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The Ca 2+ -activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca 2+ regulates TRPM5 activity remains elusive. We report cryo-EM structures of the zebrafish TRPM5 in an apo closed state, a Ca 2+ -bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca 2+ site (Ca ICD ) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The Ca ICD site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca 2+ binding to the Ca TMD site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca 2+ sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1–S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents. Cryo-EM structures of zebrafish TRPM5 reveal closed and Ca 2+ -bound open states, a unique Ca 2+ binding site that modulates voltage sensitivity and the mechanism of antagonist action.

Tuft cells are solitary chemosensory epithelial cells that can sense lumenal stimuli at mucosal barriers and secrete effector molecules to regulate the physiology and immune state of their surrounding tissue. In the small intestine, tuft cells detect parasitic worms (helminths) and microbe-derived succinate, and signal to immune cells to trigger a Type 2 immune response that leads to extensive epithelial remodeling spanning several days. Acetylcholine (ACh) from airway tuft cells has been shown to stimulate acute changes in breathing and mucocilliary clearance, but its function in the intestine is unknown. Here we show that tuft cell chemosensing in the intestine leads to release of ACh, but that this does not contribute to immune cell activation or associated tissue remodeling. Instead, tuft cell-derived ACh triggers immediate fluid secretion from neighboring epithelial cells into the intestinal lumen. This tuft cell-regulated fluid secretion is amplified during Type 2 inflammation, and helminth clearance is delayed in mice lacking tuft cell ACh. The coupling of the chemosensory function of tuft cells with fluid secretion creates an epithelium-intrinsic response unit that effects a physiological change within seconds of activation. This response mechanism is shared by tuft cells across tissues, and serves to regulate the epithelial secretion that is both a hallmark of Type 2 immunity and an essential component of homeostatic maintenance at mucosal barriers.

In summary, a concise method for the preparation of diverse α-amino alcohols and α-amino acids in both enantiomeric series from readily available achiral starting materials has been developed. Included in the analogs is 4-methoxyhomophenylalanine, a compound of current interest. The precursor vinyl amino alcohols, and vinyl oxazolidines were obtained via lipase-catalyzed kinetic resolutions. Key in this convergent approach was Pd mediated Suzuki cross-coupling. The vinyloxazolidines which were exploited in chapter 1 in the synthesis of amino acids were cross-coupled under Suzuki conditions to an advanced intermediate of cis-dihydrobromodiol. Through a series of transformations a variety of manno-azasugar amino acids and manno -azasugar amino alcohols were synthesized. Additionally a key advanced intermediate was transformed into a highly elaborated quinolizidine. The chiral building blocks, vinylglycinols were additionally utilized for the synthesis of derivatives of trans-4-hydroxypipecolic acids. Key transformations include ring closing metathesis to construct the piperidine ring and the Prevost reaction to install the 4-hydroxy regio- and stereoselectively as determined by X-ray structure analysis. A key intermediate was used to synthesize the hydrochloride of the natural product (−)-SS20846A. N-Tosyl-N-benzylvinylglycinol was utilized in the synthesis of advanced intermediates which constituted formal syntheses of the natural products 1-Deoxymannojirimycin and (+)-swainsonine.

The Ca2+-activated TRPM5 channel plays an essential role in the perception of sweet, bitter, and umami stimuli in type II taste cells and in insulin secretion by pancreatic beta cells1–3. Interestingly, the voltage dependence of TRPM5 in taste bud cells depends on the intracellular Ca2+ concentration4, yet the mechanism remains elusive. Here we report cryo-electron microscopy structures of the zebrafish TRPM5 in an apo closed state, a Ca2+-bound open state, and an antagonist-bound inhibited state, at resolutions up to 2.3 Å. We defined two novel ligand binding sites: a Ca2+ binding site (CaICD) in the intracellular domain (ICD), and an antagonist binding site in the transmembrane domain (TMD) for a drug (NDNA) that regulates insulin and GLP-1 release5. The CaICD site is unique to TRPM5 and has two roles: shifting the voltage dependence toward negative membrane potential, and promoting Ca2+ binding to the CaTMD site that is conserved throughout Ca2+-sensitive TRPM channels6. Replacing glutamate 337 in the CaICD site with an alanine not only abolished Ca2+ binding to CaICD but also reduced Ca2+ binding affinity to CaTMD, suggesting a cooperativity between the two sites. We have defined mechanisms underlying channel activation and inhibition. Conformational changes initialized from both Ca2+ sites, 70 Å apart, are propagated to the ICD–TMD interface and cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the TMD in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents to treat metabolic disorders. Competing Interest Statement The authors have declared no competing interest.

Histone Deacetylase 1 (HDAC1) removes acetyl groups from lysine residues on the core histones, a critical step in the regulation of chromatin accessibility. Despite histone deacetylation being an apparently repressive activity, suppression of HDACs causes both up- and down-regulation of gene expression. Here we exploited the degradation tag (dTAG) system to rapidly degrade HDAC1 in embryonic stem cells (ESCs) lacking its paralog, HDAC2. Unlike HDAC inhibitors that lack isoform specificity, the dTAG system allowed specific degradation and removal of HDAC1 in <1 hour (100x faster than genetic knockouts). This rapid degradation caused increased histone acetylation in as little as 2 hours, with H2BK5 and H2BK11 being the most sensitive. The majority of differentially expressed genes following 2 hours of HDAC1 degradation were upregulated (275 genes up vs 15 down) with increased proportions of downregulated genes observed at 6 (1,153 up vs 443 down) and 24 hours (1,146 up vs 967 down) respectively. Upregulated genes showed increased H2BK5ac and H3K27ac around their transcriptional start site (TSS). In contrast, decreased acetylation of super-enhancers (SEs) was linked to the most strongly downregulated genes. These findings suggest a paradoxical role for HDAC1 in the maintenance of histone acetylation levels at critical enhancer regions required for the pluripotency-associated gene network.