A Chimeric NaV1.8 Channel Expression System Based on HEK293T Cell Line.

Xi Zhou,Zhonghua Liu,Songping Liang,Dongfang Tang,Ping Chen,Cheng Tang,Yunxiao Zhang

doi:10.3389/fphar.2018.00337

Abstract

Among the nine voltage-gated sodium channel (NaV) subtypes, NaV1.8 is an attractive therapeutic target for pain. The heterologous expression of recombinant NaV1.8 currents is of particular importance for its electrophysiological and pharmacological studies. However, NaV1.8 expresses no or low-level functional currents when transiently transfected into non-neuronal cell lines. The present study aims to explore the molecular determinants limiting its functional expression and accordingly establish a functional NaV1.8 expression system. We conducted screening analysis of the NaV1.8 intracellular loops by constructing NaV chimeric channels and confirmed that the NaV1.8 C-terminus was the only limiting factor. Replacing this sequence with that of NaV1.4, NaV1.5, or NaV1.7 constructed functional channels (NaV1.8/1.4L5, NaV1.8/1.5L5, and NaV1.8/1.7L5, respectively), which expressed high-level NaV1.8-like currents in HEK293T cells. The chimeric channel NaV1.8/1.7L5 displayed much faster inactivation of its macroscopic currents than NaV1.8/1.4L5 and NaV1.8/1.5L5, and it was the most similar to wild-type NaV1.8 expressed in ND7/23 cells. Its currents were very stable during repetitive depolarizations, while its repriming kinetic was different from wild-type NaV1.8. Most importantly, NaV1.8/1.7L5 pharmacologically resembled wild-type NaV1.8 as revealed by testing their susceptibility to two NaV1.8 selective antagonists, APETx-2 and MrVIB. NaV chimeras study showed that at least the domain 2 and domain 4 of NaV1.8 were involved in binding with APETx-2. Our study provided new insights into the function of NaV1.8 intracellular loops, as well as a reliable and convenient expression system which could be useful in NaV1.8 studies.

Highlights

Mammalian voltage-gated sodium channel (NaV) is composed of four homologous domains, each domain contains six transmembrane segments, in which the first four segments (S1 – 4) construct the voltage sensing module (VSM) and the last two (S5 – 6) constitute the pore module (PM) (Catterall, 2012, 2014b)
The ion channels and receptors represented two primary drug targets in the cell membrane, and their high-level functional expression in mammalian cell lines was of particular importance for their pharmacological and biophysical researches
As in the case of M3 muscarinic acetylcholine receptor (M3R), its functional expression in mammalian cells were substantially increased by codon optimization, fusing tags with the protein, and using virus as the transfection vector (Romero-Fernandez et al, 2011)

Summary

Introduction

Mammalian NaV is composed of four homologous domains, each domain contains six transmembrane segments, in which the first four segments (S1 – 4) construct the VSM and the last two (S5 – 6) constitute the PM (Catterall, 2012, 2014b). PMs from all four domains of the channel construct the central ion conducting pathway, which is surrounded by VSMs. When the membrane is depolarized, the S4 segment, which carries the gating charges, moves outward to Abbreviations: NaV, voltage-gated sodium channel; PM: pore module; VSM: voltage sensing module. Nine NaV subtypes have been characterized so far, and their roles in pathological conditions such as epilepsy, pain, ataxia and so on, have been verified by numerous studies (Catterall, 2014a; Emery et al, 2016; Schwarz et al, 2016). Several NaV1.8 selective antagonists characterized in recent years showed analgesic effect in animal pain models (Jarvis et al, 2007; Bagal et al, 2015; Payne et al, 2015)

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Results

Conclusion