Electrophysiological and Pharmacological Analyses of Nav1.9 Voltage-Gated Sodium Channel by Establishing a Heterologous Expression System

Abstract

Nav1. 9 voltage-gated sodium channel is preferentially expressed in peripheral nociceptive neurons. Recent progresses have proved its role in pain sensation, but our understanding of Nav1.9, in general, has lagged behind because of limitations in heterologous expression in mammal cells. In this work, functional expression of human Nav1.9 (hNav1.9) was achieved by fusing GFP to the C-terminal of hNav1.9 in ND7/23 cells, which has been proved to be a reliable method to the electrophysiological and pharmacological studies of hNav1.9. By using the hNav1.9 expression system, we investigated the electrophysiological properties of four mutations of hNav1.9 (K419N, A582T, A842P, and F1689L), whose electrophysiological functions have not been determined yet. The four mutations significantly caused positive shift of the steady-state fast inactivation and therefore increased hNav1.9 activity, consistent with the phenotype of painful peripheral neuropathy. Meanwhile, the effects of inflammatory mediators on hNav1.9 were also investigated. Impressively, histamine was found for the first time to enhance hNav1.9 activity, indicating its vital role in hNav1.9 modulating inflammatory pain. Taken together, our research provided a useful platform for hNav1.9 studies and new insight into mechanism of hNav1.9 linking to pain.

Discovery of a Novel Nav1.7 Inhibitor From Cyriopagopus albostriatus Venom With Potent Analgesic Efficacy

“…Additionally, plasmids β1- and β2-eGFP encoding the human β1 and β2 subunits, respectively, were co-transfected with those encoding WT Na v 1.7 and Na v 1.7 mutations in HEK293T cells. Human Na v 1.9 was transfected into ND7/23 cells according to a previous report ( Zhou et al, 2017 ). HEK293T and ND7/23 cells were grown under standard tissue culture conditions (5% CO 2 , 37°C) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS).…”

Newly Discovered Action of HpTx3 from Venom of Heteropoda venatoria on Nav1.7 and Its Pharmacological Implications in Analgesia

“…For heterologous expression, the vector plasmids containing wild-type (WT) Na v 1.2-Na v 1.7 and mutant plasmids were transiently transfected into HEK293T cells (China Center for Type Culture Collection, Wuhan, China) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The vectors containing rNa v 1.8 and hNa v 1.9 were transiently transfected into the neuron-like ND7/23 cells (Spring Bioscience, Shanghai, China), which were more suitable for their heterologous expression [75,76]. In addition, the plasmids β1and β2-eGFP, which encode the β1-subunit and β2-subunit, respectively, were co-transfected with those encoding WT Na v 1.7 and its mutants into the HEK293T cells.…”

Purification and Characterization of JZTx-14, a Potent Antagonist of Mammalian and Prokaryotic Voltage-Gated Sodium Channels

“…We tested the activity of JZTx-14 on mammalian Na V s Na V 1.2–1.9 heterologously expressed in HEK293T or ND7/23 cells. The Na V 1.8 and Na V 1.9 channels were chimeras, as described in our previous studies [ 30 , 31 ]. Their currents were elicited by 50-ms depolarizations to 0 mV from the holding potential of −80 mV.…”

“…The mammalian Na V cDNA clones (Na V 1.2–Na V 1.8) were from professor Theodore Cummins lab (Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA), and were cloned in the pCDNA3.1 or pCMV-blank vectors. The Na V 1.8/1.7L5 channel and the Na V 1.9-EGFP channel were as described in our previous studies [ 30 , 31 ]. Mutations were made using the site-directed mutation method.…”

“…Twenty-four hours after transfection, cells were ready for patch-clamp analysis. ND7/23 cells were used for Na V 1.9-EGFP chimeric channel expression and the conditions were as previously described [ 31 ].…”