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  • 1
    Online Resource
    Online Resource
    Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac
    Proceedings of the National Academy of Sciences ; 2016
    In:  Proceedings of the National Academy of Sciences Vol. 113, No. 48 ( 2016-11-29), p. 13762-13767
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 113, No. 48 ( 2016-11-29), p. 13762-13767
    Abstract: Voltage-gated sodium channels (Na V ) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit Na V by stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in Na V , but experimental binding data are lacking. Here we use site-directed placement of 19 F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. 19 F probes were introduced individually to S129 and L150 near the S4–S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of 19 F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: ( i ) as a channel blocker at the base of the selectivity filter, and ( ii ) as a modulator to restrict the pivot motion at the S4–S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1609939113
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2016
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
    Location Call Number Limitation Availability
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  • 2
    Online Resource
    Online Resource
    Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4–S5 linker in activation and slow inactivation
    Proceedings of the National Academy of Sciences ; 2021
    In:  Proceedings of the National Academy of Sciences Vol. 118, No. 28 ( 2021-07-13)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 118, No. 28 ( 2021-07-13)
    Abstract: Voltage-gated sodium (Na V ) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4–S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4–S5 linker and S6 helix in a conformation-dependent manner. Using 19 F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4–S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic Na V channels seems to have a mechanism similar to the recently proposed “door-wedge” action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic Na V channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.2102285118
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2021
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 3
    Online Resource
    Online Resource
    NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA
    Proceedings of the National Academy of Sciences ; 2008
    In:  Proceedings of the National Academy of Sciences Vol. 105, No. 43 ( 2008-10-28), p. 16537-16542
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 105, No. 43 ( 2008-10-28), p. 16537-16542
    Abstract: Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E ) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0805501105
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2008
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
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