@article {70302, title = {Phenotypic CRISPR screens identify NLRX1 as an essential activator of the human mitochondrial permeability transition}, journal = {Proceedings of the National Academy of Sciences}, volume = {123}, year = {2026}, month = {2026 Feb 25}, pages = {e2535298123}, abstract = {

Mitochondria utilize calcium to increase ATP synthesis. However, excessive matrix calcium activates the mitochondrial permeability transition (mPT), a process that permeabilizes the mitochondrial inner membrane and leads to cell death. While initially characterized 50 y ago, the proteins underlying the process are unclear, although integral membrane proteins were expected to be the porous entities during calcium overload. Here, we designed two assays to study the mPT using high-throughput methodologies. By surveying 19,113 proteins in human cells, we identified four proteins that sensitize the human mPT, but only one that was essential for mPT activation, mitochondrial-localized NRLX1. Surprisingly, NLRX1 is not an integral membrane protein, and our work did not identify any essential integral membrane proteins for the human mPT. The mitochondrial permeability transition (mPT) is an evolutionarily conserved destructive process that permeabilizes the inner mitochondrial membrane in response to calcium overload. The molecular mechanism underlying the mPT is not established. To unambiguously identify essential proteins, we designed two phenotypic assays for mitochondrial calcium overload and applied them to FACS-based CRISPR screening in human cells, ultimately evaluating 19,113 genes. The first screen studied mitochondrial membrane potential (MMP) collapse in response to calcium overload. Top-ranked genes were the essential proteins of the mitochondrial calcium uniporter complex, MCU and EMRE, reflecting that the calcium-induced MMP collapse results from mitochondrial calcium entry and not the mPT. The second screen measured the permeability of the inner mitochondrial membrane. Here, the fluorescent interaction of a membrane impermeant 600 Da dye and a mitochondrial-targeted HaloTag protein was studied under mPT activating conditions; calcium overload and the thiol-reactive molecule phenylarsine oxide. With secondary validation, we identified four protein-encoding genes that delayed or prevented the mPT under knockout: NF2, REST, BPTF, and NRLX1. Knockout of the nonmitochondrial proteins BPTF, NF2, or REST increased mitochondrial calcium retention capacity (CRC). However, calcium release or sensitivity to cyclosporin A (CsA) persisted, indicative of mPT sensitizers. Only knockout of the mitochondrial matrix protein, NLRX1, increased CRC, abolished calcium release, and was CsA-insensitive. This top-ranked hit of the mitochondrial permeability screen meets the definition of an essential mPT activator. Integral membrane proteins, including all previously proposed mPT candidates, were not essential activators.

}, doi = {10.1073/pnas.2535298123}, url = {https://www.pnas.org/doi/abs/10.1073/pnas.2535298123}, author = {William C. Valinsky and Robert P. Ray and Kathy S. Schaefer and Jonathan B. Grimm and Carla Nicolini and Luke D. Lavis and David E. Clapham} } @article {68316, title = {The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins}, journal = {eLife}, year = {2023}, month = {2023 Jan 25}, abstract = {

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

}, doi = {10.7554/eLife.84477}, url = {https://doi.org/10.7554/eLife.84477}, author = {Alexandria N. Miller and Patrick R. Houlihan and Ella Matamala and Deny Cabezas-Bratesco and Gi Young Lee and Ben Cristofori-Armstrong and Tanya L. Dilan and Silvia Sanchez-Martinez and Doreen Matthies and Rui Yan and Zhiheng Yu and Dejian Ren and Sebastian E. Brauchi and David E. Clapham} } @article {67810, title = {A serotonergic axon-cilium synapse drives nuclear signaling to maintain chromatin accessibility}, journal = {Cell}, volume = {185}, year = {2022}, month = {2022 Sep 01}, pages = {3390-3407}, abstract = {

Chemical synapses between axons and dendrites mediate much of the brain{\textquoteright}s intercellular communication. Here we describe a new kind of synapse {\textendash} the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam {\textendash} scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

}, doi = {10.1016/j.cell.2022.07.026}, author = {Shu-Hsien Sheu and Srigokul Upadhyayula and Vincent Dupuy and Song Pang and Andrew L. Lemire and Deepika Walpita and H. Amalia Pasolli and Fei Deng and Jinxia Wan and Lihua Wang and Justin Houser and Silvia Sanchez-Martinez and Sebastian E. Brauchi and Sambashiva Banala and Melanie Freeman and C. Shan Xu and Tom Kirchhausen and Harald F. Hess and Luke Lavis and Yu-Long Li and S{\'e}verine Chaumont-Dubel and David E. Clapham} } @article {65000, title = {Cryo-EM structure of the receptor-activated TRPC5 ion channel at 2.9 angstrom resolution.}, year = {2018}, month = {2018 Nov 11}, abstract = {

The transient receptor potential canonical subfamily member 5 (TRPC5) is a non-selective calcium-permeant cation channel. As a depolarizing channel, its function is studied in the central nervous system and kidney. TRPC5 forms heteromultimers with TRPC1, but also forms homomultimers. It can be activated by reducing agents through reduction of the extracellular disulfide bond. Here we present the 2.9 {\r A} resolution electron cryo-microscopy (cryo-EM) structure of TRPC5. The structure of TRPC5 in its apo state is partially open, which may be related to the weak activation of TRPC5 in response to extracellular pH. We also report the conserved negatively charged residues of the cation binding site located in the hydrophilic pocket between S2 and S3. Comparison of the TRPC5 structure to previously determined structures of other TRPC and TRP channels reveals differences in the extracellular pore domain and in the length of the S3 helix. Together, these results shed light on the structural features that contribute to the specific activation mechanism of the receptor-activated TRPC5.

}, doi = {10.1101/467969}, author = {Jingjing Duan and Jian Li and Gui-Lan Chen and Bo Zeng and Kechen Xie and Xiaogang Peng and Wei Zhou and Jianing Zhong and Yixing Zhang and Jie Xu and Changhu Xue and Lan Zhu and Wei Liu and Xiao-Li Tian and Jianbin Wang and David E. Clapham and Zongli Li and Jin Zhang} }