Abstract
Abstract: :
Purpose: Previous studies of heterozygous connexin knockout mouse lenses found that coupling conductance in the outer shell of differentiating fibers (DF) was reduced when the amount of either Cx46 or Cx50 was reduced, whereas the coupling conductance in the inner core of mature fibers (MF) appeared to depend only on the presence of Cx46. These studies suggested that each connexin contributes about half of the coupling conductance in DF, whereas Cx46 appears to form all the functional channels in MF. To further test these hypotheses, we have studied coupling conductance in double heterozygous connexin knockout mouse lenses (DH), which have just one allele for Cx46 and one for Cx50. Methods: Whole lens impedance studies were used to determine gap junction coupling conductance between fiber cells; 100% CO2 was bubbled in the bath solution to evaluate pH gating. Results: Quantitative Western blotting suggested that DH expressed approximately half as much Cx46 and Cx50 protein as wild type lenses (WT). DH were not significantly different in size from WT (radii: DH = 0.101 ± 0.003 cm, n = 21; WT = 0.102 ± 0.005 cm, n = 14). In addition, DH maintained clarity comparable to that of WT. Surprisingly, when the impedance data on coupling conductance from DH were compared to WT, there were no significant differences. Based on these data, we calculated the coupling conductance per unit area of cell to cell contact in S/cm2 (GDF WT=0.85 ± 0.09, n = 19, DH = 0.76 ± 0.11, n = 13; GMF WT = 0.41 ± 0.09, n = 10, DH = 0.51 ± 0.04, n = 10). DH coupling conductance had the same pH sensitivity as WT. Conclusions: In the individual heterozygous knockout lenses, when half of either Cx46 or Cx50 was removed, the value of GDF was clearly reduced to about 70% of normal, but in DH when half of both Cx46 and Cx50 was removed, the value of GDF was indistinguishable from WT. The results in MF are even more perplexing. When half of Cx46 was removed, GMF was reduced by about half, whereas when half of Cx50 was removed, GMF did not change. However, when half of both Cx46 and Cx50 were removed in DH, there was no change in GMF. It appears that the stoichiometry of Cx49:Cx50 is more important to coupling conductance than the amount of protein.
Keywords: gap junctions/coupling • cell–cell communication • transgenics/knock–outs