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First published online 5 May 2004
doi: 10.1242/jcs.01084

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Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells

Joanna Gemel^1,*, Virginijus Valiunas^2,*, Peter R. Brink² and Eric C. Beyer^1,

¹ Department of Pediatrics, Section of Hematology/Oncology and Stem Cell Transplantation, University of Chicago, Chicago, IL 60637-1470, USA
² Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794, USA

View larger version (16K): [in a new window]   Fig. 1. Immunoblot detection of Cx43 and Cx26 expression in parental and stably transfected HeLa, HEK, and N2a cells. Triton X-100-soluble extracts prepared from HeLa cells (20 µg protein) or HEK cells (20 µg protein for Cx43 detection or 100 µg protein for Cx26 detection) or whole cell lysates prepared from N2a cells (75 µg protein) were resolved by SDS-PAGE, transferred to membranes and blotted with anti-Cx43 (top) or anti-Cx26 (bottom) antibodies. Migration of molecular mass markers is indicated to the left of the blot.

View larger version (118K): [in a new window]   Fig. 2. Detection of the distributions of Cx26 (red) and Cx43 (green) in HEKCx26 cells (A-H) or Cx40 (red) and Cx43 (green) in HeLa-Cx43/Cx40 cells (I-K) by immunofluorescence microscopy. (A-C) Conventional immunofluorescence images of cells double labeled for Cx26 (shown alone in B) and for Cx43 (shown alone in C). Merged image of both immunoreactivities is shown in A. (D) Stacked z-series from double label confocal analysis of a single large plaque. (E-H) Z-series of slices obtained at different depths (separated by 0.3-1.2 µm) from confocal analysis of plaques in HEK-Cx26 cells. (I-K) Z-series of slices similarly obtained at different depths from plaques in pairs of HeLa-Cx43/Cx40 cells where simultaneously obtained images show precise co-localization of Cx43 (red) and Cx40 (green). Bar: 27 µm (A-C); 1 µm (D); 5 µm (E-H); 3 µm (I,J).

View larger version (29K): [in a new window]   Fig. 3. Macroscopic currents recorded from N2a cell pairs containing different combinations of Cx26 and Cx43 and their dependence on transjunctional voltage (Vj). (A) Gap junction currents (Ij) elicited by bipolar pulse protocol from different N2A cell pairs: co-transfected cell pair, N2aCx26/Cx43-N2aCx26/Cx43 (top left panel); homotypic N2aCx43 cell pair (bottom left panel); co-transfected cell N2aCx26/Cx43 heterotypically paired with N2aCx26 cell (top right panel) and homotypic N2aCx26 cell pair (top right panel). (B) Summary plots of normalized instantaneous (open circles) and steady-state (solid circles) gj versus Vj from 7 co-transfected N2aCx26/Cx43 cell pairs. The continuous curves represent the best fit of gj,ss(l) data to the Boltzman equation (Vj,0=–97 mV/90 mV, gj,min=0.3/0.4, z=1.4/1.1 for negative/positive Vj, respectively) and 2nd order regression (r2=0.63) fit of gj,ss (O) data.

View larger version (22K): [in a new window]   Fig. 4. Single channel properties of gap junctions recorded from co-transfected N2aCx26/Cx43 cells compared to those from a heterotypic N2aCx26/Cx43-N2aCx43 cell pair. (A) Single channel properties of events in pairs of co-transfected N2aCx26/Cx43 cells. (Top panel) Multichannel recording during maintained Vj of 90 mV. Solid line represents zero current level; dashed lines represent discrete current steps indicative of opening and closing of channels. The current histograms indicate operation of at least two different channels with conductances of 135 pS and 73 pS (arrows) that resemble homotypic Cx26 and homotypic Cx43 channels, respectively. (Bottom panel) Multichannel recording during maintained Vj of 70 mV. The current histograms revealed a conductance of 139 pS (resembling homotypic Cx26 channels). (B) Bipolar pulse protocol (V1 and V2) and associated single channel currents (Ij) recorded from a N2aCx26/Cx43-N2aCx43 cell pair. Current histograms yielded similar conductances for positive and negative Vj: 69 pS and 72 pS for the main state and 16 pS-17pS for the residual state (dashed line) (which correspond to homotypic Cx43 channels).

View larger version (21K): [in a new window]   Fig. 5. Single channel properties of gap junctions recorded from co-transfected N2aCx26/Cx43 cells compared to homotypic Cx26 channels. (A) Single channel recordings from pairs of co-transfected N2aCx26/Cx43 cells. A voltage ramp protocol (top panel)Vj=±100 mV, ramp duration 2 seconds) evoked single channel currents (middle panel) with a slope conductance (dashed line) corresponding to a unitary conductance gj of 132-134 pS, which resembles homotypic Cx26 channels. The bottom panel shows another recording of one operational channel with channel rectification (gj=94 pS at Vj=–100 mV and gj=130 pS at Vj=100 mV). (B) Single channel recordings from pairs of homotypic N2aCx26 cells. (Middle panel) Single channel current elicited by voltage ramp protocol (Vj=±100 mV, 2 seconds) exhibited a unitary conductance, gj of 136 pS. (Bottom panel) Single channel currents of homotypic Cx26 showed rectification (gj=102 pS at Vj=–100 mV and gj=140 pS at Vj=100 mV). (C) Superposition of gap junction currents from a co-transfected N2aCx26/Cx43-N2aCx26/Cx43 pair and from a homotypic N2aCx26-N2aCx26 cell pair suggests that the same type of channel was recorded in both cases (i.e. Cx26).

View larger version (27K): [in a new window]   Fig. 6. Event histograms of single channel conductances from different types of N2a cell pairs: co-transfected N2aCx26/Cx43-N2aCx26/Cx43 (top left panel); homotypic N2aCx26-N2aCx26 (middle left panel); homotypic N2aCx43-N2aCx43 (bottom left panel); heterotypic N2aCx26/Cx43-N2aCx26 (top right panel) and heterotypic N2aCx26/Cx43-N2aCx43 (lower right panel).

View larger version (16K): [in a new window]   Fig. 7. Analysis of oligomeric association of Cx26 and/or Cx43 in Hela-Cx26 (A,B), HeLa-Cx43 (C), and HeLa-Cx43/Cx26 cells (D). (A) Immunoblot of fractions collected after centrifugation of Triton X-100 soluble extract from Hela-Cx26 cells through a 5-20% sucrose gradient. Prior to centrifugation, samples were incubated in buffer alone (top panel) or buffer containing 0.6% SDS to disrupt oligomers (bottom panel). The numbers under the lanes indicate the corresponding sucrose concentration as determined by refractometry. (B-D) Graphs of the densitometric values obtained after quantitation of Cx43 (triangles) or Cx26 (open and solid squares) immunoblots. For HeLa-Cx26 cells, the Triton-soluble material was untreated () or treated () with 0.6% SDS for 1 hour at 4°C before layering on the top of the sucrose gradient.

View larger version (25K): [in a new window]   Fig. 8. Affinity purification of Cx43 and potential associated proteins from N2a-Cx43(His)6/Cx26 cells. A Triton X-100-soluble extract was purified using a Ni-NTA resin and eluted with a series of increasing concentrations of L-histidine (as indicated). The presence and abundance of Cx43 and Cx26 were analyzed by immunoblotting in the cell extract, washes and eluted material using anti-Cx43 and anti-Cx26 antibodies.

View larger version (85K): [in a new window]   Fig. 9. Effects of brefeldin A or nocodazole (20 hour treatment) on the distribution of Cx26 and Cx43 in HEK-Cx26 cells. Immunofluorescent images of cells labeled for Cx26 (A-C) and for Cx43 (D-F). (A,D) Control cells; (B,E) cells treated with brefeldin A; (C,F) cells treated with nocodazole. Bar: 20 µm.

View larger version (56K): [in a new window]   Fig. 10. Cross-linking of connexins in HeLa-Cx43/Cx26 cells untreated or treated with brefeldin A for 4 hours or 20 hours. Triton X-100 soluble extracts (containing connexons) were reacted with disuccinimyl suberate (DSS) cross-linker dissolved in DMSO (+) or were incubated with DMSO solvent alone (–). Samples containing 100 µg protein were resolved by SDS-PAGE, and Cx26 (A) or Cx43 (B) were detected by immunoblot analysis using anti-Cx26 or anti-Cx43 antibodies. Migration of molecular mass markers is indicated to the left of the blot.