Biology subjects

Osei-Owusu, J.

Publications and source records attributed to Osei-Owusu, J..

3 recordsLinked to original sources

bioRxiv2025

The proton-activated chloride channel inhibits SARS-CoV-2 spike protein-mediated viral entry through the endosomal pathway.

SARS-CoV-2 binds to its obligatory receptor, angiotensin-converting enzyme 2 (ACE2) and capitalizes on decreasing endosomal acidity and cathepsin-mediated spike protein cleavage to enter cells. Endosomal acidification is driven by V-ATPase which pumps protons (H+) into the lumen. The driving force for H+ is maintained by the import of chloride (Cl-) which is mediated by intracellular CLC transporters. We have recently identified the Proton-Activated Chloride (PAC) channel as a negative regulator of endosomal acidification. PAC responds to low pH and releases Cl- from the lumen to prevent endosomal hyperacidification. However, its role in SARS-CoV-2 viral entry remains unexplored. Here, we show that overexpressing the PAC channel in ACE2 expressing HEK 293T cells markedly inhibited the SARS-CoV-2 spike-mediated viral entry. Several lines of evidence suggest that this effect was due to the suppression of the endosomal entry pathway. First, the abilities of PAC to regulate endosomal acidification and inhibit pseudoviral entry were both dependent on its endosomal localization and channel activity. Second, the inhibitory effect on viral entry was similar to the suppression mediated by E64-d, a cathepsin inhibitor, while no major additive effect for both treatments was observed. Third, this inhibition was also attenuated in cells expressing TMPRSS2, which provides the alternative entry pathway through cell surface. Importantly, PAC overexpression also inhibited the number and size of plaques formed by two live SARS-CoV-2 isolates (B.1 and Omicron XBB.1.16) in Vero E6 cells. Altogether, our data indicates that PAC plays a vital role in inhibiting SARS-CoV-2 viral entry and identifies this endosomal channel as a potential novel target against the infection of SARS-CoV-2 and other viruses, which rely on the endosomal pathway.

physiology↗

bioRxiv2022

Inhibition of the proton-activated chloride channel PAC by PIP2

Proton-Activated Chloride (PAC) channel is a ubiquitously expressed pH-sensing ion channel that regulates endosomal acidification and macropinosome shrinkage by releasing chloride from the organelle lumens. It is also found at the cell surface, where it is activated under pathological conditions related to acidosis and contributes to acid-induced cell death. However, the pharmacology of the PAC channel is poorly understood. Here, we report that phosphatidylinositol (4,5)-bisphosphate (PIP2) potently inhibits PAC channel activity. We solved the cryo-electron microscopy structure of PAC with PIP2 at pH 4.0 and identified its binding site, which, surprisingly, locates on the extracellular side of the transmembrane domain (TMD). While the overall conformation resembles the previously resolved PAC structure in the desensitized state, the TMD undergoes remodeling upon PIP2-binding. Structural and electrophysiological analyses suggest that PIP2 inhibits the PAC channel by stabilizing the channel in a desensitized-like conformation. To our knowledge, PAC is the first chloride channel reported to be inhibited by PIP2. Our findings identify PIP2 as a new pharmacological tool for the PAC channel and lay the foundation for future drug discovery targeting this channel.

biochemistry↗

bioRxiv2022

Molecular mechanism underlying desensitization of the proton-activated chloride channel PAC

Desensitization is a common property of membrane receptors, including ion channels. The newly identified proton-activated chloride (PAC) channel plays an important role in regulating the pH and size of organelles in the endocytic pathway, and is also involved in acid-induced cell death. However, how the PAC channel desensitizes is largely unknown. Here, we show by patch-clamp electrophysiological studies that PAC undergoes pH-dependent desensitization upon prolonged acid exposure. Through structure-guided and comprehensive mutagenesis, we identified several residues critical for PAC desensitization, including histidine (H) 98, glutamic acid (E) 94, and aspartic acid (D) 91 at the extracellular extension of the transmembrane helix 1 (TM1), as well as E107, D109, and E250 at the extracellular domain (ECD)-transmembrane domain (TMD) interface. Structural analysis and molecular dynamic simulations revealed extensive interactions between residues at the TM1 extension and those at the ECD-TMD interface. These interactions likely facilitate PAC desensitization by stabilizing the desensitized conformation of TM1, which undergoes a characteristic rotational movement from the resting and activated states to the desensitized state. Our studies establish a new paradigm of channel desensitization in this ubiquitously expressed ion channel and pave the way for future investigation of its relevance in cellular physiology and disease.

biophysics↗