Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43

Abstract

Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B(dye)) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (-1). The ratio of dye rate constants (B(NBD-M-TMA)/B(Alexa 350)) indicated that Cx40 junctions are cation selective (10.7 +/- 0.5), whereas Cx43 junction are nonselective (1.22 +/- 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.

Fig. 1.

Representative results and calculated fits from connexin 43 (Cx43) and Cx40 junctional dye-charge selectivity experiments. Shown are the results of the analysis of 2 dye-charge selectivity experiments. Total fluorescence intensity for N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; A and C) or Alexa 350 (B and D) for both donor (injected cell; top trace in each panel) and recipient (bottom trace in each panel) cells as a function of time for a Rin43 cell pair (A and B) and a Rin40 cell pair (C and D). Dashed lines represent the best fit of the data [using an exponential decay model (23)] that yielded the indicated junctional permeance rate constants (B_dye) and a junctional charge selectivity ratio (B_NBD-M-TMA/B_{Alexa 350}) of 0.91 for the Rin43 cell pair and 11.3 for the Rin40 cell pair.

Fig. 2.

Selectivity of Cx43 wild-type (Cx43_wt) and Cx40_wt junctions is invariant. Shown are the dye-charge selectivity results for Cx43 junctions (Rin43 cells), Cx40 junctions (Rin40 cells), and cytoplasmic bridges (incompletely divided cells). A: plot of permeance rate constants of NBD-M-TMA vs. Alexa 350 for Cx43 junctions (•), Cx40 junctions (□), and cytoplasmic bridges (▴). These parameters were significantly correlated for all 3 groups with correlation coefficients of 0.98 (n = 17) for Cx43 junctions, 0.99 (n = 15) for Cx40 junctions, 0.97 (n = 5) for bridges. B: average charge selectivities (B_NBD-M-TMA/B_{Alexa 350} ratio) for the experiments plotted in A for bridges 0.91 ± 0.07 (n = 5), Rin43 1.22 ± 0.14 (n = 17), and Rin40 10.7 ± 0.5 (n = 15). *Selectivity of Cx40 junctions was significantly different from Cx43 junctions (Student's t-test).

Fig. 3.

Cx43 dye-charge selectivity is not affected by the carboxy terminal (CT). The average junctional charge selectivity (B_NBD-M-TMA/B_{Alexa 350}) for cells expressing Cx43 truncated at residue 257 (Cx43tr) (0.98 ± 0.07; n = 5) was not different from Cx43 functioning in the presence of excess CT (Cx43+CT) (1.03 ± 0.08; n = 6).

Fig. 4.

Electrophoretic mobility of Cx43, but not Cx40, is altered by phorbol 12-myristate 13-acetate (TPA) treatment of Rin43, Rin40, or A7r5 cells. Total protein was isolated from Rin43, Rin40, and A7r5 cells treated for 0, 15, 30, or 60 min with 100 ng/ml TPA at 37°C. Mobility of Cx43 (left) was altered in both Cx43-expressing cell types, consistent with PKC-induced phosphorylation of Cx43. No changes in the mobility of Cx40 were observed (right).

Fig. 5.

Incidence and extent of dye coupling are reduced following PKC activation in Rin43 but not Rin40 cells. A: the percentage of dye-coupled cell pairs for untreated (light shaded bars) vs. TPA-treated (dark shaded bar 50 ng/ml; solid bars 100 ng/ml) Rin43 and Rin40 cells. Coupling in treated pairs is compared with untreated pairs from cells plated simultaneously on the same day; the percentage of coupled pairs on a coverslip was determined, and the mean across multiple coverslips was determined. TPA reduced dye coupling in the Rin43 cells (untreated: 6 coverslips, 37 cell pairs; treated: 3 coverslips, 18 cell pairs) but had no effect at the highest concentration on Rin40 junctions (untreated: 5 coverslips, 31 cell pairs; treated: 5 coverslips, 18 cell pairs). B: NBD-M-TMA and Alexa 350 permeances decreased in parallel as a function of TPA (50 ng/ml) treatment duration in treated Rin43 cells. C: there was a downward trend in average permeance (B_dye) for NBD-M-TMA and Alexa 350 for Rin43 cells that were TPA treated (50 ng/ml, dark shaded bars; untreated controls, light shaded bars). D: no differences were detected in the average permeances for NBD-M-TMA and Alexa 350 (B_dye) in Rin40 cells untreated (light shaded bars) or treated with TPA (100 ng/ml; solid bars). *Dye coupling in TPA-treated cells significantly different from untreated cells (Student's t-test).

Fig. 6.

Charge selectivity of Cx43 and Cx40 junctions is unaffected by PKC activation. The dye-charge selectivity results for TPA-treated Rin43 and Rin40 cells are shown as a plot of permeance rate constants for NBD-M-TMA vs. Alexa 350 (only TPA treated shown) (A) and as the average charge selectivities (B_NBD-M-TMA/B_{Alexa 350}, treated and untreated shown) (B). For the experiments plotted in A, the parameters were significantly correlated [Cx43: r = 0.98 (n = 5); Cx40: r = 0.88 (n = 4)]. TPA had no detectable effect on selectivity of either the Rin43 or Rin40 junctions.

Fig. 7.

Charge selectivity of junctions formed by cells coexpressing Cx43 and Cx40 at different expression ratios. A: plot of B_NBD-M-TMA vs. B_{Alexa 350} for A7r5C3 () and A7r5 (•) cell junctions (dotted lines are the linear regression fits for Cx40 and Cx43 junctions as shown in Fig. 2A). Inset shows data near graph origin at expanded scaling of both axes. B: the average charge selectivities (B_NBD-M-TMA/B_{Alexa 350}) of A7r5 and A7r5-C3 junctions are significantly different, P < <0.001. *Selectivity of A7r5-C3 junctions significantly greater than A7r5 junctions (Student's t-test).

Fig. 8.

Charge selectivity of junctions formed by cells coexpressing Cx43 and Cx40 is regulated by PKC in a manner requiring the CT of Cx43. A: plot of B_NBD-M-TMA vs. B_{Alexa 350} for A7r5 (solid circle) and A7r5 cells treated with TPA (shaded triangle). Dotted lines are linear regression fits to comparable data for Cx40 and Cx43 junctions (from the data in Fig. 2A). Inset shows data near graph origin at expanded scaling of both axes. B: average charge selectivity (B_NBD-M-TMA/B_{Alexa 350}) of A7r5 cell junctions treated (shaded bars) or not (solid bars) with TPA (100 ng/ml, 15 min at 37°C followed by up to 80 min at room temperature). Selectivity of treated A7r5 cells was significantly increased, P < 0.045. C: plot of B_NBD-M-TMA vs. B_{Alexa 350} for Rin40 coexpressing Cx43tr treated (shaded circles) or not (solid circles) with TPA. Dotted lines are linear regression fits to comparable data for Cx40 and Cx43 junctions (from the data in Fig. 2A). Inset shows data near graph origin at expanded scaling of both axes. D: average charge selectivity (B_NBD-M-TMA/B_{Alexa 350}) of Cx40+Cx43tr (40+43tr) junctions treated (shaded bars) or not (solid bars) with TPA (100 ng/ml, 15 min at 37°C followed by up to 80 min at room temperature). Selectivity of treated and untreated cells was not different. *Selectivity of A7r5 cell junctions significantly increased following TPA treatment.

Fig. 9.

Variable charge selectivity and its regulation in Cx43- and Cx40-coexpressing cells. Individual charge selectivities (B_NBD-M-TMA/B_{Alexa 350}) of the differently comprised junctions studied herein treated (shaded symbols) or not (solid symbols) with TPA (as described in previous figures) and plotted on a logarithmic scale to emphasize the ∼15-fold range in junctional selectivity of coexpressing cells. Note the decreased range of selectivity values in TPA-treated A7r5 cells and absence of similar decrease in Rin40+Cx43tr cells, indicating the necessity for the CT domain for such regulation.