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      Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins

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          Abstract

          Voltage-gated sodium channel Nav1.7 represents a promising target for pain relief. Here we report the cryo-EM structures of the human Nav1.7-β1-β2 complex bound to two combinations of pore blockers and gating modifier toxins (GMTs), tetrodotoxin with Protoxin-II and saxitoxin with Huwentoxin-IV, both determined at overall resolutions of 3.2 Å. The two structures are nearly identical except for minor shifts of VSDII, whose S3-S4 linker accommodates the two GMTs in a similar manner. One additional Protoxin-II sits on top of the S3-S4 linker in VSDIV. The structures may represent an inactivated state with all four VSDs "up" and the intracellular gate closed. The structures illuminate the path toward mechanistic understanding of the function and disease of Nav1.7 and establish the foundation for structure-aided development of analgesics.

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          Most cited references32

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          Electroporation and RNA interference in the rodent retina in vivo and in vitro.

          The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain- and loss-of-function studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.
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            Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.

            Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.
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              A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation.

              The inward Na+ current underlying the action potential in nerve is terminated by inactivation. The preceding report shows that deletions within the intracellular linker between domains III and IV remove inactivation, but mutation of conserved basic and paired acidic amino acids has little effect. Here we show that substitution of glutamine for three clustered hydrophobic amino acids, Ile-1488, Phe-1489, and Met-1490, completely removes fast inactivation. Substitution of Met-1490 alone slows inactivation significantly, substitution of Ile-1488 alone both slows inactivation and makes it incomplete, and substitution of Phe-1489 alone removes inactivation nearly completely. These results demonstrate an essential role of Phe-1489 in Na(+)-channel inactivation. It is proposed that the hydrophobic cluster of Ile-1488, Phe-1489, and Met-1490 serves as a hydrophobic latch that stabilizes the inactivated state in a hinged-lid mechanism of Na(+)-channel inactivation.
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                Author and article information

                Journal
                Science
                Science
                American Association for the Advancement of Science (AAAS)
                0036-8075
                1095-9203
                February 14 2019
                : eaaw2493
                Article
                10.1126/science.aaw2493
                30765606
                65eace1c-a30b-491b-a695-ee279e59e4df
                © 2019
                History

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