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      Mapping protein interactions of sodium channel Na V1.7 using epitope‐tagged gene‐targeted mice

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          The voltage‐gated sodium channel Na V1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na V1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na V1.7 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 V1.7 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 V1.7 channel.

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          Most cited references 60

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          A highly efficient recombineering-based method for generating conditional knockout mutations.

          Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.
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            Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems.

             K Terpe (2002)
            In response to the rapidly growing field of proteomics, the use of recombinant proteins has increased greatly in recent years. Recombinant hybrids containing a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. Many different proteins, domains, or peptides can be fused with the target protein. The advantages of using fusion proteins to facilitate purification and detection of recombinant proteins are well-recognized. Nevertheless, it is difficult to choose the right purification system for a specific protein of interest. This review gives an overview of the most frequently used and interesting systems: Arg-tag, calmodulin-binding peptide, cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase, FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, SBP-tag, Strep-tag, and thioredoxin.
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              The Na(V)1.7 sodium channel: from molecule to man.

              The voltage-gated sodium channel Na(V)1.7 is preferentially expressed in peripheral somatic and visceral sensory neurons, olfactory sensory neurons and sympathetic ganglion neurons. Na(V)1.7 accumulates at nerve fibre endings and amplifies small subthreshold depolarizations, poising it to act as a threshold channel that regulates excitability. Genetic and functional studies have added to the evidence that Na(V)1.7 is a major contributor to pain signalling in humans, and homology modelling based on crystal structures of ion channels suggests an atomic-level structural basis for the altered gating of mutant Na(V)1.7 that causes pain.

                Author and article information

                EMBO J
                EMBO J
                The EMBO Journal
                John Wiley and Sons Inc. (Hoboken )
                15 January 2018
                01 February 2018
                15 January 2018
                : 37
                : 3 ( doiID: 10.1002/embj.v37.3 )
                : 427-445
                [ 1 ] Molecular Nociception Group WIBR University College London London UK
                [ 2 ] TDI Mass Spectrometry Laboratory Target Discovery Institute University of Oxford Oxford UK
                [ 3 ] Center for Integrative Physiology and Molecular Medicine Saarland University Homburg Germany
                [ 4 ] Université Clermont Auvergne Inserm U1107 Neuro‐Dol, Pharmacologie Fondamentale et Clinique de la Douleur Clermont‐Ferrand France
                [ 5 ] Neuroscience IMED Biotech Unit AstraZeneca Cambridge UK
                [ 6 ] Division of Bioscience University College London London UK
                [ 7 ] Department of Neuroscience, Physiology and Pharmacology University College London London UK
                Author notes
                [* ] Corresponding author. Tel: +44 207 6796 954; E‐mail: j.wood@ 123456ucl.ac.uk

                Corresponding author. Tel: +44 207 6790 959; E‐mail: jing02.zhao@ 123456ucl.ac.uk


                These authors contributed equally to this work

                © 2018 The Authors. Published under the terms of the CC BY 4.0 license

                This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 9, Tables: 1, Pages: 19, Words: 13774
                Funded by: Wellcome
                Award ID: 200183/Z/15/Z
                Award ID: 101054/Z/13/Z
                Funded by: Deutsche Forschungsgemeinschaft (DFG)
                Award ID: SFB 894/A17
                Award ID: PY90/1‐1
                Funded by: RCUK|Medical Research Council (MRC)
                Award ID: G091905
                Award ID: G1100340
                Custom metadata
                01 February 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.3.2 mode:remove_FC converted:01.02.2018


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