title: Properties of wild-type and fluorescent protein-tagged mouse tetrodotoxin-resistant sodium channel (Na V 1.8) heterologously expressed in rat sympathetic neurons.
abstract: The tetrodotoxin (TTX)-resistant Na(+) current arising from Na(V)1.8-containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. Here, we show that injection of cDNA encoding mouse Na(V)1.8 into the nuclei of rat superior cervical ganglion (SCG) neurons results in TTX-resistant Na(+) currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed Na(V)1.8 Na(+) channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed Na(V)1.8 channels was shifted about 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of Na(V)1.8 did not substantially affect functional expression in SCG neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged Na(V)1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum, thus providing a basis for poor functional expression in clonal cell lines. Our results establish SCG neurons as a favorable surrogate for the expression and study of molecularly defined Na(V)1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of Na(V)1.8 with a biophysical phenotype identical to that found in sensory neurons.
authors.affiliation: Department of Physiology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
authors.forename: Geoffrey G
authors.initials: GG
authors.lastname: Schofield
authors.forename: Henry L
authors.initials: HL
authors.lastname: Puhl
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Ikeda
mesh_terms: D000818:Animals; D002469:Cell Separation; D002999:Clone Cells / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004351:Drug Resistance; D004594:Electrophysiology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D062559:NAV1.8 Voltage-Gated Sodium Channel; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D015222:Sodium Channels / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000378:metabolism; D013564:Sympathetic Nervous System / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology*; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D062559:NAV1.8 Voltage-Gated Sodium Channel; C568317:SCN10A protein, human; C568318:Scn10a protein, mouse; C568319:Scn10a protein, rat; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D049452:Green Fluorescent Proteins; D013779:Tetrodotoxin