International Journal of Molecular Sciences Article Differential Action of Connexin Hemichannel and Pannexin Channel Therapeutics for Potential Treatment of Retinal Diseases Mohd N. Mat Nor 1,2, Ilva D. Rupenthal 3 , Colin R. Green 4 and Monica L. Acosta 5,* 1 School of Optometry and Vision Science, University of Auckland, Auckland 1142, New Zealand; [email protected] 2 Faculty of Medicine, Universiti Sultan Zainal Abidin, Kuala Terengganu 20400, Malaysia 3 Buchanan Ocular Therapeutics Unit, Department of Ophthalmology and New Zealand National Eye Centre, University of Auckland, Auckland 1142, New Zealand; [email protected] 4 Department of Ophthalmology and New Zealand National Eye Centre, University of Auckland, Auckland 1142, New Zealand; [email protected] 5 School of Optometry and Vision Science, New Zealand National Eye Centre, Centre for Brain Research, Brain Research New Zealand—Rangahau Roro Aotearoa, University of Auckland, Auckland 1142, New Zealand * Correspondence: [email protected]; Tel.: +64-9923-6069 Abstract: Dysregulation of retinal function in the early stages of light-induced retinal degeneration involves pannexins and connexins. These two types of proteins may contribute to channels that release ATP, leading to activation of the inflammasome pathway, spread of inflammation and reti- nal dysfunction. However, the effect of pannexin channel block alone or block of both pannexin channels and connexin hemichannels in parallel on retinal activity in vivo is unknown. in this study, the pannexin channel blocker probenecid and the connexin hemichannel blocker tonabersat were used in the light-damaged rat retina. Retinal function was evaluated using electroretinography (ERG), Citation: Mat Nor, M.N.; retinal structure was analyzed using optical coherence tomography (OCT) imaging and the tissue Rupenthal, I.D.; Green, C.R.; response to light-induced injury was assessed immunohistochemically with antibodies against glial Acosta, M.L. Differential Action of fibrillary acidic protein (GFAP), Ionized calcium binding adaptor molecule 1 (Iba-1) and Connexin43 Connexin Hemichannel and Pannexin Channel Therapeutics for Potential (Cx43). Probenecid did not further enhance the therapeutic effect of connexin hemichannel block Treatment of Retinal Diseases. Int. J. in this model, but on its own improved activity of certain inner retina neurons. The therapeutic Mol. Sci. 2021, 22, 1755. https:// benefit of blocking connexin hemichannels was further evaluated by comparing these data against doi.org/10.3390/ijms22041755 results from our previously published studies that also used the light-damaged rat retina model. The analysis showed that treatment with tonabersat alone was better than probenecid alone at Academic Editor: Georg Zoidl restoring retinal function in the light-damaged retina model. The results assist in the interpretation Received: 22 January 2021 of the differential action of connexin hemichannel and pannexin channel therapeutics for potential Accepted: 7 February 2021 treatment of retinal diseases. Published: 10 February 2021 Keywords: retina; light damage; connexin43; pannexin; tonabersat; probenecid; inflammation; elec- Publisher’s Note: MDPI stays neutral troretinogram with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Tissue inflammation involves cellular communication and the roles of connexin chan- nels in this process have been elucidated using a variety of experimental tools and animal Copyright: © 2021 by the authors. models [1,2]. Two large membrane pores have been proposed to play a role in response Licensee MDPI, Basel, Switzerland. to injury or disease—the connexin hemichannel and the pannexin channel. a connexin This article is an open access article distributed under the terms and hemichannel (or connexon) is comprised of six connexin subunits, with two connexons conditions of the Creative Commons from adjacent cells docking together to form a gap junction channel linking the cytoplasm Attribution (CC BY) license (https:// of the two cells. Most organs express different types of connexins and form heterotypic or creativecommons.org/licenses/by/ homotypic gap junction channels. Connexin43 (Cx43) is expressed in various cell types 4.0/). and contributes to normal physiology. in contrast to the gap junction, opening of Cx43 Int. J. Mol. Sci. 2021, 22, 1755. https://doi.org/10.3390/ijms22041755 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1755 2 of 19 undocked hemichannels implicates injury, extracellular cell signaling and a mechanical stimulation response in various tissues [3,4]. Prior to docking, hemichannels need to remain primarily closed or they form a large, non-specific membrane pore exposing the cell cyto- plasm to the extracellular milieu [5]. The opening of connexin hemichannels is the result of molecular and physical insults that disrupt normal homeostasis [4–6]. Pannexins share sequence homology with the gap junction proteins of invertebrates (innexins) and have a similar topological structure to connexins, but do not share sequence homology with connexins. in the body, pannexin channels are vital for cell homeostasis and long-term blockage of these pores causes dysregulated tissue function [7]. in most cases, pannexins do not form cell-cell communication channels, but are instead primarily tightly regulated membrane channels [8,9]. in the eye, Cx43 is expressed in the lens and in the glia of the retina, blood vessels and epithelial cells [10]. The distribution of connexin isoforms expressed in the retina across species is listed in detail in Table1 of Danesh-Meyer et al. [ 4]. Pannexin1 (Panx1) is expressed in the anterior part of the eye but in the retina Panx1 and Panx2 are widely distributed including ganglion cells and various cells of the inner retina [11]. a role for pannexin channels in the homeostasis of the retina has been suggested given their wide distribution in the eye [11]. In the light-damaged rat retina, a model of retinal degeneration, hemichannels formed by Cx43 in the choroid, retinal pigment epithelium (RPE) and retina significantly con- tribute to the inflammatory response [12–16]. It is now understood that a substantial component of inflammation is mediated by the opening of unopposed Cx43 hemichan- nels [2,15,17,18], involved in the initiation stages and progression of the inflammatory reaction. However, the role of pannexins in the light-damaged retina is unknown. Pan- nexin channels function release ATP and metabolites for cellular communication and for paracrine signaling [19–23]. Pannexin proteins are also upregulated in tissues with chronic inflammation [24,25], and have been proposed to enhance ATP release in the retinal and choroidal environment leading to cell death by apoptotic mechanisms [21,22,26]. Pannexin channels have been suggested as the source of retinal damage in other retinal inflammation conditions. For example, in an in vitro ischemia/reperfusion model, about a third of ATP release during ischemia was found to be pannexin-channel mediated while two thirds was connexin hemichannel-mediated, although during the reperfusion phase only connexin hemichannels were responsible for ATP release [15]. Connexin hemichannels and pannexin channels have also been linked to the pathophysiology of ischemia in other organs, includ- ing the occurrence of seizures and in diseases of the eye [4,27,28]. Given the vital role of ATP in activation of the inflammasome [29] and the mixture of evidence about the roles of both pannexin channel and connexin hemichannel opening in disrupting normal home- ostasis [30], we investigated whether blocking pannexin channel opening would enhance the previously described therapeutic effect of connexin hemichannel block. One reported pannexin channel blocker is probenecid. It is used in the treatment of chronic gout or gouty arthritis caused by increased quantities of uric acid in the blood [31,32]. in the treatment of gout, inhibition of Panx1 channels did not affect con- nexin hemichannels [31]. Probenecid inhibits Panx1 channel currents in a concentration- dependent manner and by interacting with the first extracellular loop of Panx1 [31,33]. in the plasma, the t1/2 of probenecid varies from 5 h to 8 h. Int. J. Mol. Sci. 2021, 22, 1755 3 of 19 Table 1. Summary of our published data reporting a therapeutic effect in the light-damaged rat model after intervention with Cx43 hemichannel blockers (tonabersat or Peptide5) with specific delivery methods. Intervention Delivery Animals Timeline References Systemic (intraperitoneal and oral); Follow up at 24 h, 1 week, 2 weeks, Control (vehicle) 6 [13–16] ocular (intravitreal) 3 months Ocular (two intravitreal injections, Cx43 mimetic peptide one at 2 h and the other 6 Follow up for 24 h [14] (Peptide5) immediately after 24 h light exposure) Follow up at 24 h, 1 week and Peptide5 Ocular (one intravitreal injection) 6 [16] 2 weeks Peptide5 Follow up at 24 h, 1 week and Ocular (one intravitreal injection) 6 [16] in nanoparticles 2 weeks Systemic (intraperitoneal injection Follow up at 24 h, 1 week and Tonabersat at 2 h and immediately after 24 h 6 [15] 2 weeks light exposure) Systemic (oral administration Follow up at 24 h, 1 week, 2 weeks, Tonabersat immediately before starting light 6 [13] 3 months exposure) A specific connexin hemichannel blocker is the novel benzopyran compound (–)-cis-6- acetyl-4S-(3-chloro-4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3S- ol (SB-220453), also known as