Mechanism of v-Src- and mitogen-activated protein kinase-induced reduction of gap junction communication

Abstract

Connexin (Cx)43 gap junction channels are phosphorylated by numerous protein kinases, with the net effect typically being a reduction in gap junction communication (GJC). This reduction must result from a decrease in channel open probability, unitary conductance, or permselectivity, because previous results suggest that channel number is unaffected. Coexpression of v-Src with wild-type Cx43 (Cx43-wt) but not Cx43 with tyrosine to phenylalanine substitutions at 247 and 265 (Cx43-Y247,265F) resulted in reduced electrical and dye coupling but no change in single-channel amplitudes. EGF treatment of cells expressing Cx43-wt but not Cx43 with serine to alanine substitutions at 255, 279, and 282 (Cx43-S255,279,282A) resulted in reduced GJC, also with no change in single-channel amplitude. Dye coupling was reduced to a far greater extent than electrical coupling, suggesting that channel selectivity was also altered but with minimal effect on unitary conductance. The absence of Src- and MAPK-induced reductions in single-channel amplitude suggests that the decreases in GJC induced by these kinases result from reduced channel open probability and possibly altered selectivity.

Fig. 1

Macroscopic junctional conductance after 24 h of plating.A: uncorrected mean macroscopic electrical conductance (g_j) measurements (filled bars) with corresponding mean series resistance (R_s) values (open bars) for wtC1 [wild-type connexin (Cx)43 (Cx43-wt); n = 9], wtS1 (Cx43-wt and v-Src; n = 13), wtS3 (Cx43-wt and v-Src; n = 12), and dbS2 [Cx 43 with Y to F substitutions at 247, 265 (Cx43-Y247,265F) and v-Src; n = 9]cell pairs. B: corrected mean g _j values for each cell type. v-Src expression was sufficient to reduce g_j in wtS1 and wtS3 cells. *Significant difference from wtC1 (P < 0.05).

Fig. 2

Single-channel records with corresponding all-points histograms (A–D) and event-frequency histograms (E–H) derived from wtC1 (A, E), wtS1 (B, F), wtS3 (C, G), and dbS2 (D, H). In all cases halothane was used to reduce junctional conductance (P_o effect) to a level at which single-channel events could be discerned. Transjunctional driving force was 40 mV, and records were notch filtered and low-pass filtered at 100 Hz (8-pole Bessel). Calibration bars in A–D correspond to 2 pA and 2 s and are located near the zero transjunctional current level. Data from multiple cell pairs similarly analyzed indicated that no significant differences in channel amplitude existed between treatment groups (see text).

Fig. 3

Single-channel records with corresponding all-points histograms (A, B) and event-frequency histograms (C, D) derived from wtC1 cells in the absence (A, C) and presence (B, D)of 100 ng/ml EGF. Records were notch filtered and low-pass filtered at 100 Hz (8-pole Bessel). Calibration bars in A and B correspond to 2 pA and2sand are located near the zero current level. Note the absence of any differences in channel amplitude in the presence vs. absence of EGF.

Fig. 4

Single-channel records with corresponding all-points histograms (A, B) and event-frequency histograms (C, D) derived from G11 cells [expressing Cx43 with S to A substitutions at 255, 279, 282 (Cx43-S255,279,282A)] in the absence (A, C) and presence (B, D) of 100 ng/ml EGF. Records were notch filtered and low-pass filtered at 100 Hz (8-pole Bessel). Calibration bars in A and B correspond to 2 pA and 2 s and are located near the zero current level. Note the absence of any differences in channel amplitude in the presence vs. absence of EGF.

Fig. 5

Extent of dye coupling (A) and expected dye coupling (B) for wtC1, wtS1, wtS3, dbS2, and wtC1-EGF-treated cells. A: cells were injected with Lucifer yellow (filled bars), [2-(4-nitro-2,1,3-benzoxadiol-7-yl)aminoethyl]trimethylammonium (NBD-TMA; gray bars), or NBD-TMA after preincubation with 10 µM PP2 media (open bars). wtS1, wtS3, and wtC1+EGF cells displayed a significant decrease in GJC compared with wtC1 cells. GJC was significantly improved in wtS1 and wtS3 cells after incubation in PP2. The Cx43-Y247,265F mutations in dbS2 cells prevented v-Src-induced dye uncoupling between these cells. *Significant difference from wtC1; † significant difference between Lucifer yellow and NBD-TMA treatments; ‡ significant difference between NBD-TMA and NBD-TMA + PP2 treatments (P < 0.05). B: filled bars represent the expected level of coupling assuming that NBD-TMA dye coupling decreased in parallel with the decrease in junctional conductance (electrical coupling) observed in Fig. 1. Hatched bars represent the observed level of NBD-TMA coupling reproduced from A. Dark gray and light gray bars represent the expected levels of coupling assuming that Lucifer yellow coupling decreased in parallel with the decrease in junctional conductance (dark gray) or NBD-TMA coupling (light gray). Hatched gray bar is the observed level of Lucifer yellow coupling reproduced from A. In all cases, the observed levels of coupling were considerably lower than the expected levels of coupling, regardless of the predictor used.