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

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.

Pannexin 1 activation and inhibition is permeant‐selective

“…A plethora of membrane channels are reportedly regulated by pH changes and emerging evidence has suggested that Panx1 might be part of this group (Kurtenbach et al . ). Uninjected and Panx1‐expressing oocytes were exposed to a 1 unit pH increase (to pH 8.4) or decrease (to pH 6.4) to determine whether a pH change alters Panx1‐mediated ion conductance and ethidium uptake (Fig.…”

“…We detected a slight reduction in Panx1‐mediated conductance at alkaline pH and a larger pH‐dependent effect on ethidium uptake (increase with alkalization and decrease with acidification), the latter in accordance with a previous report (Kurtenbach et al . ). These results underscore the permeant‐selective states of Panx1 and specifically illustrate an activation regime that predominantly targets the ethidium flux.…”

“…A plethora of membrane channels are reportedly regulated by pH changes and emerging evidence has suggested that Panx1 might be part of this group (Kurtenbach et al 2013). Uninjected and Panx1-expressing oocytes were exposed to a 1 unit pH increase (to pH 8.4) or decrease , and mPanx1-expressing oocytes (B) exposed to control solution, high extracellular K + and high extracellular K + containing 200 µM CBX (each for 2 min), summarized for membrane potential of +60 mV in panel C (n = 18, n = 15, and n = 10, respectively).…”

Extracellular ATP Hydrolysis Inhibits Synaptic Transmission by Increasing pH Buffering in the Synaptic Cleft

“…ATP blocked the slow component of feedback, indicating the slow component depends on ATP. Note this concentration of ATP does not block Panx1 channels [20]. (C) Normalized mean feedback responses in seven cones in control (black) and in 50 µM ARL67156, a blocker of NTPDase (red).…”

Structural and Functional Consequences of Connexin 36 (Cx36) Interaction with Calmodulin

“…This assay is a variation of the ethidium bromide uptake assay previously reported (Prochnow et al, 2009; Kurtenbach et al, 2013). Transfected Neuro2a cells grown for 48 h in 3.5 cm MatTek cell culture dishes were incubated with 10 μM of ethidium bromide in complete growth medium for 10 min at 37°C and 5% CO 2.…”