{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T10:12:04Z","timestamp":1782814324748,"version":"3.54.5"},"reference-count":27,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2020,7,24]],"date-time":"2020-07-24T00:00:00Z","timestamp":1595548800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2020,7,24]],"date-time":"2020-07-24T00:00:00Z","timestamp":1595548800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100004411","name":"Heart and Stroke Foundation of Canada","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100004411","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Invertebrate LCa<jats:sub>V<\/jats:sub>3 shares the quintessential features of vertebrate Ca<jats:sub>V<\/jats:sub>3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca<jats:sup>2+<\/jats:sup> channels, the Ca<jats:sub>V<\/jats:sub>1 and Ca<jats:sub>V<\/jats:sub>2 channels. Unlike the vertebrates though, Ca<jats:sub>V<\/jats:sub>3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCa<jats:sub>V<\/jats:sub>3 channel into a higher Na<jats:sup>+<\/jats:sup> and lower Ca<jats:sup>2+<\/jats:sup> current passing channel, more resembling a classical Na<jats:sub>V<\/jats:sub>1 Na<jats:sup>+<\/jats:sup> channel. Cnidarian Ca<jats:sub>V<\/jats:sub>3 T-type channels can possess genes with alternative cysteine-rich, D4L6 extracellular loops in a manner reminiscent of the alternative cysteine-rich, D2L5 extracellular loops of non-cnidarian invertebrates. We illustrate here that the preferences for greater Na<jats:sup>+<\/jats:sup> or Ca<jats:sup>2+<\/jats:sup> ion current passing through Ca<jats:sub>V<\/jats:sub>3 T-type channels are contributed by paired cysteines within D2L5 and D4L6 extracellular loops looming above the pore selectivity filter. Swapping of invertebrate tri- and tetra-cysteine containing extracellular loops, generates higher Na<jats:sup>+<\/jats:sup> current passing channels in human Ca<jats:sub>V<\/jats:sub>3.2 channels, while corresponding mono- and di-cysteine loop pairs in human Ca<jats:sub>V<\/jats:sub>3.2 generates greater Ca<jats:sup>2+<\/jats:sup> current passing, invertebrate LCa<jats:sub>V<\/jats:sub>3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCa<jats:sub>V<\/jats:sub>3 channels increases relative monovalent ion current sizes and increases the potency of Zn<jats:sup>2+<\/jats:sup> and Ni<jats:sup>2+<\/jats:sup> block by\u2009~\u200950\u00d7\u2009and\u2009~\u200910\u00d7\u2009in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of Ca<jats:sub>V<\/jats:sub>3.2 channels, including the loss of the slowing of inactivation kinetics during Zn<jats:sup>2+<\/jats:sup> block. Charge neutralization of a ubiquitous aspartate residue of calcium passing Ca<jats:sub>V<\/jats:sub>1, Ca<jats:sub>V<\/jats:sub>2 and Ca<jats:sub>V<\/jats:sub>3 channels, in the outer pore of the selectivity filter residues in Domain II generates higher Na<jats:sup>+<\/jats:sup> current passing channels in a manner that may resemble how the unique D2L5 extracellular loops of invertebrate Ca<jats:sub>V<\/jats:sub>3 channels may confer a relatively higher peak current size for Na<jats:sup>+<\/jats:sup> ions over Ca<jats:sup>2+<\/jats:sup> The extracellular loops of Ca<jats:sub>V<\/jats:sub>3 channels are not engaged with accessory subunit binding, as the other Na<jats:sup>+<\/jats:sup> (Na<jats:sub>V<\/jats:sub>1) and Ca<jats:sup>2+<\/jats:sup> (Ca<jats:sub>V<\/jats:sub>1\/Ca<jats:sub>V<\/jats:sub>2) channels, enabling diversity and expansion of cysteine-bonded extracellular loops, which appears to serve, amongst other possibilities, to alter to the preferences for passage of Ca<jats:sup>2+<\/jats:sup> or Na<jats:sup>+<\/jats:sup> ions through invertebrate Ca<jats:sub>V<\/jats:sub>3 channels.<\/jats:p>","DOI":"10.1038\/s41598-020-69197-3","type":"journal-article","created":{"date-parts":[[2020,7,24]],"date-time":"2020-07-24T10:04:09Z","timestamp":1595585049000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions"],"prefix":"10.1038","volume":"10","author":[{"given":"Wendy","family":"Guan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert F.","family":"Stephens","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Omar","family":"Mourad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amrit","family":"Mehta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Fux","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J. David","family":"Spafford","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,7,24]]},"reference":[{"key":"69197_CR1","doi-asserted-by":"publisher","first-page":"1422","DOI":"10.1161\/01.RES.0000225862.14314.49","volume":"98","author":"ME Mangoni","year":"2006","unstructured":"Mangoni, M. E. et al. Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1\/alpha1G T-type calcium channels. Circ. Res. 98, 1422\u20131430. https:\/\/doi.org\/10.1161\/01.RES.0000225862.14314.49 (2006).","journal-title":"Circ. Res."},{"key":"69197_CR2","doi-asserted-by":"publisher","first-page":"1416","DOI":"10.1126\/science.1089268","volume":"302","author":"CC Chen","year":"2003","unstructured":"Chen, C. C. et al. Abnormal coronary function in mice deficient in alpha1H T-type Ca2+ channels. Science 302, 1416\u20131418. https:\/\/doi.org\/10.1126\/science.1089268 (2003).","journal-title":"Science"},{"key":"69197_CR3","doi-asserted-by":"publisher","first-page":"406","DOI":"10.1038\/297406a0","volume":"297","author":"R Llinas","year":"1982","unstructured":"Llinas, R. & Jahnsen, H. Electrophysiology of mammalian thalamic neurones in vitro. Nature 297, 406\u2013408 (1982).","journal-title":"Nature"},{"key":"69197_CR4","doi-asserted-by":"publisher","first-page":"2581","DOI":"10.1111\/j.1460-9568.2006.05136.x","volume":"24","author":"BE McKay","year":"2006","unstructured":"McKay, B. E. et al. Ca(V)3 T-type calcium channel isoforms differentially distribute to somatic and dendritic compartments in rat central neurons. Eur. J. Neurosci. 24, 2581\u20132594. https:\/\/doi.org\/10.1111\/j.1460-9568.2006.05136.x (2006).","journal-title":"Eur. J. Neurosci."},{"key":"69197_CR5","doi-asserted-by":"publisher","first-page":"1895","DOI":"10.1523\/JNEUROSCI.19-06-01895.1999","volume":"19","author":"EM Talley","year":"1999","unstructured":"Talley, E. M. et al. Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J. Neurosci. 19, 1895\u20131911 (1999).","journal-title":"J. Neurosci."},{"key":"69197_CR6","doi-asserted-by":"publisher","first-page":"1406","DOI":"10.1016\/j.bbamem.2007.02.017","volume":"1768","author":"A Shcheglovitov","year":"2007","unstructured":"Shcheglovitov, A., Kostyuk, P. & Shuba, Y. Selectivity signatures of three isoforms of recombinant T-type Ca2+ channels. Biochim. Biophys. Acta 1768, 1406\u20131419. https:\/\/doi.org\/10.1016\/j.bbamem.2007.02.017 (2007).","journal-title":"Biochim. Biophys. Acta"},{"key":"69197_CR7","doi-asserted-by":"publisher","first-page":"7447","DOI":"10.1074\/jbc.M109.090753","volume":"285","author":"A Senatore","year":"2010","unstructured":"Senatore, A. & Spafford, J. D. Transient and big are key features of an invertebrate T-type channel (LCav3) from the central nervous system of Lymnaea stagnalis. J. Biol. Chem. 285, 7447\u20137458. https:\/\/doi.org\/10.1074\/jbc.M109.090753 (2010).","journal-title":"J. Biol. Chem."},{"key":"69197_CR8","doi-asserted-by":"publisher","first-page":"e37409","DOI":"10.1371\/journal.pone.0037409","volume":"7","author":"A Senatore","year":"2012","unstructured":"Senatore, A. & Spafford, J. D. Gene transcription and splicing of T-type channels are evolutionarily-conserved strategies for regulating channel expression and gating. PLoS\u00a0One 7, e37409. https:\/\/doi.org\/10.1371\/journal.pone.0037409 (2012).","journal-title":"PLoS\u00a0One"},{"key":"69197_CR9","doi-asserted-by":"publisher","first-page":"11952","DOI":"10.1074\/jbc.M114.551473","volume":"289","author":"A Senatore","year":"2014","unstructured":"Senatore, A., Guan, W., Boone, A. N. & Spafford, J. D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter. J. Biol. Chem. 289, 11952\u201311969. https:\/\/doi.org\/10.1074\/jbc.M114.551473 (2014).","journal-title":"J. Biol. Chem."},{"key":"69197_CR10","doi-asserted-by":"publisher","first-page":"516","DOI":"10.1113\/jphysiol.1958.sp006034","volume":"142","author":"P Fatt","year":"1958","unstructured":"Fatt, P. & Ginsborg, B. L. The ionic requirements for the production of action potentials in crustacean muscle fibres. J. Physiol. 142, 516\u2013543 (1958).","journal-title":"J. Physiol."},{"key":"69197_CR11","first-page":"644","volume-title":"Nature of the Chemical Bond and Structure of Molecules and Crystals","author":"L Pauling","year":"1960","unstructured":"Pauling, L. Nature of the Chemical Bond and Structure of Molecules and Crystals 3rd edn, 644 (Cornell University Press, Cornell, 1960).","edition":"3"},{"key":"69197_CR12","doi-asserted-by":"publisher","first-page":"3999","DOI":"10.1074\/jbc.M008215200","volume":"276","author":"JE McRory","year":"2001","unstructured":"McRory, J. E. et al. Molecular and functional characterization of a family of rat brain T-type calcium channels. J. Biol. Chem. 276, 3999\u20134011. https:\/\/doi.org\/10.1074\/jbc.M008215200 (2001).","journal-title":"J. Biol. Chem."},{"key":"69197_CR13","doi-asserted-by":"publisher","first-page":"790","DOI":"10.1038\/nrd1197","volume":"2","author":"RJ Lewis","year":"2003","unstructured":"Lewis, R. J. & Garcia, M. L. Therapeutic potential of venom peptides. Nat. Rev. Drug Discov. 2, 790\u2013802. https:\/\/doi.org\/10.1038\/nrd1197 (2003).","journal-title":"Nat. Rev. Drug Discov."},{"key":"69197_CR14","doi-asserted-by":"publisher","first-page":"5774","DOI":"10.1016\/j.febslet.2007.11.045","volume":"581","author":"HW Kang","year":"2007","unstructured":"Kang, H. W., Moon, H. J., Joo, S. H. & Lee, J. H. Histidine residues in the IS3\u2013IS4 loop are critical for nickel-sensitive inhibition of the Cav2.3 calcium channel. FEBS Lett. 581, 5774\u20135780. https:\/\/doi.org\/10.1016\/j.febslet.2007.11.045 (2007).","journal-title":"FEBS Lett."},{"key":"69197_CR15","doi-asserted-by":"publisher","first-page":"3034","DOI":"10.1016\/S0006-3495(99)77134-1","volume":"77","author":"JH Lee","year":"1999","unstructured":"Lee, J. H., Gomora, J. C., Cribbs, L. L. & Perez-Reyes, E. Nickel block of three cloned T-type calcium channels: Low concentrations selectively block alpha1H. Biophys. J. 77, 3034\u20133042. https:\/\/doi.org\/10.1016\/S0006-3495(99)77134-1 (1999).","journal-title":"Biophys. J."},{"key":"69197_CR16","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1113\/jphysiol.2006.114496","volume":"578","author":"A Traboulsie","year":"2007","unstructured":"Traboulsie, A. et al. Subunit-specific modulation of T-type calcium channels by zinc. J. Physiol. 578, 159\u2013171. https:\/\/doi.org\/10.1113\/jphysiol.2006.114496 (2007).","journal-title":"J. Physiol."},{"key":"69197_CR17","doi-asserted-by":"publisher","first-page":"1175","DOI":"10.1529\/biophysj.106.103333","volume":"93","author":"HS Sun","year":"2007","unstructured":"Sun, H. S., Hui, K., Lee, D. W. & Feng, Z. P. Zn2+ sensitivity of high- and low-voltage activated calcium channels. Biophys. J. 93, 1175\u20131183. https:\/\/doi.org\/10.1529\/biophysj.106.103333 (2007).","journal-title":"Biophys. J."},{"key":"69197_CR18","doi-asserted-by":"publisher","first-page":"492","DOI":"10.1038\/s41586-019-1801-3","volume":"576","author":"Y Zhao","year":"2019","unstructured":"Zhao, Y. et al. Cryo-EM structures of apo and antagonist-bound human Cav3.1. Nature 576, 492\u2013497. https:\/\/doi.org\/10.1038\/s41586-019-1801-3 (2019).","journal-title":"Nature"},{"key":"69197_CR19","doi-asserted-by":"publisher","first-page":"153","DOI":"10.3389\/fphys.2015.00153","volume":"6","author":"RF Stephens","year":"2015","unstructured":"Stephens, R. F., Guan, W., Zhorov, B. S. & Spafford, J. D. Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels. Front. Physiol. 6, 153. https:\/\/doi.org\/10.3389\/fphys.2015.00153 (2015).","journal-title":"Front. Physiol."},{"key":"69197_CR20","doi-asserted-by":"publisher","first-page":"1495 e1412","DOI":"10.1016\/j.cell.2019.04.043","volume":"177","author":"Y Zhao","year":"2019","unstructured":"Zhao, Y. et al. Molecular basis for ligand modulation of a mammalian voltage-gated Ca(2+) channel. Cell 177, 1495 e1412-1506 e1412. https:\/\/doi.org\/10.1016\/j.cell.2019.04.043 (2019).","journal-title":"Cell"},{"key":"69197_CR21","doi-asserted-by":"publisher","first-page":"1303","DOI":"10.1126\/science.aaw2493","volume":"363","author":"H Shen","year":"2019","unstructured":"Shen, H., Liu, D., Wu, K., Lei, J. & Yan, N. Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins. Science 363, 1303\u20131308. https:\/\/doi.org\/10.1126\/science.aaw2493 (2019).","journal-title":"Science"},{"key":"69197_CR22","doi-asserted-by":"publisher","first-page":"9500","DOI":"10.1523\/JNEUROSCI.5803-08.2009","volume":"29","author":"WY Lee","year":"2009","unstructured":"Lee, W. Y. et al. Molecular mechanisms of lipoic acid modulation of T-type calcium channels in pain pathway. J. Neurosci. 29, 9500\u20139509. https:\/\/doi.org\/10.1523\/JNEUROSCI.5803-08.2009 (2009).","journal-title":"J. Neurosci."},{"key":"69197_CR23","doi-asserted-by":"publisher","first-page":"1015","DOI":"10.1007\/s00424-010-0874-5","volume":"460","author":"M Karmazinova","year":"2010","unstructured":"Karmazinova, M. et al. Cysteines in the loop between IS5 and the pore helix of Ca(V)3.1 are essential for channel gating. Pflugers. Arch. 460, 1015\u20131028. https:\/\/doi.org\/10.1007\/s00424-010-0874-5 (2010).","journal-title":"Pflugers. Arch."},{"key":"69197_CR24","doi-asserted-by":"publisher","first-page":"8250","DOI":"10.1523\/JNEUROSCI.1800-07.2007","volume":"27","author":"MT Nelson","year":"2007","unstructured":"Nelson, M. T. et al. Reducing agents sensitize C-type nociceptors by relieving high-affinity zinc inhibition of T-type calcium channels. J. Neurosci. 27, 8250\u20138260. https:\/\/doi.org\/10.1523\/JNEUROSCI.1800-07.2007 (2007).","journal-title":"J. Neurosci."},{"key":"69197_CR25","doi-asserted-by":"publisher","first-page":"W636","DOI":"10.1093\/nar\/gkz268","volume":"47","author":"F Madeira","year":"2019","unstructured":"Madeira, F. et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 47, W636\u2013W641. https:\/\/doi.org\/10.1093\/nar\/gkz268 (2019).","journal-title":"Nucleic Acids Res."},{"key":"69197_CR26","doi-asserted-by":"publisher","first-page":"W465","DOI":"10.1093\/nar\/gkn180","volume":"36","author":"A Dereeper","year":"2008","unstructured":"Dereeper, A. et al. Phylogeny.fr: Robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465\u2013W469. https:\/\/doi.org\/10.1093\/nar\/gkn180 (2008).","journal-title":"Nucleic Acids Res."},{"key":"69197_CR27","doi-asserted-by":"publisher","DOI":"10.3791\/2314","author":"A Senatore","year":"2011","unstructured":"Senatore, A., Boone, A. N. & Spafford, J. D. Optimized transfection strategy for expression and electrophysiological recording of recombinant voltage-gated ion channels in HEK-293T cells. J. Vis. Exp. https:\/\/doi.org\/10.3791\/2314 (2011).","journal-title":"J. Vis. Exp."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-69197-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-69197-3","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-69197-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,6]],"date-time":"2022-12-06T14:45:50Z","timestamp":1670337950000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-69197-3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,7,24]]},"references-count":27,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,12]]}},"alternative-id":["69197"],"URL":"https:\/\/doi.org\/10.1038\/s41598-020-69197-3","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,7,24]]},"assertion":[{"value":"31 March 2020","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"6 July 2020","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"24 July 2020","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"12404"}}