Fig. 4

Visualization and characterization of Cx39.4. (A) Schematic of EGFP-tagged Cx39.4. Cx39.4 C-terminal sequence from amino acids 256 to 321 was exchanged with the EGFP fragment. (B) Cx39.4exc256-321EGFP was expressed in melanophores of cx39.4−/− mutant zebrafish. The mutant phenotype was restored to the stripe pattern after expression. (C) Fluorescence image of transgenic fish skin at F0 generation in cx39.4−/− mutant zebrafish; white arrowhead indicates gap junction plaque between melanophores. (D) Control: EGFP was expressed in melanophores of cx39.4−/− mutant zebrafish; no EGFP signal was detected between melanophores. (E,E′) Transfected N2a cells: fluorescence (E) and bright-field (E′) microscopy images; white arrowhead indicates a gap junction plaque. (F) Schematic of patch-clamp experiment with spermidine (spd) treatment. (G) Both N2a cells were clamped at −40 mV and a series of transjunctional voltages (Vi, −140 to +60 mV, 20 mV increment) was applied to one of the cells (first trace), and transjunctional current (Ij) was recorded in the other cell with or without spermidine treatment (third and fourth traces). As a negative control, untransfected cells were used (second trace). (H,I) The polyamine metabolic enzyme Ssat (Sat1b) was ectopically expressed in melanophores (H) and xanthophores (I). (J) I-V plot showing the relationship between normalized steady-state junctional currents and transjunctional voltages for 0 mM, 15 mM and 30 mM spermidine treatment. Each data point represents the mean±s.d. for each Vj value (n=5). Vj=Vpre–Vpost (−40 mV). Scale bars: 10 mm in B,H,I; 100 µm in C,D; 20 µm in E,E′.