Fish-specific N-terminal domain sequence in Connexin 39.4 plays an important role in zebrafish stripe formation by regulating the opening and closing of gap junctions and hemichannels.

BACKGROUND

Connexin 39.4 (Cx39.4) is involved in zebrafish (Danio rerio) skin patterning; when mutated, zebrafish exhibit a wavy stripe/labyrinth pattern instead of stripes. Cx39.4 is unique in that it has two additional serine/arginine (SR) residues, Ser2 and Arg3, at positions 2 and 3. Here, I investigated the role of these SR residues in Cx39.4 function.

METHODS

To examine the SR residues in Cx39.4, mutants of the SR residues were generated. Voltage-clamp recordings were performed using Xenopus oocytes to characterize the channel properties of the mutants. Transgenic zebrafish expressing each mutant were generated, and the effects of each mutation on fish skin patterning were evaluated.

RESULTS

The Cx39.4R3K mutant showed essentially the same properties as the wild-type (Cx39.4WT) in both electrophysiological analyses, leading to transgenic, complete phenotype rescue. Both the Cx39.4R3A mutant and deletion mutant of SR residues (Cx39.4delSR) showed a faster decay of gap junction activity and abnormal hemichannel activity, resulting in wide stripes and interstripes that indicate instability. Although the Cx39.4R3D mutant showed no channel activity in gap junctions or hemichannels, it caused unstable phenotypes in the transgene, namely a completely rescued phenotype in some individuals and loss of melanophores in others.

CONCLUSIONS

The SR residues in the NT domain of Cx39.4 are critical for the regulation of channel function, which appears to determine skin patterning.

GENERAL SIGNIFICANCE

These results elucidate the roles of the two SR residues unique to the NT domain of Cx39.4 in its channel function, which is important for zebrafish stripe pattern formation.