Biophysical and Pharmacological Characterization of Nav1.9 Voltage Dependent Sodium Channels Stably Expressed in HEK-293 Cells.

Zhixin Lin,Sonia Santos,Neil A Castle,Karen Padilla,David Printzenhoff,Steven Barnes

doi:10.1371/journal.pone.0161450

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.

Highlights

Sodium flux through voltage-gated sodium (Nav) channels is a major contributor to action potential electrogenesis and propagation in excitatory cells [1,2,3]
Given its charge and location within the pore, we investigated if K799 influences inhibitor interaction with Nav1.9, in particular compounds that may interact with the local anesthetic binding site
To evaluate the impact of the K799N mutation on Nav1.9 pharmacology we examined potency of TC-N 1752 and the two local anesthetic agents, tetracaine and benzocaine since we have shown their effect is modulated by the local anesthetic binding site mutation, suggesting a pore interaction

Summary

Introduction

Sodium flux through voltage-gated sodium (Nav) channels is a major contributor to action potential electrogenesis and propagation in excitatory cells [1,2,3]. Several Nav channel subtypes like Nav1.7, Nav1.8 and Nav1.9 exhibit preferential expression in peripheral sensory neurons and have been reported to be important for conveying nociceptive sensory information from peripheral afferents to the central nervous system [7,8,9,10,11,12]. Because of their restricted expression profile and their proposed role in pain signaling, Nav1.7, Nav1.8 and Nav1.9 have received significant interest as potentially attractive targets for the development of novel pain therapeutics. These studies have shown that Nav1.9 has unique biophysical properties most notably activation at membrane potentials significantly more hyperpolarized than those required other neuronal Nav channels, and a much slower inactivation process results in persistent inward currents near the threshold membrane potential for action potential firing and possibly plays a role in regulating resting potential and amplifying depolarizing responses to subthreshold stimuli [9, 12, 14]

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