Elsevier

Neuroscience

Volume 162, Issue 4, 15 September 2009, Pages 1039-1054
Neuroscience

Cellular Neuroscience
Research Paper
Pannexin1 in the outer retina of the zebrafish, Danio rerio

https://doi.org/10.1016/j.neuroscience.2009.04.064Get rights and content

Abstract

In the retina, chemical and electrical synapses couple neurons into functional networks. New candidates encoding for electrical synapse proteins have recently emerged. In the present study, we determined the localization of the candidate protein pannexin1 (zfPanx1) in the zebrafish retina and studied the functional properties of zfPanx1 exogenously expressed in Neuroblastoma 2a (N2a) cells. zfPanx1 was identified on the surface of horizontal cell dendrites invaginating deeply into the cone pedicle near the glutamate release sites of the cones, providing in vivo evidence for hemichannel formation at that location. This strategic position of zfPanx1 in the photoreceptor synapse could potentially allow modulation of cone output. Using whole cell voltage clamp and excised patch recordings of transfected N2a cells, we demonstrated that zfPanx1 forms voltage-activated hemichannels with a large unitary conductance in vitro. These channels can open at physiological membrane potentials. Functional channels were not formed following mutation of a single amino acid within a conserved protein motif recently shown to be N-glycosylated in rodent Panx1. Together, these findings indicate that zfPanx1 displays properties similar to its mammalian homologues and can potentially play an important role in functions of the outer retina.

Section snippets

Animals

Zebrafish were kept at 28 °C in aerated tanks filled with tap water circulating through a bacterial filter system. The fish were kept on a 12-h light/dark cycle (12 h ON/12 h OFF). Animal experiments were carried out according to the guidelines of the German Animal Protection Law in its present version (1998) and under the responsibility of the ethical committee of the Royal Netherlands Academy of Arts and Sciences acting in accordance with the European Communities Council Directive of 24

Cloning of the zfPanx1 gene

A survey of public genome databases led to the discovery of three annotated zfPanx1-like sequences. These sequences were predicted to derive from two distinct chromosomal localizations on zebrafish chromosomes (chr) 5 and chr 15. Initial attempts to clone the two candidate open reading frames (ORF) predicted for chr 5 encoding for proteins of 402 aa (ENSDART00000016625) and 419 aa (ENSDART00000097738) size using a PCR-based cloning technique failed as did RT-PCR based expression analysis using

Discussion

The data presented here build on the previously reported cloning and expression studies demonstrating Panx1 mRNA expression in the retina of mouse, rat and fish (Ray et al 2005, Dvoriantchikova et al 2006, Zoidl et al 2008). Due to the well-established importance of electrical coupling in the retina, the focus of the present study was the putative role of Panx1 in the zebrafish retina. In this paper, we describe the cloning of zfPanx1, generated a polyclonal antibody against zfPanx1, determined

Acknowledgments

We thank Sabine Peuckert, Sabine Schreiber-Minjoli, Jeanette Willms, and Christiane Zoidl for technical assistance. Furthermore, we thank Dr. D. Krause-Finkeldey and Dr. K. Ladage for help with the antibody production. The work was sponsored by grants from the Deutsche Forschungsgemeinschaft to R.D. and G.Z. (DFG 292/11-3 and SFB 509).

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