Functional Expression of Rat Nav1.6 Voltage-Gated Sodium Channels in HEK293 Cells: Modulation by the Auxiliary β1 Subunit

The Nav1.6 voltage-gated sodium channel α subunit isoform is abundantly expressed in the adult rat brain. To assess the functional modulation of Nav1.6 channels by the auxiliary β1 subunit we expressed the rat Nav1.6 sodium channel α subunit by stable transformation in HEK293 cells either alone or in combination with the rat β1 subunit and assessed the properties of the reconstituted channels by recording sodium currents using the whole-cell patch clamp technique. Coexpression with the β1 subunit accelerated the inactivation of sodium currents and shifted the voltage dependence of channel activation and steady-state fast inactivation by approximately 5–7 mV in the direction of depolarization. By contrast the β1 subunit had no effect on the stability of sodium currents following repeated depolarizations at high frequencies. Our results define modulatory effects of the β1 subunit on the properties of rat Nav1.6-mediated sodium currents reconstituted in HEK293 cells that differ from effects measured previously in the Xenopus oocyte expression system. We also identify differences in the kinetic and gating properties of the rat Nav1.6 channel expressed in the absence of the β1 subunit compared to the properties of the orthologous mouse and human channels expressed in this system.


Introduction
Voltage-gated sodium channels open and close on a millisecond time scale in response to changes in cell membrane potential. This activation/inactivation cycle mediates the transient influx of sodium ions that underlies the electrical action potential in most types of excitable cells [1]. Native sodium channels are thought to exist as heteromultimers comprising one large (,260 kDa) a subunit and either one or two smaller (33-36 kDa) auxiliary b subunits [2,3]. The a and b subunits of voltage-gated sodium channels are encoded by multi-gene families. Mammalian genomes contain nine genes for sodium channel a subunit isoforms, designated Na v 1.1 -Na v 1.9 [4,5], and four genes for sodium channel b subunits, designated b1-b4 [3].
Heterologous expression studies in Xenopus oocytes and transfected mammalian cells have identified the discrete functional roles of sodium channel a and b subunits. The a subunit forms the ion pore and contains structural domains that confer voltagedependent gating and the pharmacological properties of the channel [2]. The b subunits modify channel gating, regulate channel expression in the plasma membrane, and contribute to cell adhesion and cell-cell communication [3]. Individual neurons express multiple sodium channel a and b subunit isoforms and contain multiple functionally and pharmacologically distinct sodium channel subunit complexes [6,7,8]. However, the subunit compositions of native sodium channel complexes remain to be established.
The Na v 1.6 isoform is widely expressed in the central and peripheral nervous systems [4] and is the most abundant a subunit isoform in the brains of adult rats [9]. Na v 1.6 is the predominant isoform at nodes of Ranvier and in regions of brain axons associated with action potential initiation, as well as in presynaptic and postsynaptic membranes of the neocortex and cerebellum [10,11]. This pattern of expression implies important roles for Na v 1.6 sodium channels in both electrical and chemical signaling in the brain. A null mutation of the Na v 1.6 ( = Scn8a) gene in mice, termed ''motor endplate disease'' (med), impairs synaptic transmission at neuromuscular junctions and causes severe paralysis, muscle atrophy and juvenile death [12]. The coincident expression of the Na v 1.6 and b1 sodium channel subunits in many brain regions [7,13,14] suggests that Na v 1.6 sodium channels may coassemble in heteromultimeric complexes with the b1 subunit in vivo. Moreover, the reciprocal interaction between the Na v 1.6 and b1 subunits to promote neurite outgrowth and determine sodium channel localization [15] identifies a specific functional association between these two subunits.
The only previous studies of the modulatory effects of b subunits on rat Na v 1.6 sodium channels employed the Xenopus oocyte expression system [16,17]. Whereas the Xenopus oocyte system readily permits manipulation of the subunit structure of heteromultimeric channel complexes, the properties of channels in the oocyte membrane environment often differ from the properties of the same channels in native cells, presumably due to differences in membrane structure and post-translational modification [18].
Expression in human embryonic kidney-derived cell lines such as HEK293 offers an alternative system for the functional reconstruction of ion channel complexes that overcomes many of the limitations of the Xenopus oocyte system [19]. In light of the importance of the b1 subunit as a modulator of the function and pharmacology of rat Na v 1.6 sodium channels in the oocyte expression system [16,17,20] we undertook the present study to characterize the impact of coexpression with the rat b1 subunit on the functional properties of rat Na v 1.6 channels expressed in HEK293 cells. Here we describe the functional expression rat Na v 1.6 sodium channels in HEK293 cells alone or in combination with the rat b1 subunit and compare the properties of the resulting Na v 1.6 and Na v 1.6b1 channels. Our results identify modulatory effects of the b1 subunit on the kinetics and gating of Na v 1.6 sodium channels when expressed in HEK293 cells that differ from its effects on Na v 1.6 sodium channels expressed in the Xenopus oocyte system.

Sodium Channel Subunit cDNAs
The cloned rat Na v 1.6 voltage-gated sodium channel a subunit cDNA was provided by L. Sangameswaran (Roche Bioscience, Palo Alto, CA) and the cloned rat sodium channel b1 subunit cDNA was provided by W.A. Catterall (University of Washington, Seattle, WA). Each cDNA insert was subcloned into the vector pcDNA3.1 (Invitrogen, Carlsbad, CA) and the integrity of each clone was confirmed by DNA sequencing.

HEK-Na v 1.6 Cell Lines
HEK293 cells (CRL-1573, lot number 7681666) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured at 37uC in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from ATCC) in a humidified atmosphere of 5% CO 2 /95% air. Upon receipt cells were passaged twice and then frozen in DMEM+FBS with 5% dimethyl sulfoxide (DMSO) for future use; these stocks were considered to be at ''laboratory passage one.'' One day before transfection, cells (passage five, 0.5610 5 cells/100 ml growth medium without antibiotics) were transferred to a well of a 96-well plate and grown until ,80% confluent. Cells were transfected using Lipofectamine TM 2000 (Invitrogen) according to the manufacturer's protocol and either 254 ng of the Na v 1.6 plasmid or 200 ng of a mixture of the Na v 1.6 and b1 plasmids (1:2 molar ratio). Cells were diluted 1:10 into 6-well plates 24 h after transfection, incubated in culture medium for an additional 24 h, and then selected for 15 days with culture medium containing G418 (Invitrogen; 800 mg/ml). Clonal colonies (derived from a single cell; ,50 cells/colony) of G418-selected cells were isolated using cloning rings (Sigma-Aldrich, St. Louis, MO) and maintained in continuous culture under G418 selection (400 mg/ml) for electrophysiological characterization. Clonal cell lines giving cells with whole-cell peak transient sodium currents amplitudes $2000 pA were saved as frozen stocks for further use.

Analysis of Sodium Channel Subunit Expression
First-strand cDNA from transfected sodium current-positive cell lines, synthesized using the SuperScript TM III CellsDirect cDNA synthesis system (Invitrogen), was employed as the template in polymerase chain reaction (PCR) amplifications using pairs of oligonucleotide primers specific for the rat Na v 1.6 a subunit and the rat b1 subunits as described previously [21].

Electrophysiology
On the day prior to assay, cells were plated at low density in 35mm Petri dishes. For electrophysiological assays, cells (24-48 h after plating) were rinsed three times with extracellular perfusion medium that contained (mM): NaCl (140), KCl (5), CaCl 2 (2), MgCl 2 (1), and HEPES (10) at pH 7.40 (adjusted with 2 M NaOH). Whole-cell patch clamp recordings were conducted at room temperature (23-27uC) using an Axopatch 200B amplifier (Molecular Devices, Foster City, CA). Cells were perfused at ,350 ml/min with extracellular medium using a custom-fabricated passive perfusion manifold and a disposable plastic recording chamber insert (,240 ml volume; Warner Instruments, Hamden, CT). A stock solution of tetrodotoxin (TTX; Sigma Chemical Co., St. Louis, MO) was diluted to a final concentration of 0.5 mM in extracellular medium and applied through the perfusion system. The intracellular solution contained (in mM): NaCl (35), CsF (105), MgCl 2 (2), EGTA (10), and HEPES (10) at pH 7.20 (adjusted with 2 M CsOH). The final osmolarity of both solutions was 295-305 mOsm. Fire-polished patch electrodes were fabricated from borosilicate glass capillaries (1.5 mm O.D.; 1.0 mm I.D.; World Precision Instruments Inc., Sarasota, FL) using a P-87 puller (Sutter Instruments, Novato, CA) to give a resistance of 1-2 MV when filled with intracellular solution. The ground electrode was a bridge of 1% agar in extracellular medium in a glass pipet. Output signals were filtered at 2 kHz and sampled at 50 kHz (DigiData 1322A; Molecular Devices). Voltage errors were minimized using 70-80% series resistance compensation. Leak currents were corrected using the P/4 method [22]. Data were acquired using pClamp 10.2 (Molecular Devices) software. Following the establishment of a stable holding potential (2120 mV) under voltage clamp and measurement of cell capacitance, sodium currents were sampled using 40-ms step depolarizations to 215 mV at a frequency of 0.05 Hz for ,20 min to achieve stable sodium current amplitudes prior to initiating other protocols. To determine the voltage dependence of activation, cells were clamped at a membrane potential of 2120 mV and currents were measured during 40-ms depolarizing test pulses to potentials from 280 mV to 65 mV in 5-mV increments. Persistent currents, operationally defined as the residual current remaining at the end of a 40-ms depolarizing test pulse, were measured at test potentials giving maximal peak transient sodium current in each cell and normalized to the amplitude of the peak current in the same depolarization [23]. Efforts to detect and characterize resurgent currents involved 20ms step depolarizations from a holding potential of 2120 mV to a test potential of 30 mV followed by partial repolarizations for 100 ms to potentials from 20 mV to 280 mV in 5-mV increments. To determine the voltage dependence of steady-state fast inactivation, cells were clamped at a membrane potential of 2120 mV followed by a 100-ms conditioning prepulse to potentials from 2120 mV to 0 mV in 5-mV increments and then a 40-ms test pulse to 215 mV. For determinations of use dependence, cells were given trains of up to 100 5-ms conditioning prepulses from 2120 mV to 10 mV at 20 or 66.7 Hz followed by a 40-ms test pulse from 2120 mV to 215 mV.

Data Analysis
Data were acquired and analyzed using pClamp 10.2 (Molecular Devices) and Origin 8.1 (OriginLab Corp., Northampton, MA). For each cell, currents from activation experiments were converted to sodium conductances and plotted as a function of test potential using the Boltzmann equation [y = (A 1 -A 2 )/(1+e (x-x0)/ dx )+A 2 ] to give values for V 0.5 (potential causing half-maximal activation) and K (slope factor). Similarly, currents from steadystate inactivation experiments with each cell were plotted as a function of prepulse potential and fitted to the Boltzmann equation. Statistically significant effects of the b1 subunit were identified using Student's unpaired t-test by analysis in Prism 5.0 (GraphPad Software, La Jolla, CA).

Results
We obtained clonal cell lines that were stably transformed to express the rat Na v 1.6 sodium channel a subunit isoform either alone (HEK-Na v 1.6) or in combination with the rat b1 subunit (HEK-Na v 1.6b1). We confirmed the translational expression of the desired sodium channel subunits in each cell line by RT-PCR (data not shown). Table 1 summarizes the mean sodium current amplitudes and densities for each cell line. The amplitudes of peak transient sodium currents varied from 1 nA to 20 nA between individual cells in each cell line, with the majority of cells expressing currents in the 2-5 nA range. Fig. 1A shows representative sodium current traces recorded from HEK-Na v 1.6 and HEK-Na v 1.6b1 cells. Table 2 summarizes the kinetic parameters of sodium currents, such as those illustrated in Fig. 1A, obtained in recordings from multiple cells derived from each cell line. We used the time interval from membrane depolarization to the peak current as an indirect index of the rate of channel activation. Coexpression of the Na v 1.6 a subunit with the b1 subunit had no effect on the time to peak current. We obtained inactivation time constants (t inact ) for currents in each cell line by fitting the falling phase of peak transient currents to a firstorder decay model. Coexpression of Na v 1.6 with the b1 subunit significantly accelerated the decay of the peak current.
Previous studies of human [23,24] and mouse [25] Na v 1.6 sodium channels expressed in mammalian cell lines in the absence of auxiliary b subunits identified a significant persistent component of the sodium current ($10% of the peak current) that did not inactivate upon prolonged depolarization. In HEK293 cells  Table 2. Effects of coexpression with the b1 subunit on the kinetics of sodium current activation and inactivation and amplitudes of persistent currents in HEK293 cells expressing Na v 1.6 sodium channels. expressing the rat Na v 1.6 channel alone, the persistent component of current (measured at the end of a 40-ms depolarizing pulse as in Fig. 1A) was small (,1.5% of the peak transient current; Table 2). Coexpression of the Na v 1.6 a subunit with the b1 subunit had no effect on the amplitude of the persistent current. Figure 1B shows the effect of TTX (0.5 mM) on a representative sodium current recorded from HEK-Na v 1.6b1 cells. In this cell, which is typical of all cells in which the action of TTX was examined, TTX blocked the persistent current but did not completely block the peak current. The small residual peak currents measured in this and other cells in the presence of 0.5 mM TTX correspond to the TTX-resistant component of the endogenous multicomponent voltage-gated cation current described previously in the parental HEK293 cell line [26]. Figure 2 describes the effects of the b1 subunit on the voltagedependent activation of Na v 1.6 sodium channels expressed in HEK293 cells. Figure 2A shows families of sodium currents, recorded using the indicated pulse protocol, from an individual Figure 2. Voltage-dependent activation of Na v 1.6 and Na v 1.6b1 sodium channels expressed in HEK293 cells. (A) Representative current traces recorded from a HEK-Na v 1.6 cell using the indicated pulse protocol (left) and the plot of peak sodium current in these traces as a function of test potential (right). (B) Representative current traces recorded from a HEK-Na v 1.6b1 cell using the pulse protocol shown in Panel A and the plot of peak sodium current in these traces as a function of test potential. (C) Conductance -voltage plots for the activation of Na v 1.6 and Na v 1.6b1 channels. Peak sodium currents such as those in Panels A and B were transformed to conductances (G) using the equation G = I/(V t -V rev ), where I is the peak current, V rev is the reversal potential, and V t is the voltage of the test potential; conductances were then normalized to the maximum conductance (G max ) for that cell. Values are means of 64 (Na v 1. 6)  representative cell expressing rat Na v 1.6 sodium channels and current -voltage plot of the peak transient sodium currents in these traces. Figure 2B shows sodium current traces and currentvoltage relationships from an individual representative cell expressing Na v 1.6b1 sodium channels. Figure 2C shows conductance -voltage plots of data obtained from multiple cells expressing rat Na v 1.6 sodium channels either in the absence or presence of the b1 subunit, and Table 3 summarizes the statistical analysis of these data. The b1 subunit caused a statisticallysignificant 6.6-mV depolarizing shift in the midpoint potential (V 0.5 ) for channel activation that was accompanied by a significant increase in the slope factor (K) of the activation curve. Figure 3 shows the effects of the b1 subunit on the voltage dependence of steady-state fast inactivation of Na v 1.6 sodium channels expressed in HEK293 cells, and Table 3 summarizes the statistical analysis of these data. The b1 subunit caused a statistically-significant 5.2-mV depolarizing shift in the midpoint potential (V 0.5 ) for steady-state inactivation that was accompanied by a significant decrease in the slope factor (K) of the activation curve.
Evidence implicating Na v 1.6 sodium channels in high-frequency firing and the production of resurgent currents in Purkinje neurons [27] led us to assess the impact of coexpression with b1 subunit on these properties in HEK-293 cells expressing rat Na v 1.6 sodium channels. Figure 4 shows the effect of the b1 subunit on the stability of peak transient sodium currents following highfrequency stimulation. We applied 0-100 brief (5-ms) depolarizing pulses to 10 mV prior to a standard 40-ms test depolarization to 215 mV. Currents carried by Na v 1.6 sodium channels in the absence of b subunits declined rapidly to ,95% of the control current within the first 10 prepulses and then stabilized. Coexpression of Na v 1.6 with the b1 subunit did not affect the stability of the peak current measured using this protocol. We obtained similar results using a prepulse frequency of 66.7 Hz (data not shown). We also employed a conventional pulse protocol, involving partial membrane repolarization following a depolarizing pulse [28], in an effort to identify and characterize resurgent currents in these cells. However, we found no evidence for resurgent currents in either the absence or presence of the b1 subunit.

Discussion
HEK293 cells and cell lines derived from them are employed extensively as platforms for the transient or stable heterologous expression of neuroreceptor and ion channel proteins [19].
HEK293 cells were derived from human embryonic kidney cells by transformation with sheared adenovirus type 5 DNA [29]. Despite their origin HEK293 cells exhibit some characteristics of neurons, expressing more than 60 neuronal genes such as neurofilament proteins and neuroreceptor and ion channel subunits [19,30], including low levels of the sodium channel b1A auxiliary subunit [31]. Moreover, our previous electrophysiological studies confirmed the existence of small endogenous voltage-   gated cation currents in HEK293 cells. The TTX-sensitive component of the endogenous current, a presumptive sodium current, was associated primarily with the expression of the human Na v 1.7 sodium channel isoform [26].
We overexpressed the rat Na v 1.6 a subunit and the rat b1 subunit in HEK293 cells by transformation with the corresponding plasmids and selecting stably-transformed clonal cell lines that exhibited voltage-gated sodium currents under whole-cell patch clamp conditions with amplitudes .1 nA. Each of the cell lines described here exhibited sodium current densities more than tenfold greater than the density of the endogenous multicomponent cation currents in the parental HEK293 cell line [26]. Currents measured in the presence of 0.5 mM TTX (as in Fig. 1B) represent the TTX-resistant component of this current, which constitutes ,50% of the total endogenous current. The relative amplitudes of peak transient currents measured in the absence and presence of TTX therefore provide a direct assessment of the contamination of the total current by elements of the endogenous cation current. The high level of current expression in HEK-Na v 1.6 and HEK-Na v 1.6b1 cells ensured that the whole-cell sodium currents described here were carried predominantly by heterologously-expressed rat Na v 1.6 sodium channels rather than by endogenous channels. Previous studies showed that the low level of expression of the endogenous human b1A auxiliary subunit in HEK293 cells is insufficient to affect the properties of overexpressed heterologous sodium channel a subunits [32]. Thus, we attribute the sodium currents described here to the heterologously-expressed rat sodium channel subunits or subunit complexes rather than to endogenous channel complexes present in the parental cell line.
This study is the first description of the properties of rat Na v 1.6 sodium channels in a mammalian cell expression system and the modulation of these properties by the rat sodium channel b1 auxiliary subunit. It is also the first description of the modulation of any Na v 1.6 ortholog by the b1 subunit of the same species. Coexpression with the rat b1 subunit accelerated the inactivation of peak transient sodium currents carried by Na v 1.6 channels in HEK293 cells and shifted the voltage dependence of both channel activation and steady-state inactivation in the direction of depolarization. By contrast, we found no effect of the b1 subunit on the latency of the peak current (an indirect measure of rate of activation) or the stability of peak sodium currents following repeated, high-frequency stimulation.
The effects of the rat b1 subunit on the rat Na v 1.6 sodium channel reported here differ from its effects on the human Na v 1.6 ortholog in the HEK293 cell expression system [33]. Contrary to our findings, the rat b1 subunit had no effect on the voltage dependence of either activation or steady-state inactivation of the human Na v 1.6 channel. This result suggests that the rat b1 subunit is not able to interact in an equivalent manner with the rat and human Na v 1.6 orthologs to modulate voltage-dependent gating and underscores the importance of employing subunits from the same species in the reconstitution of channel function in vitro.
Comparison of the results of the present study with those for rat Na v 1.6 sodium channels expressed in the Xenopus oocyte system [17] reveals substantial differences in voltage-dependent gating between expression systems. In the absence of auxiliary b subunits the midpoint potentials for the voltage dependence of both activation and steady-state inactivation of rat Na v 1.6 sodium channels in HEK293 cells were shifted by 17-22 mV in the direction of hyperpolarization when compared to channels expressed in oocytes. Moreover, in HEK293 cells coexpression with the b1 subunit produced depolarizing shifts in the voltage dependence of activation and steady-state inactivation, whereas in oocytes the b1 subunit had no effect on the voltage dependent gating of rat Na v 1.6 channels. Coexpression of Na v 1.6 with the b1 subunit in oocytes enhanced the persistent component of the sodium current, whereas in HEK293 cells the b1 subunit had no effect on persistent current. In both expression systems, however, the b1 subunit accelerated the rate of sodium current inactivation.
Comparison of our results using the HEK-Na v 1.6b1 cell line with the results of our previous study using the corresponding HEK-Na v 1.6b1b2 cell line [21] permits us to infer the distinctive contributions of the rat b2 subunit to the properties of heterotrimeric Na v 1.6b1b2 sodium channel complexes. Such comparisons reveal no discernable effect of the b2 subunit on the kinetics of peak current decay or the voltage dependence of channel activation and steady-state fast inactivation. Thus, we conclude that the b1 subunit is the principal modulator of the properties of Na v 1.6b1b2 sodium channel complexes based on aspects of channel function that have been examined to date. However, this does not preclude important effects of the b2 subunit in channel trafficking or other channel properties, such as slow inactivation, that remain to be investigated.
The modulatory effects of the b1 subunit on sodium channel function are neither consistent nor predictable across sodium channel a subunit isoforms from the same species. The effects of the rat b1 subunit on the voltage dependence of various rat sodium channel subunit isoforms expressed in HEK293-based cell systems exemplify these isoform-specific effects. Consistent with our results using the rat Na v 1.6 ortholog, coexpression with the rat b1 subunit shifted the voltage dependence of activation and inactivation of rat Na v 1.2 channels in the direction of depolarization [34]. By contrast, coexpression with the rat b1 subunit did not significantly affect the voltage dependence of activation and inactivation of rat Na v 1.3 channels [35] or rat Na v 1.4 channels expressed in HEK293 cells [36].
Finally, comparison of our data for the rat Nav1.6 channel expressed in the absence of b subunits with published data for the expression the human Na v 1.6 channel expressed in HEK293 cells [23,24,33] and the mouse Na v 1.6 channel expressed in tsA-201 cells (a subclone of HEK293 cells) [25], reveals apparent species differences in both the persistent component of the sodium current and the voltage dependence of channel gating. Both human and mouse Na v 1.6 channels gave prominent persistent currents that did not inactivate during a typical depolarizing pulse. The amplitudes of these persistent currents, expressed as a percentage Table 4. Comparison of the voltage dependence of activation and steady-state fast inactivation of rat, human and mouse Na v 1.6 sodium channels expressed in human embryonic kidney-derived cell lines in the absence of auxiliary b subunits. of the peak transient current in the same depolarization, were approximately 12% for mouse channels expressed in TsA-201 cells [25] and up to 26% for human channels expressed by transient transfection in HEK293 cells [23]. By contrast the persistent current in our assays with rat Na v 1.6 channels was barely detectable at approximately 1.5% of the peak transient current, a value similar to that reported previously for rat Na v 1.2 channels expressed in TsA-201 cells [25] but much lower than those found in equivalent assays with the orthologous mouse and human channels. The rat Na v 1.6 channel, similar to the human ortholog, also differed from the mouse ortholog in its voltage dependence of activation and steady-state fast inactivation. Table 4 summarizes available data for the voltage dependent gating of rat, human and mouse Na v 1.6 channels expressed in human embryonic kidneyderived cell lines in the absence of b subunits. Rat Na v 1.6 channels gave midpoint potentials for activation and steady-state fast inactivation that were similar to or somewhat more than more negative than those measured in two separate studies with the orthologous human Na v 1.6 channels [23,33] but approximately 21 mV more negative than those measured for the orthologous mouse Na v 1.6 channels [25]. The relatively depolarized activation gating of the mouse Na v 1.6 ortholog is confirmed by expression studies of mouse Na v 1.6 R (mutated to confer resistance to TTX) in ND7/23 cells, a dorsal root ganglion-derived cell line [37] in which sodium currents carried by the heterologously-expressed Na v 1.6 R channels are isolated from endogenous sodium currents by recording in the presence of TTX. These results suggest that intrinsic species differences in the gating properties of orthologous Na v 1.6 channels may exist despite their high amino acid sequence identity. Differences in channel structure capable of conferring these differences remain to be determined.