The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs shared a dimeric stoichiometry following detergent solubilization, their structures revealed divergence in their relative subunit orientation. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrate that mTTYH1 and mTTYH3 form tetramers at the plasma membrane, stabilized by interactions between their extracellular domains. Using blue-native PAGE, fluorescence-detection size-exclusion chromatography, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we reveal that detergent solubilization results in tetramers destabilization, leading to their dissolution into dimers. Moreover, HDX-MS demonstrates that the extracellular domains are stabilized in the context of the tetrameric mTTYH complex. Together, our results expose the innate tetrameric organization of TTYH complexes at the cell membrane. Future structural analyses of these assemblies in native membranes are required to illuminate their long-sought cellular function.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 128 43 Prague 2 Czech Republic

Department of Physiology and Pharmacology Sackler Faculty of Medicine Tel Aviv University Tel Aviv 6997801 Israel

Institute of Microbiology of the Czech Academy of Sciences Division BioCeV Prumyslova 595 252 50 Vestec Czech Republic

Sagol School of Neuroscience Tel Aviv University Tel Aviv 6997801 Israel

Tel Aviv Sourasky Medical Center Tel Aviv 6423906 Israel

Zobrazit více v PubMed

Campbell HD, et al. The Drosophila melanogaster flightless-I gene involved in gastrulation and muscle degeneration encodes gelsolin-like and leucine-rich repeat domains and is conserved in Caenorhabditis elegans and humans. Proc. Natl Acad. Sci. USA. 1993;90:11386–11390. doi: 10.1073/pnas.90.23.11386. PubMed DOI PMC

Maleszka R, De Couet HG, Gabor Miklos GL. Data transferability from model organisms to human beings: Insights from the functional genomics of the flightless region of Drosophila. Proc. Natl Acad. Sci. USA. 1998;95:3731–3736. doi: 10.1073/pnas.95.7.3731. PubMed DOI PMC

Campbell HD, et al. Human and mouse homologues of the Drosophila melanogaster tweety (tty) gene: A novel gene family encoding predicted transmembrane proteins. Genomics. 2000;68:89–92. doi: 10.1006/geno.2000.6259. PubMed DOI

Rae FK, et al. TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is located on 17q24 and upregulated in renal cell carcinoma. Genomics. 2001;77:200–207. doi: 10.1006/geno.2001.6629. PubMed DOI

Nagase T, et al. Prediction of the coding sequences of unidentified human genes. XIX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res. 2000;7:347–355. doi: 10.1093/dnares/7.6.347. PubMed DOI

Suzuki M, Mizuno A. A novel human Cl− channel family related to Drosophila flightless locus. J. Biol. Chem. 2004;279:22461–22468. doi: 10.1074/jbc.M313813200. PubMed DOI

Matthews CA, et al. Expression and evolution of the mammalian brain gene Ttyh1. J. Neurochem. 2007;100:693–707. doi: 10.1111/j.1471-4159.2006.04237.x. PubMed DOI

Uhlén M, et al. Tissue-based map of the human proteome. Science. 2015;347:1260419. doi: 10.1126/science.1260419. PubMed DOI

Han YE, et al. Tweety-homolog (Ttyh) family encodes the pore-forming subunits of the swelling-dependent volume-regulated anion channel (VRACswell) in the brain. Exp. Neurobiol. 2019;28:183–215. doi: 10.5607/en.2019.28.2.183. PubMed DOI PMC

Suzuki M, Morita T, Iwamoto T. Diversity of Cl− channels. Cell. Mol. Life Sci. 2006;63:12–24. doi: 10.1007/s00018-005-5336-4. PubMed DOI PMC

Hussy N. Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons. J. Neurophysiol. 1992;68:2042–2050. doi: 10.1152/jn.1992.68.6.2042. PubMed DOI

Fahmi M, et al. Recording of a large-conductance in normal rat lactotrophs chloride channel. Signals. 1995;269:E969–E976. PubMed

Suzuki M. The Drosophila tweety family: molecular candidates for large-conductance Ca2+-activated Cl− channels. Exp. Physiol. 2006;91:141–147. doi: 10.1113/expphysiol.2005.031773. PubMed DOI

Sukalskaia A, Straub MS, Deneka D, Sawicka M, Dutzler R. Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions. Nat. Commun. 2021;12:1–12. doi: 10.1038/s41467-021-25106-4. PubMed DOI PMC

Li B, Hoel CM, Brohawn SG. Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization. Nat. Commun. 2021;12:1–9. doi: 10.1038/s41467-020-20314-w. PubMed DOI PMC

Sabirov RZ, Okada Y. The maxi-anion channel: a classical channel playing novel roles through an unidentified molecular entity. J. Physiol. Sci. 2009;59:3–21. doi: 10.1007/s12576-008-0008-4. PubMed DOI PMC

Nalamalapu, R. R., Yue, M., Stone, A. R., Murphy, S. & Saha, M. S. The tweety gene family: from embryo to disease. Front. Mol. Neurosci. 14, 672511 (2021). PubMed PMC

Wiernasz E, et al. Ttyh1 protein is expressed in glia in vitro and shows elevated expression in activated astrocytes following status epilepticus. Neurochem. Res. 2014;39:2516–2526. doi: 10.1007/s11064-014-1455-3. PubMed DOI PMC

Jung E, et al. Tweety-homolog 1 drives brain colonization of gliomas. J. Neurosci. 2017;37:6837–6850. doi: 10.1523/JNEUROSCI.3532-16.2017. PubMed DOI PMC

Kleinman CL, et al. Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR. Nat. Genet. 2014;46:39–44. doi: 10.1038/ng.2849. PubMed DOI

Chorev DS, Robinson CV. The importance of the membrane for biophysical measurements. Nat. Chem. Biol. 2020;16:1285–1292. doi: 10.1038/s41589-020-0574-1. PubMed DOI PMC

Ulbrich MH, Isacoff EY. Subunit counting in membrane-bound proteins. Nat. Methods. 2007;4:319–321. doi: 10.1038/nmeth1024. PubMed DOI PMC

Kawate T, Gouaux E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure. 2006;14:673–681. doi: 10.1016/j.str.2006.01.013. PubMed DOI

Lee S-Y, Letts JA, Mackinnon R. Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1. Proc. Natl Acad. Sci. USA. 2008;105:7692–7695. doi: 10.1073/pnas.0803277105. PubMed DOI PMC

Clatot J, et al. Voltage-gated sodium channels assemble and gate as dimers. Nat. Commun. 2017;8:2077. doi: 10.1038/s41467-017-02262-0. PubMed DOI PMC

Whicher JR, MacKinnon R. Structure of the voltage-gated K+ channel Eag1 reveals an alternative voltage sensing mechanism. Science. 2016;353:664–669. doi: 10.1126/science.aaf8070. PubMed DOI PMC

Siebert AP, et al. Structural and functional similarities of calcium homeostasis modulator 1 (CALHM1) ion channel with connexins, pannexins, and innexins. J. Biol. Chem. 2013;288:6140–6153. doi: 10.1074/jbc.M112.409789. PubMed DOI PMC

Eubel H, Braun HP, Millar AH. Blue-native PAGE in plants: a tool in analysis of protein-protein interactions. Plant Methods. 2005;1:1–13. doi: 10.1186/1746-4811-1-11. PubMed DOI PMC

Hattori M, Hibbs RE, Gouaux E. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure. 2012;20:1293–1299. doi: 10.1016/j.str.2012.06.009. PubMed DOI PMC

Konermann L, Pan J, Liu YH. Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem. Soc. Rev. 2011;40:1224–1234. doi: 10.1039/C0CS00113A. PubMed DOI

Jumper J, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Thorsen TSS, et al. Modified T4 lysozyme fusion proteins facilitate G protein-coupled receptor crystallogenesis. Structure. 2014;22:1657–1664. doi: 10.1016/j.str.2014.08.022. PubMed DOI PMC

He Y, et al. N-glycosylation analysis of the human tweety family of putative chloride ion channels supports a penta-spanning membrane arrangement: impact of N-glycosylation on cellular processing of tweety homologue 2 (TTYH2) Biochem. J. 2008;412:45–55. doi: 10.1042/BJ20071722. PubMed DOI

Stefaniuk M, Swiech L, Dzwonek J, Lukasiuk K. Expression of Ttyh1, a member of the tweety family in neurons in vitro and in vivo and its potential role in brain pathology. J. Neurochem. 2010;115:1183–1194. doi: 10.1111/j.1471-4159.2010.07023.x. PubMed DOI

Borroto-Escuela DO, Fuxe K. Oligomeric receptor complexes and their allosteric receptor-receptor interactions in the plasma membrane represent a new biological principle for integration of signals in the CNS. Front. Mol. Neurosci. 2019;12:230. doi: 10.3389/fnmol.2019.00230. PubMed DOI PMC

Isom LL, De Jongh KS, Catterall WA. Auxiliary subunits of voltage-gated ion channels. Neuron. 1994;12:1183–1194. doi: 10.1016/0896-6273(94)90436-7. PubMed DOI

Schmidt N, et al. Neuroplastin and basigin are essential auxiliary subunits of plasma membrane Ca2+-ATPases and key regulators of Ca2+ clearance. Neuron. 2017;96:827–838.e9. doi: 10.1016/j.neuron.2017.09.038. PubMed DOI

Schwenk J, et al. Native GABAB receptors are heteromultimers with a family of auxiliary subunits. Nature. 2010;465:231–235. doi: 10.1038/nature08964. PubMed DOI

He Y, et al. The ubiquitin-protein ligase Nedd4-2 differentially interacts with and regulates members of the Tweety family of chloride ion channels. J. Biol. Chem. 2008;283:24000–24010. doi: 10.1074/jbc.M803361200. PubMed DOI PMC

Salussolia CL, et al. A eukaryotic specific transmembrane segment is required for tetramerization in AMPA receptors. J. Neurosci. 2013;33:9840–9845. doi: 10.1523/JNEUROSCI.2626-12.2013. PubMed DOI PMC

Mayer ML. Structure and mechanism of glutamate receptor ion channel assembly, activation and modulation. Curr. Opin. Neurobiol. 2011;21:283–290. doi: 10.1016/j.conb.2011.02.001. PubMed DOI PMC

Gong X, et al. Structural basis for the recognition of Sonic Hedgehog by human Patched1. Science. 2018;361:eaas8935. doi: 10.1126/science.aas8935. PubMed DOI

Zhang Y, et al. Structural basis for cholesterol transport-like activity of the hedgehog receptor patched. Cell. 2018;175:1352–1364.e14. doi: 10.1016/j.cell.2018.10.026. PubMed DOI PMC

Qian, H. et al. Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm. Nat. Commun. 10, 2320 (2019). PubMed PMC

Cirri E, et al. Consensus designs and thermal stability determinants of a human glutamate transporter. Elife. 2018;7:e40110. doi: 10.7554/eLife.40110. PubMed DOI PMC

Yu X, et al. Dimeric structure of the uracil:proton symporter UraA provides mechanistic insights into the SLC4/23/26 transporters. Cell Res. 2017;27:1020–1033. doi: 10.1038/cr.2017.83. PubMed DOI PMC

Lu F, et al. Structure and mechanism of the uracil transporter UraA. Nature. 2011;472:243–247. doi: 10.1038/nature09885. PubMed DOI

Gibson DG, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods. 2009;6:343–345. doi: 10.1038/nmeth.1318. PubMed DOI

Trcka F, et al. Human stress-inducible Hsp70 has a high propensity to form ATP-dependent antiparallel dimers that are differentially regulated by cochaperone binding. Mol. Cell. Proteom. 2019;18:320–337. doi: 10.1074/mcp.RA118.001044. PubMed DOI PMC

Kavan D, Man P. MSTools - Web based application for visualization and presentation of HXMS data. Int. J. Mass Spectrom. 2011;302:53–58. doi: 10.1016/j.ijms.2010.07.030. DOI

Ferofontov A, Vankova P, Man P, Giladi M, Haitin Y. Conserved cysteine dioxidation enhances membrane interaction of human Cl− intracellular channel 5. FASEB J. 2020;34:9925–9940. doi: 10.1096/fj.202000399R. PubMed DOI

Zhang Z, Smith DL. Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation. Protein Sci. 1993;2:522–531. doi: 10.1002/pro.5560020404. PubMed DOI PMC

Emerging roles of prominin-1 (CD133) in the dynamics of plasma membrane architecture and cell signaling pathways in health and disease