Person: Retamal, Mauricio A.
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Retamal
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Mauricio A.
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Publication KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells(2023) Chang, Cheng-Wei; Poudyal, Naveena; Peña, Francisca; Verdugo, Daniel; Stehberg, Jimmy; Retamal, Mauricio A.Background: Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in E. coli, which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells. Methods: We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD). Results: We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC50 close to 10 μM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel. Results: These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors.Publication SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics(2023) Prieto, Juan; Lucero, Claudia; Rovegno, Maximiliano; Gómez, Gonzalo; Retamal, Mauricio A.; Orellana, JuanBackground: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels. Results: We found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP. Conclusions: We hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.