Heterologous expression of NaV1.9 chimeras in various cell systems
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SCN11A encodes the voltage-gated sodium channel NaV1.9, which deviates most strongly from the other eight NaV channels expressed in mammals. It is characterized by resistance to the prototypic NaV channel blocker tetrodotoxin and exhibits slow activation and inactivation gating. Its expression in dorsal root ganglia neurons suggests a role in motor or pain signaling functions as also recently demonstrated by the occurrence of various mutations in human SCN11A leading to altered pain sensation syndromes. The systematic investigation of human NaV1.9, however, is severely hampered because of very poor heterologous expression in host cells. Using patch-clamp and two-electrode voltage-clamp methods, we show that this limitation is caused by the C-terminal structure of NaV1.9. A chimera of NaV1.9 harboring the C terminus of NaV1.4 yields functional expression not only in neuronal cells but also in non-excitable cells, such as HEK 293T or Xenopus oocytes. The major functional difference of the chimeric channel with respect to NaV1.9 is an accelerated activation and inactivation. Since the entire transmembrane domain is preserved, it is suited for studying pharmacological properties of the channel and the functional impact of disease-causing mutations. Moreover, we demonstrate how mutation S360Y makes NaV1.9 channels sensitive to tetrodotoxin and saxitoxin and that the unusual slow open-state inactivation of NaV1.9 is also mediated by the IFM (isoleucine–phenylalanine–methionine) inactivation motif located in the linker connecting domains III and IV.
KeywordsSodium channel TTX resistance STX resistance Pain Heterologous expression Patch clamp SCN11A
- NaV channel
Voltage-gated sodium channel
R. Blum (Würzburg, Germany) generously provided the coding sequence of SCN11A. We also thank C. Nau (Lübeck, Germany) and Ch. Fahlke (Jülich, Germany) for providing us with ND7/23 and HEK 293T cells, respectively, and A. Roßner and A. Kessler for the technical assistance.
E.L. was partially supported by German Research Foundation (DFG, LE2338/3-1).
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.
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