Pannexin1 Channel Proteins in the Zebrafish Retina Have Shared and Unique Properties

Sarah Kurtenbach,Jan Klooster,Nora Prochnow,Maarten Kamermans,Christiane Zoidl,Stefan Kurtenbach,Georg Zoidl,Rolf Dermietzel,Peter Hitchcock

doi:10.1371/journal.pone.0077722

Sarah Kurtenbach, Jan Klooster + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0077722

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Abstract

In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.

Highlights

Pannexin1 (Panx1) shares topological similarities with gap junction forming connexins (Cx), and functions as a largeconductance channel
We demonstrate pH dependent Panx1 channel modulation, which is a novel form of Panx1 modulation. drPanx1b differs from drPanx1a and mouse Panx1 (mPanx1) due to their more complex glycosylation putatively involving three N-glycosylation sites and by different electrophysiological gating kinetics
A phylogenetic tree was calculated including all pannexin sequences, selected innexin, connexin and LRRC8 sequences, the latter being a novel class of putative channel proteins sharing similarities to pannexins (Figure 1; for full tree containing bootstrap values see Figure S1)

Summary

Introduction

Pannexin (Panx1) shares topological similarities with gap junction forming connexins (Cx), and functions as a largeconductance channel (for review see [1]). In the murine visual system, Panx is expressed in the inner and outer retinae. We identified an ortholog of mammalian Panx in zebrafish (zfPanx1/ drPanx1a) and described its expression in the fish retina [9,10,11]. Initial analysis of the distribution of the two drPanx1s revealed that the drpanx1a gene seems to be expressed ubiquitously, similar to the mammalian Panx, whereas drpanx1b shows a highly specific expression pattern in the brain. Both genes are expressed in equal amounts in the retina

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