{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:26:19Z","timestamp":1760235979261,"version":"build-2065373602"},"reference-count":32,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2021,10,12]],"date-time":"2021-10-12T00:00:00Z","timestamp":1633996800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1R15GM124606"],"award-info":[{"award-number":["1R15GM124606"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.<\/jats:p>","DOI":"10.3390\/toxins13100722","type":"journal-article","created":{"date-parts":[[2021,10,13]],"date-time":"2021-10-13T21:48:39Z","timestamp":1634161719000},"page":"722","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties"],"prefix":"10.3390","volume":"13","author":[{"given":"Ryan V.","family":"Schroder","sequence":"first","affiliation":[{"name":"Department of Chemistry, College of Staten Island, University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA"},{"name":"The Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, NY 10016, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leah S.","family":"Cohen","sequence":"additional","affiliation":[{"name":"Department of Chemistry, College of Staten Island, University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ping","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Chemistry, College of Staten Island, University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4406-5597","authenticated-orcid":false,"given":"Joekeem D.","family":"Arizala","sequence":"additional","affiliation":[{"name":"Department of Chemistry, College of Staten Island, University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA"},{"name":"The Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, NY 10016, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1724-3386","authenticated-orcid":false,"given":"S\u00e9bastien F.","family":"Poget","sequence":"additional","affiliation":[{"name":"Department of Chemistry, College of Staten Island, University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA"},{"name":"The Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, NY 10016, USA"},{"name":"The Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, NY 10016, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"506","DOI":"10.1016\/S1359-6446(00)01570-1","article-title":"Voltage-gated sodium channels as therapeutic targets","volume":"5","author":"Clare","year":"2000","journal-title":"Drug Discov. Today"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/S0896-6273(00)81133-2","article-title":"From Ionic Currents to Molecular Mechanisms","volume":"26","author":"Catterall","year":"2000","journal-title":"Neuron"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"12706","DOI":"10.1073\/pnas.0404915101","article-title":"Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain","volume":"101","author":"Nassar","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.toxicon.2006.09.022","article-title":"Voltage-gated ion channels and gating modifier toxins","volume":"49","author":"Catterall","year":"2007","journal-title":"Toxicon"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6623","DOI":"10.1021\/jm500687u","article-title":"Studies Examining the Relationship between the Chemical Structure of Protoxin II and Its Activity on Voltage Gated Sodium Channels","volume":"57","author":"Park","year":"2014","journal-title":"J. Med. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"702","DOI":"10.1016\/j.cell.2018.12.018","article-title":"Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin","volume":"176","author":"Xu","year":"2019","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.1126\/science.aaw2493","article-title":"Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins","volume":"363","author":"Shen","year":"2019","journal-title":"Science"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2299","DOI":"10.1021\/jm501765v","article-title":"Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the NaV1.7 Sodium Channel","volume":"58","author":"Murray","year":"2015","journal-title":"J. Med. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Deuis, J.R., Wingerd, J.S., Winter, Z., Durek, T., Dekan, Z., Sousa, S.R., Zimmermann, K., Hoffmann, T., Weidner, C., and Nassar, M.A. (2016). Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain. Toxins, 8.","DOI":"10.3390\/toxins8030078"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"10442","DOI":"10.1021\/bi2012965","article-title":"In Vitro Folding of KvAP, a Voltage-Gated K+ Channel","volume":"50","author":"Devaraneni","year":"2011","journal-title":"Biochemistry"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"953","DOI":"10.3389\/fphar.2019.00953","article-title":"Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human Nav1.4 Channel","volume":"10","author":"Myshkin","year":"2019","journal-title":"Front. Pharmacol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1822","DOI":"10.1002\/pro.5560031021","article-title":"Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor","volume":"3","author":"Staley","year":"1994","journal-title":"Protein Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1002\/bip.10567","article-title":"Sexual conjugation in yeast: A paradigm to study G-protein-coupled receptor domain structure","volume":"76","author":"Naider","year":"2004","journal-title":"Biopolymers"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4854","DOI":"10.1016\/S0021-9258(18)62873-X","article-title":"The specific nonenzymatic cleavage of bovine ribonuclease with hydroxylamine","volume":"245","author":"Bornstein","year":"1970","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1093\/jb\/mvm075","article-title":"Stable Supply of Large Amounts of Human Fab from the Inclusion Bodies in E. coli","volume":"141","author":"Fujii","year":"2007","journal-title":"J. Biochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1146\/annurev.bb.09.060180.002343","article-title":"Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes)","volume":"9","author":"Szoka","year":"1980","journal-title":"Annu. Rev. Biophys. Bioeng."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.molstruc.2014.03.009","article-title":"MicroScale Thermophoresis: Interaction analysis and beyond","volume":"1077","author":"Wanner","year":"2014","journal-title":"J. Mol. Struct."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"12687","DOI":"10.1074\/jbc.M610462200","article-title":"Molecular interactions of the gating modifier toxin ProTx-II with Nav1.5: Implied existence of a novel toxin binding site coupled to activation","volume":"282","author":"Smith","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/j.ab.2015.12.013","article-title":"On the acquisition and analysis of microscale thermophoresis data","volume":"496","author":"Scheuermann","year":"2016","journal-title":"Anal. Biochem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1476","DOI":"10.1124\/mol.108.047670","article-title":"ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors","volume":"74","author":"Schmalhofer","year":"2008","journal-title":"Mol. Pharmacol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1002\/pst.1943","article-title":"Equilibrium, affinity, dissociation constants, IC5O: Facts and fantasies","volume":"18","author":"Barbet","year":"2019","journal-title":"Pharm. Stat."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1038\/nature02632","article-title":"A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom","volume":"430","author":"Lee","year":"2004","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"9041","DOI":"10.1074\/jbc.RA118.002553","article-title":"Gating modifier toxins isolated from spider venom: Modulation of voltage-gated sodium channels and the role of lipid membranes","volume":"293","author":"Agwa","year":"2018","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"10071","DOI":"10.1021\/bi049463y","article-title":"Localization of the voltage-sensor toxin receptor on KvAP","volume":"43","author":"Ruta","year":"2004","journal-title":"Biochemistry"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"srep34333","DOI":"10.1038\/srep34333","article-title":"Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels","volume":"6","author":"Lau","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"14226","DOI":"10.1038\/srep14226","article-title":"Structural basis for the inhibition of volt-age-dependent K+ channel by gating modifier toxin","volume":"5","author":"Ozawa","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4495","DOI":"10.1073\/pnas.1720185115","article-title":"Spider toxin inhibits gating pore currents underlying periodic paralysis","volume":"115","author":"Shenkarev","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Katz, D., Sindhikara, D., DiMattia, M., and Leffler, A.E. (2021). Potency-Enhancing Mutations of Gating Modifier Toxins for the Voltage-Gated Sodium Channel NaV1.7 Can be Predicted Using Accurate Free-Energy Calculations. Toxins, 13.","DOI":"10.3390\/toxins13030193"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1423","DOI":"10.1007\/s00424-021-02610-6","article-title":"CNG channel structure, function, and gating: A tale of conformational flexibility","volume":"473","author":"Napolitano","year":"2021","journal-title":"Pflug. Arch.-Eur. J. Physiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1006\/jmbi.1996.0399","article-title":"Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels","volume":"260","author":"Miroux","year":"1996","journal-title":"J. Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5655","DOI":"10.1021\/ac000811l","article-title":"A Robust, Detergent-Friendly Method for Mass Spectrometric Analysis of Integral Membrane Proteins","volume":"72","author":"Cadene","year":"2000","journal-title":"Anal. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1124\/mol.110.066332","article-title":"The Tarantula Toxins ProTx-II and Huwentoxin-IV Differentially Interact with Human Nav1.7 Voltage Sensors to Inhibit Channel Activation and Inactivation","volume":"78","author":"Xiao","year":"2010","journal-title":"Mol. Pharmacol."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/13\/10\/722\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:12:14Z","timestamp":1760166734000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/13\/10\/722"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,12]]},"references-count":32,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2021,10]]}},"alternative-id":["toxins13100722"],"URL":"https:\/\/doi.org\/10.3390\/toxins13100722","relation":{},"ISSN":["2072-6651"],"issn-type":[{"type":"electronic","value":"2072-6651"}],"subject":[],"published":{"date-parts":[[2021,10,12]]}}}