Amantadine has potential for the treatment of COVID-19 because it inhibits known and novel ion channels encoded by SARS-CoV-2

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19.

Identifying the major lactate transporter of Toxoplasma gondii tachyzoites

Toxoplasma gondii and Plasmodium falciparum parasites both extrude l-lactate, a byproduct of glycolysis. The P. falciparum Formate Nitrite Transporter, PfFNT, mediates l-lactate transport across the plasma membrane of P. falciparum parasites and has been validated as a drug target. The T. gondii genome encodes three FNTs that have been shown to transport l-lactate, and which are proposed to be the targets of several inhibitors of T. gondii proliferation. Here, we show that each of the TgFNTs localize to the T. gondii plasma membrane and are capable of transporting l-lactate across it, with TgFNT1 making the primary contribution to l-lactate transport during the disease-causing lytic cycle of the parasite. We use the Xenopus oocyte expression system to provide direct measurements of l-lactate transport via TgFNT1. We undertake a genetic analysis of the importance of the tgfnt genes for parasite proliferation, and demonstrate that all three tgfnt genes can be disrupted individually and together without affecting the lytic cycle under in vitro culture conditions. Together, our experiments identify the major lactate transporter in the disease causing stage of T. gondii, and reveal that this transporter is not required for parasite proliferation, indicating that TgFNTs are unlikely to be targets for anti-Toxoplasma drugs.

A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae)

Feeding and oviposition deterrents help phytophagous insects to identify host plants. The taste organs of phytophagous insects contain bitter gustatory receptors (GRs). To explore their function, the GRs in Plutella xylostella were analyzed. Through RNA sequencing and qPCR, we detected abundant PxylGr34 transcripts in the larval head and adult antennae. Functional analyses using the Xenopus oocyte expression system and 24 diverse phytochemicals showed that PxylGr34 is tuned to the canonical plant hormones brassinolide (BL) and 24-epibrassinolide (EBL). Electrophysiological analyses revealed that the medial sensilla styloconica of 4th instar larvae are responsive to BL and EBL. Dual-choice bioassays demonstrated that BL inhibits larval feeding and female oviposition. Knock-down of PxylGr34 by RNAi attenuates the taste responses to BL, and abolishes BL-induced feeding inhibition. These results increase our understanding of how herbivorous insects detect compounds that deter feeding and oviposition, and may be useful for designing plant hormone-based pest management strategies.

A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae)

Feeding and oviposition deterrents help phytophagous insects to identify host plants. The taste organs of phytophagous insects contain bitter gustatory receptors (GRs). To explore their function, we focused on PxylGr34, a bitter GR in Plutella xylostella (L.). We detected abundant PxylGr34 transcripts in the larval head and specific expression of PxylGr34 in the antennae of females. Analyses using the Xenopus oocyte expression system and two-electrode voltage-clamp recording showed that PxylGr34 is specifically tuned to the plant hormone brassinolide (BL) and its analog 24-epibrassinolide. Electrophysiological analyses revealed that the medial sensilla styloconica on the maxillary galea of the 4th instar larvae are responsive to BL. Dual-choice bioassays demonstrated that BL inhibits larval feeding and female ovipositing. Knock-down of PxylGr34 by RNAi abolished BL-induced feeding inhibition. These results shed light on gustatory coding mechanisms and deterrence of insect feeding and ovipositing, and may be useful for designing plant hormone-based pest management strategies.

Novel Mutations in the TMPRSS3 Gene May Contribute to Taiwanese Patients with Nonsyndromic Hearing Loss

A previous study indicated that mutations in the transmembrane protease serine 3 (TMPRSS3) gene, which encodes a transmembrane serine protease, cause nonsyndromic hearing loss (NSHL). This was the first description of a serine protease involved in hearing loss (HL). In Taiwan, however, data on the TMPRSS3 gene’s association with NSHL is still insufficient. In this study, we described 10 mutations of TMPRSS3 genes found in 14 patients after screening 230 children with NSHL. The prevalence of the TMPRSS3 mutation appeared to be 6.09% (14/230). Of the 10 mutations, three were missense mutations: c.239G>A (p.R80H), c.551T>C (p.L184S), and 1253C>T (p.A418V); three were silent mutations, and four were mutations in introns. To determine the functional importance of TMPRSS3 mutations, we constructed plasmids carrying TMPRSS3 mutations of p.R80H, p.L184S, and p.A418V. TMPRSS3 function can be examined by secretory genetic assay for site-specific proteolysis (sGASP) and Xenopus oocyte expression system. Our results showed that p.R80H, p.L184S, and p.A418V TMPRSS3 mutations gave ratios of 19.4%, 13.2%, and 27.6%, respectively, via the sGASP system. Moreover, these three TMPRSS3 mutations failed to activate the epithelial sodium channel (ENaC) in the Xenopus oocyte expression system. These results indicate that the p.R80H, p.L184S, and p.A418V missense mutations of TMPRSS3 resulted in greatly diminishing the proteolytic activity of TMPRSS3. Our study provides information for understanding the importance of TMPRSS3 in the NSHL of Taiwanese children and provides a novel molecular explanation for the role of TMPRSS3 in HL.

Cellular encoding of Cy dyes for single-molecule imaging

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.

Ca2+ permeability and Na+ conductance in cellular toxicity caused by hyperactive DEG/ENaC channels

Hyperactivated DEG/ENaC channels cause neuronal death mediated by intracellular Ca2+ overload. Mammalian ASIC1a channels and MEC-4(d) neurotoxic channels in Caenorhabditis elegans both conduct Na+ and Ca2+, raising the possibility that direct Ca2+ influx through these channels contributes to intracellular Ca2+ overload. However, we showed that the homologous C. elegans DEG/ENaC channel UNC-8(d) is not Ca2+ permeable, yet it is neurotoxic, suggesting that Na+ influx is sufficient to induce cell death. Interestingly, UNC-8(d) shows small currents due to extracellular Ca2+ block in the Xenopus oocyte expression system. Thus, MEC-4(d) and UNC-8(d) differ both in current amplitude and Ca2+ permeability. Given that these two channels show a striking difference in toxicity, we wondered how Na+ conductance vs. Ca2+ permeability contributes to cell death. To address this question, we built an UNC-8/MEC-4 chimeric channel that retains the calcium permeability of MEC-4 and characterized its properties in Xenopus oocytes. Our data support the hypothesis that for Ca2+-permeable DEG/ENaC channels, both Ca2+ permeability and Na+ conductance contribute to toxicity. However, for Ca2+-impermeable DEG/ENaCs (e.g., UNC-8), our evidence shows that constitutive Na+ conductance is sufficient to induce toxicity, and that this effect is enhanced as current amplitude increases. Our work further refines the contribution of different channel properties to cellular toxicity induced by hyperactive DEG/ENaC channels.