title: Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.
abstract: The voltage dependent sodium channel Nav1.9, is expressed preferentially in peripheral sensory neurons and has been linked to human genetic pain disorders, which makes it target of interest for the development of new pain therapeutics. However, characterization of Nav1.9 pharmacology has been limited due in part to the historical difficulty of functionally expressing recombinant channels. Here we report the successful generation and characterization of human, mouse and rat Nav1.9 stably expressed in human HEK-293 cells. These cells exhibit slowly activating and inactivating inward sodium channel currents that have characteristics of native Nav1.9. Optimal functional expression was achieved by coexpression of Nav1.9 with β1/β2 subunits. While recombinantly expressed Nav1.9 was found to be sensitive to sodium channel inhibitors TC-N 1752 and tetracaine, potency was up to 100-fold less than reported for other Nav channel subtypes despite evidence to support an interaction with the canonical local anesthetic (LA) binding region on Domain 4 S6. Nav1.9 Domain 2 S6 pore domain contains a unique lysine residue (K799) which is predicted to be spatially near the local anesthetic interaction site. Mutation of this residue to the consensus asparagine (K799N) resulted in an increase in potency for tetracaine, but a decrease for TC-N 1752, suggesting that this residue can influence interaction of inhibitors with the Nav1.9 pore. In summary, we have shown that stable functional expression of Nav1.9 in the widely used HEK-293 cells is possible, which opens up opportunities to better understand channel properties and may potentially aid identification of novel Nav1.9 based pharmacotherapies.
authors.affiliation: Neuroscience and Pain Research Unit, Pfizer Inc., Durham, North Carolina, United States of America.
authors.forename: Zhixin
authors.identifier: http://orcid.org/0000-0002-5458-3817
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Neuroscience and Pain Research Unit, Pfizer Inc., Durham, North Carolina, United States of America.
authors.forename: Sonia
authors.initials: S
authors.lastname: Santos
authors.affiliation: Neuroscience and Pain Research Unit, Pfizer Inc., Durham, North Carolina, United States of America.
authors.forename: Karen
authors.initials: K
authors.lastname: Padilla
authors.affiliation: Neuroscience and Pain Research Unit, Pfizer Inc., Durham, North Carolina, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Printzenhoff
authors.affiliation: Neuroscience and Pain Research Unit, Pfizer Inc., Durham, North Carolina, United States of America.
authors.forename: Neil A
authors.initials: NA
authors.lastname: Castle
mesh_terms: D000595:Amino Acid Sequence; D000779:Anesthetics, Local / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D015640:Ion Channel Gating / Q000187:drug effects; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D062558:NAV1.9 Voltage-Gated Sodium Channel / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D062687:Sodium Channel Agonists / Q000737:chemistry / Q000494:pharmacology; D026941:Sodium Channel Blockers / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000779:Anesthetics, Local; D062558:NAV1.9 Voltage-Gated Sodium Channel; D062687:Sodium Channel Agonists; D026941:Sodium Channel Blockers; D008239:Lysine