagenesis. After cloning of PCR products into pBlueScript cloning vector, location and insertion of mutations were verified by sequencing (Macrogen Europe, The Netherlands). Then, Cx26-WT, D50A and A88V cDNAs were subcloned into pIRES2EGFP2 and pCS2+ mammalian expression vectors.

Cell culture

HeLa and neuro-2A (N2A) (ATCC, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Scientific HyClone, USA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel) and 1% penicillin/streptomycin (GIBCO, USA) in a humidified chamber with 5% CO₂ and 37°C. Cells were plated in 6 well plates for transfection with Lipofectamine 2000 reagent (Invitrogen, USA) on the following day with 1:2 DNA to Lipofectamine 2000 ratio following the manufacturer’s protocol. 3.2 mM CaCI₂ was added in medium to close prospective hemichannels and cells were used within 24–36 h after transfection.

Neurobiotin dye uptake assay

N2A cells transfected with pIRES2EGFP2 clones were used for fluorescent dye uptake assays with neurobiotin (NB, 287 Da, +1 charge, Vector Labs, USA). Twenty four hours after transfection, cells were washed with PBS and incubated 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 washed with PBS containing 3.2 mM CaCl₂ three times for 10 min. After fixation of cells with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT), cells were permeabilized with 0.1% Triton-X 100 for 10 min, blocked with 3% BSA-0.1% TritonX-100 for 15 min and then were incubated with tetra-methyl rhodamine isothiocyanate (TRIT-C) conjugated streptavidin (1:1000 dilution, Pierce, USA) for 30 min at RT in dark. Images were acquired with a fluorescence microscope (IX71, Olympus, Japan) using the same exposure times for all conditions. Image analysis for signal intensity was performed with ImageJ (NIH, USA) program. Background subtraction was applied to all images before thresholding and signal intensities of fluorescence within cells were determined.

Immunofluorescent staining

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 and blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature (RT). A 1:500 dilution of a polyclonal rabbit anti-Cx26 antibody (Invitrogen, USA) was applied for 1 h at RT followed by an incubation with 1:200 dilution of Alexa555-conjugated goat anti-rabbit antibody (Invitrogen, USA), 1:200 dilution of Alexa Fluor 488 conjugated Phalloidin (Invitrogen, USA) and 1 μM DAPI for 45 min at RT in dark. Antibodies and DAPI was removed by PBS wash and coverslips were dipped in distilled water, dried and mounted on glass slides. Staining was verified under fluorescence microscope (IX83, Olympus, Japan) with ×40 objective and images were taken with a CCD digital camera [27].

For co-immunofluorescent staining of golgin-97 and Cx26, 1 h incubation at room temperature with primary antibodies [rabbit anti-Cx26 antibody (1:500) and mouse anti-golgin-97 (1:1000) (Invitrogen, USA)] was followed by the application of secondary antibodies [Alexa555-conjugated 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 with PBS, coverslips were dipped in distilled water, dried and mounted on glass slides. Staining was verified under fluorescence microscope (IX83, Olympus, Japan) with ×40 objective and images were taken with a CCD digital camera.

Measurement of intracellular calcium levels

Intracellular calcium content in N2A cells (5×10⁵) transfected with pCS2+ clones was determined by using Fluo-3AM Ca²⁺ indicator (Invitrogen, USA) and flow cytometry [23]. After washing cells with Ca²⁺ free PBS twice for 5 min, cells were incubated with 5 μM Fluo-3AM at 37°C for 30 min. Then, cells were washed with PBS, trypsinized, pelleted and resuspended in 300 μL Hank’s Balanced Salt Solution (HBSS) [28] and analyzed with FACSCANTO (BD Biosciences, USA).

Statistical analysis

All results were expressed as mean (±standard deviation). Groups were compared using ANOVA followed by Student-Newman-Keuls post-hoc comparison analysis. Statistical significance was considered for p<0.05.

Verification of the ability of Cx26-D50A and A88V mutations to form aberrant hemichannels

Cx26-D50A and A88V mutations were previously shown to cause elevated membrane currents in cRNA injected Xenopus oocytes and in HeLa cells, suggesting the presence of active hemichannels [26]. We wanted to confirm if these mutations were also able to facilitate the uptake of fluorescent dyes in transfected N2A cells (Figure 1). In sample images, neurobiotin fluorescent intensities were observed to increase in cells with Cx26-WT relative to the negative control, pIRES2EGFP2 transfected cells. Further, cells with Cx26-D50A and A88V had higher neurobiotin signals than cells with Cx26-WT (Figure 1A). Fluorescent signal intensity analysis from at least 15 images from three different experiments suggested that the fluorescent intensity in cells transfected with Cx26-WT increased by 2-fold (p<0.001) compared to cells with pIRES2EGFP2 (Figure 1B). Further, Cx26-D50A and Cx26-A88V resulted in 3.2 and 3.6-fold (p<0.001) increase in neurobiotin uptake relative to pIRES2EGFP2 cells and 1.6 and 1.8-fold (p<0.001) increase compared to cells with Cx26-WT, respectively. Neurobiotin uptake assay confirmed the ability of Cx26-D50A and A88V mutations to form aberrant hemichannels and also showed that these hemichannels allow the passage of larger molecules than ions across the plasma membrane [26].

Figure 1:

Aberrant hemichannel activities of Cx26-D50A and A88V.

(A) Representative images from neurobiotin uptake assays. pIRES2EGFP2 transfected cells were used as a negative control. Green is for GFP expression and red is for neurobiotin signal. Scale bar 90 μm. (B) Comparison of fluorescent dye intensities in transfected cells. Cx26-D50A and A88V were compared with both negative control pIRES2EGFP2 and Cx26-WT (p-value *<0.001 comparison between pIRES2EGFP2 and Cx26-WT, D50Y, A88V; p**<0.001 comparison between Cx26-WT and mutants D50Y and A88V).

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 pIRES2EGFP2 Cx26-WT, D50A and A88V constructs were used for immunofluorescent staining (Figure 2). Cells expressing 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). Furthermore, Cx26-WT expressing cells had gap junction plaques 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].

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.

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 were detected to extensively overlap with golgin-97 in transfected cells as observed by yellow signals in transfected cells (Figure 2B). These suggested that a fraction of proteins retained in the Golgi apparatus while others formed hemichannels on the plasma membrane.

Effect of Cx26-D50A and A88V mutations on intracellular calcium signals

Calcium signals are indispensable for proper functioning of keratinocytes and abnormalities in calcium homeostasis have been shown to lead skin disorders [25]. In order to investigate whether Cx26-D50A and A88V mutations cause any change in the internal calcium levels, we determined cellular calcium content by a calcium indicator Fluo-3AM using flow cytometry (Figure 3). Comparison of Fluo-3AM signals among groups suggested that calcium amount in cells with Cx26-WT and cells transfected with the negative control, pCS2+ were similar (p=0.12). However, cells with Cx26-D50A and A88V had 1.6-fold increase in the intracellular 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.

Figure 3:

Effect of Cx26-D50A and A88V hemichannels on intracellular calcium levels.

Intracellular calcium content was compared among mutants, Cx26-WT and pCS2+ containing cells (p-value *<0.05).