Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice
The voltage‐gated sodium channel Na V 1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na V 1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na V 1.7 complexes affinity‐purified under native conditions by mass spectrometry r...
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| Veröffentlicht in: | The EMBO journal 2018-02, Vol.37 (3), p.427-445 |
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| creator | Kanellopoulos, Alexandros H Koenig, Jennifer Huang, Honglei Pyrski, Martina Millet, Queensta Lolignier, Stéphane Morohashi, Toru Gossage, Samuel J Jay, Maude Linley, John E Baskozos, Georgios Kessler, Benedikt M Cox, James J Dolphin, Annette C Zufall, Frank Wood, John N Zhao, Jing |
| description | The voltage‐gated sodium channel Na
V
1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na
V
1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na
V
1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7
in vivo
. The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel Na
V
1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the Na
V
1.7 channel.
Synopsis
Numerous novel protein‐protein interactors of voltage‐gated sodium channel Na
V
1.7 were identified using mouse genetics combined with proteomic approaches.
An epitope‐tagged (TAP tag) Na
V
1.7 knock‐in mouse line was generated to investigate protein‐protein interactions of Na
V
1.7. The TAP tag was inserted at the C‐terminus of Na
V
1.7.
The TAP‐tagged Na
V
1.7 knock‐in mice show a normal Na
V
1.7 expression pattern and normal pain behaviour.
Na
V
1.7 complexes were isolated from neuronal tissues from TAP‐tagged Na
V
1.7 mice using affinity purification.
Two hundred and sixty‐seven protein interactors of Na
V
1.7 were identified by mass spectrometry (LC‐MS/MS) analyses following affinity purification.
Graphical Abstract
Combining mouse genetics and proteomics reveals various new interactors of a voltage‐gated channel with key roles in pain perception, including direct links to opoid signalling and potential targets for better pain treatment. |
| doi_str_mv | 10.15252/embj.201796692 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_5793798</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>1993021802</sourcerecordid><originalsourceid>FETCH-LOGICAL-p2782-fa30ad424770c5e53cfec4f380b1827baf4fd91216884bf699b684bed31dde843</originalsourceid><addsrcrecordid>eNptkctOwzAQRS0EgvJYs43EOsWPJLZZIEFVXiqwAbaWk0yCq8YOTgLqjk_gG_kSAqkqkFjNXM3cMyNdhA4JHpOYxvQYqnQ-pphwmSSSbqARiRIcUszjTTTCNCFhRITcQbtNM8cYx4KTbbRDJWMxFXiE9K2ua2PLoPauBWMDY1vwOmuNs03giqBxuemqIHvW1sIiuNNPZMyDrvn2QG1aV8Pn-0eryxLyoAQ7KF9C2-vKZLCPtgq9aOBgVffQ48X0YXIVzu4vrydns7CmXNCw0AzrPKIR5ziLIWZZAVlUMIFTIihPdREVuSSUJEJEaZFImSZ9AzkjeQ4iYnvodODWXVpBnoFtvV6o2ptK-6Vy2qi_E2ueVeleVcwl41L0gKMVwLuXDppWzV3nbf-zIlIyTInAtN86GbbezAKWazzB6icQ9R2IWgeiprfnN2vVm_FgbnqfLcH_OvE_gH0BYzOTxQ</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1993021802</pqid></control><display><type>article</type><title>Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice</title><source>Wiley Free Content</source><source>Wiley Online Library Journals Frontfile Complete</source><source>EZB-FREE-00999 freely available EZB journals</source><source>PubMed Central</source><source>Free Full-Text Journals in Chemistry</source><source>Springer Nature OA Free Journals</source><creator>Kanellopoulos, Alexandros H ; Koenig, Jennifer ; Huang, Honglei ; Pyrski, Martina ; Millet, Queensta ; Lolignier, Stéphane ; Morohashi, Toru ; Gossage, Samuel J ; Jay, Maude ; Linley, John E ; Baskozos, Georgios ; Kessler, Benedikt M ; Cox, James J ; Dolphin, Annette C ; Zufall, Frank ; Wood, John N ; Zhao, Jing</creator><creatorcontrib>Kanellopoulos, Alexandros H ; Koenig, Jennifer ; Huang, Honglei ; Pyrski, Martina ; Millet, Queensta ; Lolignier, Stéphane ; Morohashi, Toru ; Gossage, Samuel J ; Jay, Maude ; Linley, John E ; Baskozos, Georgios ; Kessler, Benedikt M ; Cox, James J ; Dolphin, Annette C ; Zufall, Frank ; Wood, John N ; Zhao, Jing</creatorcontrib><description>The voltage‐gated sodium channel Na
V
1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na
V
1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na
V
1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7
in vivo
. The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel Na
V
1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the Na
V
1.7 channel.
Synopsis
Numerous novel protein‐protein interactors of voltage‐gated sodium channel Na
V
1.7 were identified using mouse genetics combined with proteomic approaches.
An epitope‐tagged (TAP tag) Na
V
1.7 knock‐in mouse line was generated to investigate protein‐protein interactions of Na
V
1.7. The TAP tag was inserted at the C‐terminus of Na
V
1.7.
The TAP‐tagged Na
V
1.7 knock‐in mice show a normal Na
V
1.7 expression pattern and normal pain behaviour.
Na
V
1.7 complexes were isolated from neuronal tissues from TAP‐tagged Na
V
1.7 mice using affinity purification.
Two hundred and sixty‐seven protein interactors of Na
V
1.7 were identified by mass spectrometry (LC‐MS/MS) analyses following affinity purification.
Graphical Abstract
Combining mouse genetics and proteomics reveals various new interactors of a voltage‐gated channel with key roles in pain perception, including direct links to opoid signalling and potential targets for better pain treatment.</description><identifier>ISSN: 0261-4189</identifier><identifier>EISSN: 1460-2075</identifier><identifier>DOI: 10.15252/embj.201796692</identifier><identifier>PMID: 29335280</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>Affinity ; Amino acids ; Analgesics ; Axonogenesis ; C-Terminus ; EMBO24 ; EMBO27 ; Epitope mapping ; Gene mapping ; Genetics ; Immunoprecipitation ; Mass spectrometry ; Mass spectroscopy ; Mediator protein ; Membrane proteins ; Membrane trafficking ; Mice ; Narcotics ; NaV1.7 ; Opioid receptors ; Pain ; Peptide mapping ; Protein interaction ; Protein purification ; Protein transport ; Proteins ; protein–protein interactor ; Purification ; Resource ; Scientific imaging ; sensory neuron ; Signal transduction ; Signaling ; Sodium ; sodium channel ; Sodium channels (voltage-gated) ; Spectroscopy ; Synaptotagmin ; Synaptotagmin II</subject><ispartof>The EMBO journal, 2018-02, Vol.37 (3), p.427-445</ispartof><rights>The Authors. Published under the terms of the CC BY 4.0 license 2018</rights><rights>2018 The Authors. Published under the terms of the CC BY 4.0 license</rights><rights>2018 EMBO</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-p2782-fa30ad424770c5e53cfec4f380b1827baf4fd91216884bf699b684bed31dde843</cites><orcidid>0000-0002-5751-178X ; 0000-0003-2587-1198 ; 0000-0001-9237-5878</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793798/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793798/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,1411,1427,27901,27902,41096,42165,45550,45551,46384,46808,51551,53766,53768</link.rule.ids></links><search><creatorcontrib>Kanellopoulos, Alexandros H</creatorcontrib><creatorcontrib>Koenig, Jennifer</creatorcontrib><creatorcontrib>Huang, Honglei</creatorcontrib><creatorcontrib>Pyrski, Martina</creatorcontrib><creatorcontrib>Millet, Queensta</creatorcontrib><creatorcontrib>Lolignier, Stéphane</creatorcontrib><creatorcontrib>Morohashi, Toru</creatorcontrib><creatorcontrib>Gossage, Samuel J</creatorcontrib><creatorcontrib>Jay, Maude</creatorcontrib><creatorcontrib>Linley, John E</creatorcontrib><creatorcontrib>Baskozos, Georgios</creatorcontrib><creatorcontrib>Kessler, Benedikt M</creatorcontrib><creatorcontrib>Cox, James J</creatorcontrib><creatorcontrib>Dolphin, Annette C</creatorcontrib><creatorcontrib>Zufall, Frank</creatorcontrib><creatorcontrib>Wood, John N</creatorcontrib><creatorcontrib>Zhao, Jing</creatorcontrib><title>Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice</title><title>The EMBO journal</title><addtitle>EMBO J</addtitle><description>The voltage‐gated sodium channel Na
V
1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na
V
1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na
V
1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7
in vivo
. The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel Na
V
1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the Na
V
1.7 channel.
Synopsis
Numerous novel protein‐protein interactors of voltage‐gated sodium channel Na
V
1.7 were identified using mouse genetics combined with proteomic approaches.
An epitope‐tagged (TAP tag) Na
V
1.7 knock‐in mouse line was generated to investigate protein‐protein interactions of Na
V
1.7. The TAP tag was inserted at the C‐terminus of Na
V
1.7.
The TAP‐tagged Na
V
1.7 knock‐in mice show a normal Na
V
1.7 expression pattern and normal pain behaviour.
Na
V
1.7 complexes were isolated from neuronal tissues from TAP‐tagged Na
V
1.7 mice using affinity purification.
Two hundred and sixty‐seven protein interactors of Na
V
1.7 were identified by mass spectrometry (LC‐MS/MS) analyses following affinity purification.
Graphical Abstract
Combining mouse genetics and proteomics reveals various new interactors of a voltage‐gated channel with key roles in pain perception, including direct links to opoid signalling and potential targets for better pain treatment.</description><subject>Affinity</subject><subject>Amino acids</subject><subject>Analgesics</subject><subject>Axonogenesis</subject><subject>C-Terminus</subject><subject>EMBO24</subject><subject>EMBO27</subject><subject>Epitope mapping</subject><subject>Gene mapping</subject><subject>Genetics</subject><subject>Immunoprecipitation</subject><subject>Mass spectrometry</subject><subject>Mass spectroscopy</subject><subject>Mediator protein</subject><subject>Membrane proteins</subject><subject>Membrane trafficking</subject><subject>Mice</subject><subject>Narcotics</subject><subject>NaV1.7</subject><subject>Opioid receptors</subject><subject>Pain</subject><subject>Peptide mapping</subject><subject>Protein interaction</subject><subject>Protein purification</subject><subject>Protein transport</subject><subject>Proteins</subject><subject>protein–protein interactor</subject><subject>Purification</subject><subject>Resource</subject><subject>Scientific imaging</subject><subject>sensory neuron</subject><subject>Signal transduction</subject><subject>Signaling</subject><subject>Sodium</subject><subject>sodium channel</subject><subject>Sodium channels (voltage-gated)</subject><subject>Spectroscopy</subject><subject>Synaptotagmin</subject><subject>Synaptotagmin II</subject><issn>0261-4189</issn><issn>1460-2075</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>24P</sourceid><recordid>eNptkctOwzAQRS0EgvJYs43EOsWPJLZZIEFVXiqwAbaWk0yCq8YOTgLqjk_gG_kSAqkqkFjNXM3cMyNdhA4JHpOYxvQYqnQ-pphwmSSSbqARiRIcUszjTTTCNCFhRITcQbtNM8cYx4KTbbRDJWMxFXiE9K2ua2PLoPauBWMDY1vwOmuNs03giqBxuemqIHvW1sIiuNNPZMyDrvn2QG1aV8Pn-0eryxLyoAQ7KF9C2-vKZLCPtgq9aOBgVffQ48X0YXIVzu4vrydns7CmXNCw0AzrPKIR5ziLIWZZAVlUMIFTIihPdREVuSSUJEJEaZFImSZ9AzkjeQ4iYnvodODWXVpBnoFtvV6o2ptK-6Vy2qi_E2ueVeleVcwl41L0gKMVwLuXDppWzV3nbf-zIlIyTInAtN86GbbezAKWazzB6icQ9R2IWgeiprfnN2vVm_FgbnqfLcH_OvE_gH0BYzOTxQ</recordid><startdate>20180201</startdate><enddate>20180201</enddate><creator>Kanellopoulos, Alexandros H</creator><creator>Koenig, Jennifer</creator><creator>Huang, Honglei</creator><creator>Pyrski, Martina</creator><creator>Millet, Queensta</creator><creator>Lolignier, Stéphane</creator><creator>Morohashi, Toru</creator><creator>Gossage, Samuel J</creator><creator>Jay, Maude</creator><creator>Linley, John E</creator><creator>Baskozos, Georgios</creator><creator>Kessler, Benedikt M</creator><creator>Cox, James J</creator><creator>Dolphin, Annette C</creator><creator>Zufall, Frank</creator><creator>Wood, John N</creator><creator>Zhao, Jing</creator><general>Nature Publishing Group UK</general><general>Springer Nature B.V</general><general>John Wiley and Sons Inc</general><scope>C6C</scope><scope>24P</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-5751-178X</orcidid><orcidid>https://orcid.org/0000-0003-2587-1198</orcidid><orcidid>https://orcid.org/0000-0001-9237-5878</orcidid></search><sort><creationdate>20180201</creationdate><title>Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice</title><author>Kanellopoulos, Alexandros H ; Koenig, Jennifer ; Huang, Honglei ; Pyrski, Martina ; Millet, Queensta ; Lolignier, Stéphane ; Morohashi, Toru ; Gossage, Samuel J ; Jay, Maude ; Linley, John E ; Baskozos, Georgios ; Kessler, Benedikt M ; Cox, James J ; Dolphin, Annette C ; Zufall, Frank ; Wood, John N ; Zhao, Jing</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p2782-fa30ad424770c5e53cfec4f380b1827baf4fd91216884bf699b684bed31dde843</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Affinity</topic><topic>Amino acids</topic><topic>Analgesics</topic><topic>Axonogenesis</topic><topic>C-Terminus</topic><topic>EMBO24</topic><topic>EMBO27</topic><topic>Epitope mapping</topic><topic>Gene mapping</topic><topic>Genetics</topic><topic>Immunoprecipitation</topic><topic>Mass spectrometry</topic><topic>Mass spectroscopy</topic><topic>Mediator protein</topic><topic>Membrane proteins</topic><topic>Membrane trafficking</topic><topic>Mice</topic><topic>Narcotics</topic><topic>NaV1.7</topic><topic>Opioid receptors</topic><topic>Pain</topic><topic>Peptide mapping</topic><topic>Protein interaction</topic><topic>Protein purification</topic><topic>Protein transport</topic><topic>Proteins</topic><topic>protein–protein interactor</topic><topic>Purification</topic><topic>Resource</topic><topic>Scientific imaging</topic><topic>sensory neuron</topic><topic>Signal transduction</topic><topic>Signaling</topic><topic>Sodium</topic><topic>sodium channel</topic><topic>Sodium channels (voltage-gated)</topic><topic>Spectroscopy</topic><topic>Synaptotagmin</topic><topic>Synaptotagmin II</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kanellopoulos, Alexandros H</creatorcontrib><creatorcontrib>Koenig, Jennifer</creatorcontrib><creatorcontrib>Huang, Honglei</creatorcontrib><creatorcontrib>Pyrski, Martina</creatorcontrib><creatorcontrib>Millet, Queensta</creatorcontrib><creatorcontrib>Lolignier, Stéphane</creatorcontrib><creatorcontrib>Morohashi, Toru</creatorcontrib><creatorcontrib>Gossage, Samuel J</creatorcontrib><creatorcontrib>Jay, Maude</creatorcontrib><creatorcontrib>Linley, John E</creatorcontrib><creatorcontrib>Baskozos, Georgios</creatorcontrib><creatorcontrib>Kessler, Benedikt M</creatorcontrib><creatorcontrib>Cox, James J</creatorcontrib><creatorcontrib>Dolphin, Annette C</creatorcontrib><creatorcontrib>Zufall, Frank</creatorcontrib><creatorcontrib>Wood, John N</creatorcontrib><creatorcontrib>Zhao, Jing</creatorcontrib><collection>Springer Nature OA Free Journals</collection><collection>Wiley Online Library Open Access</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The EMBO journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kanellopoulos, Alexandros H</au><au>Koenig, Jennifer</au><au>Huang, Honglei</au><au>Pyrski, Martina</au><au>Millet, Queensta</au><au>Lolignier, Stéphane</au><au>Morohashi, Toru</au><au>Gossage, Samuel J</au><au>Jay, Maude</au><au>Linley, John E</au><au>Baskozos, Georgios</au><au>Kessler, Benedikt M</au><au>Cox, James J</au><au>Dolphin, Annette C</au><au>Zufall, Frank</au><au>Wood, John N</au><au>Zhao, Jing</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice</atitle><jtitle>The EMBO journal</jtitle><stitle>EMBO J</stitle><date>2018-02-01</date><risdate>2018</risdate><volume>37</volume><issue>3</issue><spage>427</spage><epage>445</epage><pages>427-445</pages><issn>0261-4189</issn><eissn>1460-2075</eissn><abstract>The voltage‐gated sodium channel Na
V
1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na
V
1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na
V
1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7
in vivo
. The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel Na
V
1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the Na
V
1.7 channel.
Synopsis
Numerous novel protein‐protein interactors of voltage‐gated sodium channel Na
V
1.7 were identified using mouse genetics combined with proteomic approaches.
An epitope‐tagged (TAP tag) Na
V
1.7 knock‐in mouse line was generated to investigate protein‐protein interactions of Na
V
1.7. The TAP tag was inserted at the C‐terminus of Na
V
1.7.
The TAP‐tagged Na
V
1.7 knock‐in mice show a normal Na
V
1.7 expression pattern and normal pain behaviour.
Na
V
1.7 complexes were isolated from neuronal tissues from TAP‐tagged Na
V
1.7 mice using affinity purification.
Two hundred and sixty‐seven protein interactors of Na
V
1.7 were identified by mass spectrometry (LC‐MS/MS) analyses following affinity purification.
Graphical Abstract
Combining mouse genetics and proteomics reveals various new interactors of a voltage‐gated channel with key roles in pain perception, including direct links to opoid signalling and potential targets for better pain treatment.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>29335280</pmid><doi>10.15252/embj.201796692</doi><tpages>19</tpages><orcidid>https://orcid.org/0000-0002-5751-178X</orcidid><orcidid>https://orcid.org/0000-0003-2587-1198</orcidid><orcidid>https://orcid.org/0000-0001-9237-5878</orcidid><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0261-4189 |
| ispartof | The EMBO journal, 2018-02, Vol.37 (3), p.427-445 |
| issn | 0261-4189 1460-2075 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_5793798 |
| source | Wiley Free Content; Wiley Online Library Journals Frontfile Complete; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry; Springer Nature OA Free Journals |
| subjects | Affinity Amino acids Analgesics Axonogenesis C-Terminus EMBO24 EMBO27 Epitope mapping Gene mapping Genetics Immunoprecipitation Mass spectrometry Mass spectroscopy Mediator protein Membrane proteins Membrane trafficking Mice Narcotics NaV1.7 Opioid receptors Pain Peptide mapping Protein interaction Protein purification Protein transport Proteins protein–protein interactor Purification Resource Scientific imaging sensory neuron Signal transduction Signaling Sodium sodium channel Sodium channels (voltage-gated) Spectroscopy Synaptotagmin Synaptotagmin II |
| title | Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice |
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