Ion channels are the second largest class of drug targets after G protein-coupled receptors. In addition to well-recognized ones like voltage-gated Na/K/Ca channels in the heart and neurons, novel ion channels are continuously discovered in both excitable and non-excitable cells and demonstrated to play important roles in many physiological processes and diseases such as developmental disorders, neurodegenerative diseases, and cancer. However, in the field of ion channel discovery, there are an unignorable number of published studies that are unsolid and misleading. Despite being the gold standard of a functional assay for ion channels, electrophysiological recordings are often accompanied by electrical noise, leak conductance, and background currents of the membrane system. These unwanted signals, if not treated properly, lead to the mischaracterization of proteins with seemingly unusual ion-conducting properties. In the recent ten years, the technical revolution of cryo-electron microscopy (cryo-EM) has greatly advanced our understanding of the structures and gating mechanisms of various ion channels and also raised concerns about the pore-forming ability of some previously identified channel proteins. In this review, we summarize cryo-EM findings on ion channels with molecular identities recognized or disputed in recent ten years and discuss current knowledge of proposed channel proteins awaiting cryo-EM analyses. We also present a classification of ion channels according to their architectures and evolutionary relationships and discuss the possibility and strategy of identifying more ion channels by analyzing structures of transmembrane proteins of unknown function. We propose that cross-validation by electrophysiological and structural analyses should be essentially required for determining molecular identities of novel ion channels.

Morinda officinalis saponins (MOS), a traditional Chinese medicine extracted from M. officinalis roots, have been used as a health supplement. Existing evidence suggests that extracts from this plant can be used for osteoporosis treatment. However, the molecular mechanisms underlying the anti-osteoporotic effects of M. officinalis remain poorly understood. In this study, we investigated the osteogenesis-promoting effects of MOS on human umbilical cord-derived mesenchymal stem cells (HUC-MSCs). Alkaline phosphatase staining, alizarin red staining, and quantitative reverse transcription-PCR demonstrated that MOS promoted the osteogenic differentiation of HUC-MSCs in a concentration-dependent manner. RNA sequencing results showed that the expression of key osteogenic differentiation-related genes, including BMP4, as well as the activity of transforming growth factor-β and calcium signaling pathways increased following MOS treatment. Furthermore, treatment with the bone morphogenetic protein (BMP) antagonist Noggin reversed the MOS-induced pro-osteogenic differentiation effects and the upregulation of osteoblast-specific markers. Overall, the results indicate that MOS can partially promote osteogenic differentiation of HUC-MSCs by regulating the BMP-SMAD signaling pathway. These findings indicate the potential utility of MOS as a therapeutic agent for osteoporosis, particularly in the context of stem cell therapy.

Preventing the spread of SARS-CoV-2 and its variants is crucial in the fight against COVID-19. Inhibition of the main protease (M^pro) of SARS-CoV-2 is the key to disrupting viral replication, making M^pro a promising target for therapy. PF-07321332 and shikonin have been identified as effective broad-spectrum inhibitors of SARS-CoV-2 M^pro. The crystal structures of SARS-CoV-2 M^pro bound to PF-07321332 and shikonin have been resolved in previous studies. However, the exact mechanism regarding how SARS-CoV-2 M^pro mutants impact their binding modes largely remains to be investigated. In this study, we expressed a SARS-CoV-2 M^pro mutant, carrying the D48N substitution, representing a class of mutations located near the active sites of M^pro. The crystal structures of M^pro D48N in complex with PF-07321332 and shikonin were solved. A detailed analysis of the interactions between M^pro D48N and two inhibitors provides key insights into the binding pattern and its structural determinants. Further, the binding patterns of the two inhibitors to M^pro D48N mutant and wild-type M^pro were compared in detail. This study illustrates the possible conformational changes when the M^pro D48N mutant is bound to inhibitors. Structural insights derived from this study will inform the development of new drugs against novel coronaviruses.