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

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.


INTRODUCTION
Voltage-gated sodium channels (VGSCs) are a cluster of important transmembrane proteins, that play crucial roles for initiation and propagation of action potential (AP) in excitable tissues, including heart, brain and peripheral nerves (Catterall, 2000(Catterall, , 2012. All VGSCs are composed of a pore-forming α-subunit and one/two auxiliary β-subunits. To date, nine α-subunits (Na v 1.1-1.9, also referred as channels) and four β-subunits (β1-β4) have been identified in mammals (Bant and Raman, 2010;Catterall, 2014Catterall, , 2015. Of the nine VGSC isoforms, Na v 1.9 is preferentially expressed in peripheral nociceptive neurons, as well as visceral afferents (Padilla et al., 2007;Hockley et al., 2014;Dib-Hajj et al., 2015). It exhibits unique biophysical properties that include activation near resting membrane potential, slow activation and inactivation kinetics, generation of large persistent currents in low depolarizing voltages and large window current (Cummins et al., 1999;Dib-Hajj et al., 2002, 2015. These make Na v 1.9 act as a threshold channel in AP firing, amplifying sub-threshold stimulus that leads to AP burst and increases repetitive firing (Herzog et al., 2001;Dib-Hajj et al., 2002, 2015. Preclinical studies in pain animal models suggest that Na v 1.9 might be involved in pain-signaling pathway (Lolignier et al., 2011(Lolignier et al., , 2015Hockley et al., 2014). Using Na v 1.9 null or knock down rodent models, it has been proved that Na v 1.9 contributes to regulating the excitability of sensory neurons (e.g., dorsal root ganglion (DRG) neurons) and plays a crucial role in inflammation-induced hyperalgesia (Baker et al., 2003;Ostman et al., 2008). More recently, genetic and functional findings illustrate that hNa v 1.9 mutations cause human pain diseases, which provides directly clinical evidences linking hNa v 1.9 to human pain. To date, a total of 16 mutations of hNa v 1.9 have been identified in individuals with rare genetic pain disorders and a population of patients with painful peripheral neuropathy (Leipold et al., 2013Zhang et al., 2013;Huang et al., 2014Huang et al., , 2017Han et al., 2015Han et al., , 2017Okuda et al., 2016;Phatarakijnirund et al., 2016). Patch-clamp analysis revealed that these are gainof-function mutations, which are expected to correlate with the clinical phenotype of increased sensitivity to pain (Leipold et al., 2013Zhang et al., 2013;Huang et al., 2014;Han et al., 2015). However, two mutations, L811P and L1302F, are associated with congenital insensitivity to pain, a phenotype contrary to functional evidences (Leipold et al., 2013;Woods et al., 2015;Huang et al., 2017). Huang et al. suggested that L811P and L1302F evoked large depolarization of resting membrane potential and impaired action potential generation (Huang et al., 2017). Therefore, all these studies validate the direct correlation between hNa v 1.9 and human pain.
Studies have proved that inflammatory mediators released from injured and inflammatory cells modulate Na v 1.9 currents to maintain inflammation-induced hyperalgesia (Baker et al., 2003;Priest et al., 2005;Amaya et al., 2006;Ostman et al., 2008), which probably supports a role for Na v 1.9 in inflammatory and neuropathic pain. Treatment of rat DRG neurons with prostaglandin E2 (PGE 2 ) was found to increase the amplitude of Na v 1.9 current (Rush and Waxman, 2004). Maingret et al. has indicated that Na v 1.9 is up-regulated by the concerted action of inflammatory soup, a well-defined mixture of histamine, PGE 2 , bradykinin (BK), norepinephrine and ATP. Increased hNa v 1.9 current may contribute to lowering the threshold for AP firing and therefore render nociceptor hyperexcitable during peripheral inflammation (Maingret et al., 2008). It is worth noting that these results are based on acute isolation of rodent DRG neurons and inevitably affected by other VGSC subtypes expressed in the same neurons. Interestingly, anti-inflammatory medication reduces the frequency of painful events caused by Na v 1.9 mutation (Zhang et al., 2013;Leipold et al., 2015), which is consistent with the effects of inflammatory mediators.
Although, considerable progress has been made in recent years, our understanding of Na v 1.9 in general, has lagged behind. One of the primary reasons is that, it is difficult to study Na v 1.9 in heterologous expression systems, since it is only expressed at low levels and is hard to study in isolation in native neurons (Dib-Hajj et al., 2015;Goral et al., 2015;Lin et al., 2016). Meanwhile, the above findings have raised some questions. Even for the same Na v 1.9 mutations, studies in human mutant channel expressed in ND7/23 cells and in mouse mutant channel in DRG neurons have brought to striking differences in gating profiles because of specific sequence divergence, cell types used for channel expression or different recording protocols. In addition, the functions of five mutant channels have not been explored yet. The precise effect of inflammatory mediators on hNa v 1.9 and the underlying mechanisms needed to be investigated further. Generally speaking, a heterologous system expressing high level and stable Na v 1.9 current is still valuable; intensive electrophysiological analyses by using this heterologous expression system may contribute in part to answering these questions.
In the following study, we succeeded in achieving a functional expression of hNa v 1.9 channel in heterologous cells by fusing GFP to the C-terminal of hNa v 1.9 sequence, which provides a powerful platform for the electrophysiological studies of hNa v 1.9. Here, by applying this expression system, we systematically investigated the biophysical characteristics of wild type (WT) and mutant hNa v 1.9 channels, as well as elucidated the effects and mechanisms of inflammatory mediators on hNa v 1.9. This heterologous expression system of hNa v 1.9 is useful for the electrophysiological and pharmacological studies of this channel.

Plasmid Constructs and Mutagenesis
The deleted stop codon cDNA of hNa v 1.9, using KpnI and SmaI enzyme site, was subcloned into the pEGFP-N1 vector (Clontech). Mutations were constructed by using the Quick-change II XL Site-directed Mutagenesis kit (Agilent Technologies) according to the manufacturer's instruction. All mutations were verified by DNA sequencing.

Cell Culture and Transfection
ND7/23 and HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin in a 5% CO 2 incubator at 37 • C; CHO-K1 cells were maintained in 45% DMEM and 45% F12K supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin in a 5% CO 2 incubator at 37 • C. Cells were trypsinized, diluted with culture medium, and grown in 35 mm dishes. When grown to 90% confluence, cells were transfected with hNa v 1.9-GFP or hNa v 1.9-GFP mutations using the transfection kit X-treme GENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions (4 µg hNa v 1.9-GFP or hNa v 1.9-GFP mutant plasmid and 8 µl X-tree GENE HP DNA). Transfected cells were first maintained at 37 • C for 24 h, with 5% CO 2 and then incubated at 29 • C with 5% CO 2 for 20 h, before use in electrophysiology experiments. The green fluorescent was used for visual identification of individual transfected cells.
To generate activation curves, cells were held at −120 mV and stepped to potentials of −100 to +50 mV in 5-mV or 10-mV increments for 50-ms every 5-s. The G-V curves were obtained by calculating the conductance (G) at each voltage (V) using the equation G=I/(V-V rev ), with V rev being the reversal potential determined for each cell individually. G-V curves were fitted using a Boltzmann equation: y = 1/ (1 + exp[(V a − V)/κ]) in which V a , V, and κ represented midpoint voltage of kinetics, test potential and slope factor, respectively. The repetition interval was 5-s.
Voltage dependent steady-state fast inactivation was measured with a series of 1,000-ms pre-pulses (−120 to 0 mV in 10-mV increments), followed by a 50-ms depolarization to −40 mV to assess the available non-inactivated currents, and the repetition interval was 10-s. Steady-state slow-inactivation was determined with 30 s pre-pulses ranging from −120 to −40 mV followed by a 100 ms pulse to −100 mV to remove fast-inactivation. Remaining available channels were activated by a 50 ms test pulse to −40 mV. The repetition interval was 40-s. Peak inward currents at the test pulse were normalized to the maximal inward current and fit with Boltzmann functions: represents the inactivating pre-pulse potential, V h is the midpoint of the steady-state fast-inactivation or slow-inactivation, A is the minimal channel availability, and κ is the slope factor.
The ramp current was measured by a small slow ramp depolarization protocol, which started from the holding potential of −100 mV and steadily increased to 20 mV over 600-ms at the rate of 0.2 mV/ms. The repetition interval was 10-s.
The deactivation current of each channel was measured using a 25-ms depolarization to −40 mV, followed by a 100-ms repolarizing pulse to potentials ranging from −120 to −80 mV in steps of 5-mV with a repetition interval of 10-s. The deactivation currents were fitted with a single exponential function according to: where I 0 is the maximal current amplitude, I ∞ is the current remaining after infinite time, t is the time and τ d is the deactivation time constant.
Dose response curves of histamine were fitted using the following Hill logistic equation: y = f max − (f max − f min )/(1 + (x/EC 50 ) n ), where f max and f min represent the maximum and minimum response of channel to histamine, the f min was set to 0, x represents histamine concentration and n is an empirical Hill coefficient.

Drug Treatment
One micromolar TTX were applied in all experiments except special description. In measurements examining the effects of histamine receptor inhibitors on the histamine-enhanced hNa v 1.9 current, the ND7/23 cells expressing hNa v 1.9-GFP were pretreated for 30 min with 50 nM mepyramine (Abcam), 100 µM ranitidine (Abcam) or 1 µM thioperamide (Abcam), and they were also present when histamine was applied.
For electrophysiology experiments, the stock solution of drugs was diluted with fresh bath solution to a concentration of 10-fold of the interested concentration, 30 µl of the concentrated drugs was diluted into the recording chamber (containing 270 µl bath solution) far from the recording pipet (the recording cell) and was mixed by repeatedly pipetting to achieve the specified final concentration.
All compounds were dissolved in DMSO (TTX and PGE 2 ) or water (histamine, BK, 5-HT, mepyramine, ranitidine and thioperamide) to make 1 mM-1 M stock solutions. The final concentration of DMSO did not exceed 0.2%, which was found to have no significant effect on sodium currents.

Data Analysis
Data were analyzed with Fit-Master (HEKA Elektronik), Igor-Pro (WaveMetrics, Lake Oswego, OR, USA) software and Prism 5 (GraphPad Software). Data are presented as mean ± S.E.M, and n is presented as the number of the separate experimental cells. Statistical significance was assessed with GraphPad Prism using the paired Student's t-test or one-way ANOVA with Tukey's Multiple Comparison Test. Statistical significance was accepted at P < 0.05.

Functional Expression and Characterization of hNa v 1.9 Channel in Heterologous Cells
It has been a challenge for the functional expression of Na v 1.9 in heterologous cells such as HEK293T, CHO-K1, ND7/23, and Xenopus oocytes. ND7/23 cells are hybrids of neuro-blastoma and dorsal root ganglia cells. It was confirmed that >97% of macroscopic Na v currents in ND7-23 cells is carried by TTXsensitive channels (Zhou et al., 2003;Rogers et al., 2016). As shown in Figures S1A,B, the endogenous currents of ND7/23 cells were completely blocked by 200 nM TTX. Recently, Vanoye et al. found that functional expression of Na v 1.9 was achieved in ND7/23 cells when cells were cultivated at lower temperature after transient transfection although the yield was rather poor (Vanoye et al., 2013). As shown in Figure 1A, ND7/23 cells were cultivated at 29 • C after the transient transfection of hNa v 1.9-encoding DNA. The Na v 1.9 currents, in the presence of 1 µM tetrodotoxin (TTX), were distinctly recorded at 50ms depolarizing steps with 10-mV increments from the holding potential of −120 mV. However, the peak current density was only −5.74 ± 1.44 pA/pF (n = 6) at the depolarizing voltage of −40 mV ( Figure 1B), which is too small to functional analysis. Goral et al. showed that a chimera of hNa v 1.9 harboring the C-terminal of rNa v 1.4 yielded big currents in heterologous cells (Goral et al., 2015). Yet, the gating properties of activation and inactivation of the chimera channel were significantly different from those of WT-hNa v 1.9. It is not an optimal method to study Na v 1.9 gating and functions of mutant channels. In an attempt to studying the expression and translocation of hNa v 1.9, we linked GFP to the C-terminal of hNa v 1.9 sequence to construct a fusion protein channel (hNa v 1.9-GFP). After transfection in ND7/23 cells, this channel mediated large currents activated by a serial of 50-ms depolarizing pulses (ranging from −100 mV to 40 mV with 5-mV increments) from the holding potential of −120 mV in the presence of 1 µM TTX which completely blocked TTX-S currents in ND7/23 cells ( Figure 1A). The current density was −34.21 ± 8.83 pA/pF (n = 6) at the depolarizing voltage of −40 mV when cells were cultivated at 37 • C after transient transfection ( Figure 1B); lower temperature culture (29 • C) also enhanced the current density to −80.23 ± 8.4 pA/pF (n = 12) ( Figure 1B), which is consistent with its role in the perception of cold pain under normal and pathological conditions Lolignier et al., 2015). From the current-voltage (I-V) curves as shown in Figure 1C, the fusion of GFP to hNa v 1.9 and lower temperature culture did not alter I-V curve shape and the voltage-dependence of activation. Namely, the initial activation voltage (∼-70 mV), the maximum activation voltage (∼-40 mV) and the reversal potential of hNa v 1.9 current in ND7/23 cells (∼35 mV) were not changed. The current was singularly stable, because the current amplitude showed no obvious alteration within 10 min recording ( Figure 1D). In addition, the hNa v 1.9-GFP channel also had functional expression in HEK 293T and CHO-K1 cells, and both yielded current signals typical for Na v 1.9 channel (Figures 1E-G). Note that the currents in ND7/23 cells expressing hNa v 1.9-GFP channels could not affected by MrVIB that is a known blocker of Na v 1.8 (MrVIB/Control = 0.99 ± 0.01, n = 3) but not Na v 1.9, indicating that these currents were actually mediated by hNa v 1.9 without being mixed by Na v 1.8 ( Figure 1H; Ekberg et al., 2006). Because of larger currents observed and the DRG background of ND7/23 cells which contains accessory Na + channel β1and β3 subunits (Rogers et al., 2016), our studies were performed in this cell line.
After establishing a dependable heterologous expression of hNa v 1.9, we proceeded with determining more detailed biophysical properties of this channel. As shown in Figure 1A, representative voltage-dependent inward currents of hNa v 1.9 exhibited slow activation and inactivation at low potentials. The voltage-dependence of activation and steady-state fast inactivation for hNa v 1.9 generated a large overlap (window current) (Figure 2A), and the midpoints of activation (V a ) and steady-state fast inactivation (V h ) were −53.3 ± 0.9 and −69.4 ± 0.7 mV (Table 1), respectively. The steady-state slowinactivation, a process that inactivates the channel much more slowly than fast-inactivation, had the midpoint value of −77.1 ± 0.8 mV (Figure 2A and Table 1). We also measured the recovery from inactivation (repriming) of hNa v 1.9 after a 50-ms depolarization to −40 mV and found it exhibited fast priming, and the time constant was 13.7 ± 0.7 ms ( Figure 2B). The protocol was shown in Figure 2Ca. These data were consistent with previous results of WT-Na v 1.9 currents observed in DRG neurons or ND7/23 cells (see Table 1).
Inactivation is an intrinsic property for VGSCs. There are two kinds of inactivation. Open-state inactivation (OSI) may occur from the open state at strongly depolarized membrane potentials, while closed-state inactivation (CSI) may be generated from pre-open closed states at hyperpolarized membrane potentials (Armstrong, 2006;Bähring and Covarrubias, 2011). The OSI of Na v 1.9 is extremely slow, generating persistent current. Nevertheless, the CSI of Na v 1.9 still maintains unkown. In this work, we investigated for the first time the development of CSI of hNa v 1.9 using a standard development of inactivation voltage protocol (Figure 2Cb). As shown in Figure 2B, it displayed a slow rate for the development of CSI, where only 18% current was inactivated at a 1,024-ms pre-pulse at −100 mV. The slow rate for CSI development indicated that the transition from the closed state to closed-inactivated state is slow for hNa v 1.9. Consequently, the channel is less-likely to undergo closedstate inactivation during slow depolarization. Cummins et al. proposed that slow development of CSI contributes to the generation of large ramp current in VGSCs (Cummins et al., 1998). Indeed, a large ramp current of hNa v 1.9 in ND7/23 cells was elicited in response to slow ramp depolarization (0.2 mV/ms), which was 32.2 ± 1.0% (n = 16) of the peak transient current of the same cells. The voltage at which the peak of the ramp current was produced was −51.9 ± 1.1 mV (n = 16) ( Figure 2D and Table 2). These data prompted that hNa v 1.9 has capability to open at voltages close to membrane potential. Our study provided more solid evidences to support that Na v 1.9 serves as a subthreshold amplifier.
Functional Analysis of hNa v 1.9 Mutations by the hNa v 1.9-GFP Heterologous Expression System Of the 16 mutations of hNa v 1.9 causing human pain disorders, five mutations (K419N, A582T, A681D, A842P, and F1689L, their location in the Na v 1.9 sequence are shown in Figure 3A) associated with painful peripheral neuropathy were not functionally analyzed (Huang et al., 2014). We measured the biophysical characteristics of four mutants in ND7/23 cells (of the five mutants, A681D was not functionally expressed in ND7/23 cells). As shown in Figure 3, all the four mutants (K419N, A582T, A842P, and F1689L) positively shifted the steady-state fast inactivation significantly and had no obvious effect on activation, resulting in larger window currents (Figures 3B-E); they did not change the deactivation time significantly, compared with the WT-channel ( Figure 3F); except F1689L, the other three mutations had augmented ramp currents ( Figure 3G). Quantified biophysical parameters for FIGURE 1 | Functional expression and characterization of hNa v 1.9 in heterologous cells. (A) Representative inward currents of hNa v 1.9 or hNa v 1.9-GFP transiently transfected in ND7/23 cells cultivated at 29 • C. Currents were evoked by depolarization from a holding potential of −120 mV up to −10 mV in 10-mV steps. Note that the data were recorded 5 min after establishing whole-cell configuration. (B) Comparison of peak current densities of hNa v 1.9 and hNa v 1.9-GFP in ND7/23 cells cultivated at 37 • or 29 • C. *p < 0.05; **p < 0.01; ***p < 0.001. (C) Current-voltage relationships measured from channels indicated. (D) The stability of hNa v 1.9-GFP current in ND7/23 cells that elicited by a 50-ms depolarization to −50 mV from a holding potential of −120 mV. Data sweeps were acquired at 0.1 Hz. The inset shows representative current traces before (sweep a) and after (sweep b) a 10-min recording. (E) Representative current traces in HEK 293T (upper) or CHO-K1 (below) cells transfected with hNa v 1.9-GFP. Currents were activated by depolarizing up to −10 mV in 10-mV steps from a holding potential of −120 mV. (F) Current density-voltage relationship of hNa v 1.9 in CHO-K1 cells. The cells were held at −120 mV and stepped to potentials of −100 to +40 mV in 5-mV increments for 50-ms every 5-s. (G) The steady state activation and inactivation of hNa v 1.9 in CHO-K1 cells. Data points were well fitted with the Boltzmann equation. Voltage-dependent activation was derived from the data in (F). Voltage-dependent steady-state fast inactivation of hNa v 1.9 in CHO-K1 cells was measured with a series of 1,000-ms prepulse (−120 to −10 mV in 10-mV increments), followed by a 50-ms depolarization to −40 mV to assess the available non-inactivated channels. (H) During pre-incubated with MrVIB 2 min, MrVIB had no effect on the hNa v 1.9-GFP current in ND7/23 cells. The current was activated by a 50-ms depolarization of −40 mV from the holding potential of −120 mV. 1 µM TTX were applied in all experiments.
Frontiers in Pharmacology | www.frontiersin.org To determine the recovery from fast inactivation, a 50-ms prepulse at −40 mV was used to move channels into the fast inactivated state, followed by a pulse at −120 mV with increased duration to allow channels to recover from fast inactivation before a 50-ms test pulse at −40 mV to measure the available current. To determine the development of CSI, from a holding potential of −120 mV, the cells were prepulsed to −100 mV (pre-open state) for increasing durations before a 50-ms test pulse to −40 mV. The repetition interval was 10-s. (D) Representative ramp currents of hNa v 1.9-GFP in response to slow depolarization (0.2 mV/ms) were normalized to the transient peak current and plotted as a function of membrane potentials. Data points are represented as mean ± S.E.M. One micro molar TTX were applied in all experiments. these channels are provided in Table 2. These data implied that the four mutations might enhance hNa v 1.9 activity and cause increased sensitivity to pain.

Histamine Enhances hNa v 1.9 Activity
Previous studies have shown that some pro-inflammatory mediators potentiate Na v 1.9 activity, resulting in enhanced nociceptor excitability (Maingret et al., 2008). In the present study, the effects of inflammatory mediators, including histamine, bradykinin (BK), PGE 2 , and 5-TH, were elucidated by using the hNa v 1.9-GFP heterologous expression system. First of all, we found histamine directly enhanced hNa v 1.9 current, which has not been reported in previous studies. As show in Figure 4A, adding 1 mM histamine to bath solution potently increased Na v 1.9 current in ND7/23 cells by 37.9 ± 6.3% (n = 6). Figure 4B shows the time course of the action of histamine at different concentrations. In contrast to 0.1 and 0.5 mM histamine, 1 mM histamine showed a rapid onset of action. Within 5 s, the action could reach the maximum and maintain stable. The action of histamine is reversible, since the peak current would rapidly return back to control level upon washing (Figures 4A,B). There are endogenous TTX-S Na + currents in ND7/23 cells we used and it was reported that histamine also strengthened the activation of TTX-S Na + currents ( Figure S1C). Although no detectable TTX-S Na + currents in ND7/23 cells were elicited at lower depolarized voltages (e.g., −50 mV), as show in Figure S2A, we still used CHO-K1 cells (no detectable endogenous TTX-S currents were examined) for the functional expression of hNa v 1.9-GFP. Significant enhancement of hNa v 1.9 peak current was observed in the presence of 1 mM histamine, while in control cells, no obvious currents was induced by histamine ( Figure S2A). In contrast to histamine, 1 mM 5-TH (5-TH/Control = 1.0 ± 0.01, n = 4, P > 0.05), 100 µM BK (BK/Control = 1.0 ± 0.01, n = 3) and 100 µM PGE 2 (PGE 2 /Control = 0.99 ± 0.01, n = 5) did not affect hNa v 1.9 current when they were added directly to bath solution ( Figure S2F). These data confirmed the action of histamine on hNa v 1.9.
The action of histamine was dose-dependent and voltagedependent. Higher concentrations of histamine led to greater enhancement of hNa v 1.9 current ( Figure 4C). However, the enhancement of hNa v 1.9 current by histamine was negatively correlated with depolarization voltages. For example, compared with control, 10 mM histamine treatment resulted in 25.3 ± 4.2, 161.8 ± 20.5, and 285.4 ± 5.0% (n = 3) of increments of hNa v 1.9 currents at −30, −50 and −70 mV, respectively. This was also observed in I-V curves (Figure 4D). In the presence of 1 mM histamine, larger currents occurred at lower depolarization   Leipold et al. (2015) study, these temperatures are recording temperatures.
Frontiers in Pharmacology | www.frontiersin.org FIGURE 3 | Functional analysis of hNa v 1.9 mutations by the hNa v 1.9-GFP heterologous expression system. (A) Membrane topology of Na v 1.9-GFP with the position of mutants. Hollowed circles (numbered 1-6) represent amino acid substitutions in hNaV1.9 from individuals with pain disorders. Four mutant channels were functionally expressed in ND7/23 cells. Compared with WT-hNa v 1.9, the steady-state fast inactivation of the K419N (B), A582T (C), A842P (D), and F1689L (E) mutants were significantly shifted in depolarization direction, but no obvious change of the voltage dependent activation was noticed. (F) The time courses of deactivation of the mutants were not significantly different from that of the WT-channel. (G) Representative ramp currents of the four mutants in response to slow depolarization (0.2 mV/ms) were normalized to the corresponding peak current acquired during activation protocol and plotted as a function of membrane potentials. One micro molar TTX were applied in all experiments.
In addition, we found that with the pretreatment of 50 nM mepyramine (selective H1 inverse agonist), 100 µM ranitidine (selective H2 antagonist) or 1 µM thioperamide The current-voltage curves indicate that 1 mM histamine changes the current-voltage relationship of hNa v 1.9. (E) 1 mM histamine significantly enhances the steady-state activation but not fast inactivation of hNa v 1.9. (F) Compared with control, 1 mM histamine enhances hNa v 1.9 ramp current and causes a hyperpolarized shift of the peak current. (G) Representative current traces show that 0.1 µM TTX completely block the current mediated by the S360Y hNa v 1.9 channel, which can be partially rescued by histamine. (H) Bars show when TTX concentration was raised to 10 µM, the recovery effect was significantly reduced for both 1 and 5 mM histamine. Data points are represented as mean ± S.E.M. ***p < 0.001. One micro molar TTX were applied in all experiments except special description.
(H3/H4 antagonist), the enhancement of hNa v 1.9 currents in ND7/23 cells by 1 mM histamine was not significantly different from that in the absence of these reagents ( Figure S2B). Quantified data are provided in Table 3. Notably these inhibitors themselves had no effect on hNa v 1.9 current in ND7/23 cells (Figures S2C-E). These data demonstrate that histamine might increase hNa v 1.9 current independently of histamine receptors.
In our study, we found histamine was able to counteract TTX blockage on TTX-S Na + currents ( Figure S1C). We therefore mutated the 360th serine of hNa v 1.9 to tyrosine, which makes this channel sensitive to TTX. In the presence of 0.1 µM TTX which completely block the current mediated by the S360Y mutant, the treatment of 1 or 5 mM histamine led to 14.2 ± 2.6% or 51.1 ± 3.7% recovery of currents triggered by a depolarization of −50 mV from a holding potential of −120 mV (Figures 4G,H). On the other hand, when TTX concentration was increased to 10 µM, the recovery effect was significantly reduced for both 1 and 5 mM histamine. These data implied that antagonistic effect might exist between TTX and histamine.

DISCUSSION
The hNa v 1.9-GFP Heterologous Expression System Provides a Promising Method for hNa v 1.9 Studies VGSCs are essential for the initiation and propagation of AP in excitable tissues such as nerves and muscles. They also participate in many pathological processes and are targets of clinical drugs (Fozzard et al., 2005;Imbrici et al., 2016). Therefore, studies regarding to VGSCs have been attracted interests from scientific area and pharmaceutical industry. Functional expression of human VGSCs in heterologous cells is an important and widelyused method to their studies. Of the nine subtypes of VGSCs (Na v 1.1-1.9), except Na v 1.9, the other eight subtypes have been achieved functional expression in heterologous cells, such as HEK 293, CHO, ND7/23, and Xenopus oocytes. For hNa v 1.9, the only feasible method is to express it heterologous in DRG neurons of Na v 1.9 −/− mice. However, expression of large plasmids in DRG neurons is discommodious and expensive because of the need to isolate and cultivate primary neurons. Furthermore, Na v 1.8 channels natively expressed in the neurons become the major TTX-R background to compromise a faithful investigation of Na v 1.9 currents, which makes this method difficult to be used widely (Huang et al., 2014;Goral et al., 2015). Therefore, great efforts have been made to establish convenient and reproducible methods. Evidences indicate that low temperature culture of transfected cells and modification of the Na v 1.9 C-terminal are helpful for functional expression of Na v 1.9 (Vanoye et al., 2013;Goral et al., 2015), which inspires us to generate a fusion protein channel (hNa v 1.9-GFP). Compared with previous expression system, this hNa v 1.9-GFP heterologous expression system has advantages as follows.
(1) It expresses large and stable current (>1 nA) in ND7/23 cells, which is essential to experimental data collection.
(2) Compared with WT-Na v 1.9 in ND7/23 cells or DRG neurons (see Table 1), it shows similar properties with respect to the voltage dependence of activation and inactivation.
(3) It is able to express functionally in different cell types, including ND7/23, HEK293T and CHO cells. In addition, it has advantages in convenience, high-efficiency, reproducibility and low-consumption, since the fused GFP serves as a fluorescent tracer and only one plasmid is used for transfection. Thus, with the hNa v 1.9-GFP heterologous expression system, we developed a feasible method suitable for hNa v 1.9 studies. For example, it helps to study the biophysical properties of hNa v 1.9 and functional analysis of clinical mutants. It is noted that more recently, a group from Pfizer Inc. established a Na v 1.9 stably expressed HEK 293 system by coexpression of Na v 1.9 with β1/β2 subunits (Lin et al., 2016), which was used for Na v 1.9 modulator screening. We are now constructing hNa v 1.9 stably expressed ND7/23 cell lines, which will be conveniently applied to highthroughput screening of therapeutic agents.

The Electrophysiological Mechanism Underlying the Gain of Function Mutations Causing Pain
The functions of four hNa v 1.9 mutant channels have been elucidated by using the hNa v 1.9-GFP heterologous expression system. We attempted to offer electrophysiological evidences linking mutant channel functions to phenotypes through a comparative analysis. Our data, together with those reported previously, show that these mutations result in depolarized shift of inactivation of hNa v 1.9, which therefore increases the open probability of hNa v 1.9 and leads to sensitivity to pain.
Histamine Directly Modulates the Activity of hNa v 1.9 Histamine is an important inflammatory mediator in pain processing and modulation. It can be released following mast cell degranulation by some inflammatory mediators including substance P, interleukin-1 and NGF (Dray, 1995;Woolf and Ma, 2007). In the peripheral nervous system, histamine directly sensitizes nociceptors and facilitates pain transmission, or indirectly causes pain via H1 receptors (Gorelova and Reiner, 1996;Mobarakeh et al., 2000;Kajihara et al., 2010;Yu et al., 2013). Histamine was discovered to upregulate Na v 1.8 expression via H2 receptor-mediated pathway, which might contribute to neuropathic pain (Vanoye et al., 2013;Yue et al., 2014).
Recent studies indicate that histamine also has ionotropic receptors apart from metabotropic GPCRs H1-H4 (Haas and Panula, 2003). It directly activates ionotropic GABAA receptor β3 homooligomers and potentiates GABA responses in αβ heterooligomers via a site homologous to the GABA site in αβγ receptors (Saras et al., 2008;Thiel et al., 2015;Hoerbelt et al., 2016). It also selectively potentiates the response of ASIC1a homomers to acidification in CHO cells (Nagaeva et al., 2016). Interestingly, our results revealed that histamine positively modulates hNa v 1.9 activity by promoting channel activation and potentiates ramp current amplitude. These data expand our understanding for the function of histamine and mechanism of pain processing by histamine. In the present study, histamine has a lower affinity for hNa v 1.9 than the metabotropic histamine receptor, and the EC 50 was 3.3 ± 0.5 mM at the depolarizing voltage of −50 mV. In spite of this, 0.5 mM histamine potently increased hNa v 1.9 current by ∼25% ( Figure 4C). However, in inflammatory tissue, histamine concentrations could reach 1 mM at transient and local inflammatory area (Adams and Lichtenstein, 1979;Benbarek et al., 1999). Nevertheless, our finding provides clues for illuminating the mechanism of hNa v 1.9 in inflammatory and neuropathic pain. Our data also reveal that histamine could directly act on hNa v 1.9, which could be explained as follows: (1) The action of histamine on hNa v 1.9 displays rapid association and dissociation kinetics as revealed by the fast onset of action and recovery upon washing. This might rule out indirect modulations by histamine targeting other proteins which can regulate hNa v 1.9 activity through protein-protein interaction, posttranslational modification or transcriptional regulation, since these processes often take effect within extended period of time.
(2) TTX and histamine had antagonistic effect on the S360Y hNa v 1.9 channel, which implied that they might possibly have partially overlapped binding site or the binding of histamine to hNa v 1.9 might likely alter the local structure of TTX binding site and then reduce TTX binding affinity to the S360Y channel. TTX is a known VGSC blocker by docking on extracellular pore of VGSCs (Penzotti et al., 1998). We speculate that the amino acid residues in pore region might possibly constitute the receptor of histamine in hNa v 1.9.

CONCLUSION
In conclusion, we have established a hNa v 1.9 heterologous expression system, which is effective and reliable for hNa v 1.9 studies. Using this system, we attempted to elucidate the electrophysiological mechanism of hNa v 1.9 in pain signaling and made new discoveries. The biophysical properties of hNa v 1.9 were systematically investigated in ND7/23 cells, which sheds new light on the role of Na v 1.9 as a threshold channel. Histamine can upregulate hNa v 1.9 activity through direct interaction, expanding our understanding of the importance of hNa v 1.9 in inflammatory and neuropathic pain. Due to the complexity of physiological and pathological conditions, we do not expect with only electrophysiological data the mechanistic basis of hNa v 1.9 in pain signaling would be fully expounded. Undoubtedly, our study has given new insight into the electrophysiological mechanism underlying hNa v 1.9 involving in human pain sensation.

ACKNOWLEDGMENTS
We thank Professor P. Alewood for generously and kindly providing MrVIB.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2017.00852/full#supplementary-material Figure S1. | (A) Representative current traces of ND7/23 cells expression of endogenous Na v currents before and after the application of 200 nM TTX (n = 5).