JN Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Neurophysiol 99: 1917-1927, 2008. First published February 13, 2008; doi:10.1152/jn.01170.2007
0022-3077/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
99/4/1917    most recent
01170.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schofield, G. G.
Right arrow Articles by Ikeda, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schofield, G. G.
Right arrow Articles by Ikeda, S. R.

Properties of Wild-Type and Fluorescent Protein-Tagged Mouse Tetrodotoxin-Resistant Sodium Channel (NaV1.8) Heterologously Expressed in Rat Sympathetic Neurons

Geoffrey G. Schofield1, Henry L. Puhl, 3rd2 and Stephen R. Ikeda2

1Department of Physiology, Tulane University Health Sciences Center, New Orleans, Louisiana; and 2Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland

Submitted 22 October 2007; accepted in final form 12 February 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The tetrodotoxin (TTX)-resistant Na+ current arising from NaV1.8-containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. Here, we show that injection of cDNA encoding mouse NaV1.8 into the nuclei of rat superior cervical ganglion (SCG) neurons results in TTX-resistant Na+ currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed NaV1.8 Na+ channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed NaV1.8 channels was shifted about 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of NaV1.8 did not substantially affect functional expression in SCG neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged NaV1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum, thus providing a basis for poor functional expression in clonal cell lines. Our results establish SCG neurons as a favorable surrogate for the expression and study of molecularly defined NaV1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of NaV1.8 with a biophysical phenotype identical to that found in sensory neurons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Voltage-dependent Na+ channels are responsible for the upstroke of action potentials and consequently play a major role in neural information flow. Each of the nine known Na+ channel gene products (NaV1.1–1.9) share a common structure consisting of four homologous domains of six transmembrane-spanning helices (Catterall et al. 2005Go). Na+ channels associate with accessory β-subunits (NaVβ1–4) that modify the biophysical properties and membrane trafficking of the channels (Isom et al. 1992Go). Each of the Na+ channels has a particular tissue distribution. For example, NaV1.1, 1.2, and 1.3 are located primarily in central neurons (Goldin et al. 1986Go; Kayano et al. 1988Go; Lu et al. 1992Go), NaV1.4 in skeletal muscle (George et al. 1992Go), and NaV1.5 in cardiac muscle (Gellens et al. 1992Go).

Of particular interest in terms of nociceptive mechanisms are Na+ channel isoforms highly expressed in primary sensory neurons (Cummins et al. 2007Go). NaV1.7 is highly expressed in dorsal root ganglion (DRG) and sympathetic ganglia of the autonomic nervous system (Black et al. 1996Go; Felts et al. 1997Go; Sangameswaran et al. 1997Go; Toledo-Aral et al. 1997Go). NaV1.9 is found in small and medium-size DRG neurons (Dib-Hajj et al. 1998Go; Tate et al. 1998Go) and additional reports suggest NaV1.9 expression, based on the presence of mRNA, in some areas of the CNS (Blum et al. 2002Go; Jeong et al. 2000Go). Perhaps the most impressive in terms of highly restricted sensory neuron expression is NaV1.8. To date, NaV1.8-containing Na+ channels have been identified solely in primary sensory neurons (both DRG and cranial sensory ganglia such as the nodose ganglia), although ectopic expression in cerebellar Purkinje cells has been documented in experimental models of multiple sclerosis (Black et al. 2000Go). Another characteristic of NaV1.8, shared with other Na+ channels that arise from a contiguous gene cluster on human chromosome 3 (i.e., NaV1.5 and NaV1.9), is resistance to the puffer fish toxin tetrodotoxin (TTX). Most Na+ channels are blocked by TTX in the range of 4–25 nM. Conversely, NaV1.5, 1.8, and 1.9 have EC50 values for TTX block of 2, 60, and 40 µM, respectively (Catterall et al. 2005Go).

The presence of TTX-resistant Na+ channels in sensory neurons was first inferred from voltage recordings (Yoshida et al. 1978Go) and later confirmed with voltage-clamp recordings that investigated the ionic selectivity of TTX-resistant Na+ currents (INa) in neonatal rat DRG neurons (Kostyuk et al. 1981Go) and adult rat nodose ganglion neurons (Ikeda and Schofield 1987Go; Ikeda et al. 1986Go). These studies were followed by thorough biophysical studies of TTX-resistant INa from adult rat DRG neurons (Elliott and Elliott 1993Go). Although a cDNA encoding rat NaV1.8 was cloned in 1996 (Akopian et al. 1996Go), in-depth biophysical characterizations of heterologously expressed TTX-resistant INa are sparse. This deficiency arises from the inefficient heterologous expression of NaV1.8 channels in nonneuronal systems. Given the recent interest in NaV1.8 as both a mediator of specific nociceptive modalities (Zimmermann et al. 2007Go) and a potential therapeutic target for novel analgesics (Jarvis et al. 2007Go), we investigated the properties of mouse NaV1.8 expressed in a neuronal surrogate—rat sympathetic neurons.

In this report we confirm the poor functional expression of mouse NaV1.8 (mNaV1.8) in clonal HEK 293 and HeLa cells, but show that injection of mNaV1.8 cDNA directly into the nuclei of superior cervical ganglion (SCG) neurons resulted in robust TTX-resistant INa with maximal conductance similar to that observed for native TTX-resistant INa in mouse DRG neurons. Addition of a fluorescent protein (FP) tag to either the N- or C-terminus has little effect on the biophysical properties of the TTX-resistant INa, whereas insertion of an FP within an internal site (after residue 1739) within the C-terminus severely interfered with functional expression. The NaV1.8 fluorescent protein fusion constructs facilitated the study of NaV1.8 subcellular trafficking following heterologous expression in clonal and neuronal cells.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Molecular biology

Mouse NaV1.8 cDNA was amplified from DRG total RNA following reverse transcription with the Advantage RT for PCR kit (Clontech, Mountain View, CA). The following primers, based on GenBank accession number Y09108 (Souslova et al. 1997Go), were used to amplify a full-length cDNA (start codon bolded) with Kpn I and Not I sites (underlined) at the 5' and 3' termini, respectively: forward ATTA ggtacc ACC ATG GAG TTC CCC TTT GGG TC and reverse ATA GTT TA gcggccgc AGT GTC TTC ACT GAG GTC CAG. The resulting PCR product was ligated into the mammalian expression vector pCI (Promega, Madison, WI). Several clones were completely sequenced to identify a consensus sequence free of polymerase-induced mutations. The final clone selected (accession number AY538273) matched (100% identity) the mouse genomic sequence as determined with the web-based BLAT program (Kent 2002Go) and served as the basis for all subsequent constructs. The yellow fluorescent protein variant, Venus (Nagai et al. 2002Go), was inserted into an Eco NI site corresponding to residue 1739 of NaV1.8 by amplifying the Venus cDNA with the PCR using oligonucleotide primers incorporating Eco NI sites (underlined) at the 5' and 3' termini: forward AGC ACG GAG CC cctgagcgagg ACG TGA GCA AGG GCG AGG AGC TGC ATG TCAA and reverse AGT CGT cctcgctcagg GGC TTG TAC AGC TCG TCC ATG CC. The PCR product was digested with Eco NI and ligated into pCI Nav1.8 [NaV1.8–(1739)Venus]. Proper orientation of the Venus insert was confirmed by sequencing. Fusion of Venus to the C-terminus of NaV1.8 was accomplished by adding Bgl II and Hin dIII restriction sites to the NaV1.8 open reading frame (start codon bolded) with the following primers: forward GATC agatct GCC ACC ATG GAG TTC CCC TTT GGG TCC GTG and reverse GATC aagctt CTG AGG TCC AGG GCT CTT CCC TTC. The PCR product was digested with Bgl II and Hin dIII and then ligated into these sites in the Venus–N1 vector (NaV1.8–Venus). Fusion of enhanced green fluorescent protein (EGFP) to the N-terminus of NaV1.8 (EGFP–NaV1.8) was accomplished by inserting three new restriction sites (Not I, Eco RV, and Swa I) into the pEGFP–C1 vector (Clontech). The NaV1.8 open reading frame was subcloned into the modified vector after digestion with Kpn I and Not I. All PCR amplifications were performed with either Pfu or PfuUltra polymerase (Stratagene, La Jolla, CA).

Cell isolation and DNA injection

Superior cervical and nodose ganglion neurons from male Wistar rats (150–300 g) were enzymatically dissociated as described previously (Ikeda 2004Go). Rats were killed by decapitation after anesthesia by CO2 inhalation as approved by the Institutional Animal Care and Use Committee. Both SCG were dissected from the carotid bifurcation, desheathed, cut into small pieces, and then transferred into 6 ml of modified Earle's balanced salt solution containing 0.7 mg/ ml collagenase D (Roche Diagnostics, Indianapolis, IN), 0.3 mg/ml trypsin (Worthington Biochemicals, Freehold, NJ), and 0.05 mg/ ml DNase I (Sigma–Aldrich, St. Louis, MO). After incubation for 1 h in a shaking water bath at 36°C under an atmosphere of 5% CO2-95% O2, the ganglia fragments were shaken vigorously to release the neuronal somata. The dissociated neurons were washed twice and resuspended in minimal essential medium (MEM) containing 10% fetal calf serum and 1% penicillin-streptomycin, plated onto poly-L-lysine–coated tissue-culture dishes (35 mm), and placed in an incubator (95% air and 5% CO2; 100% humidity) at 37°C. After attachment, microinjection of cDNA into neuronal nuclei was performed with an Eppendorf FemtoJet microjector and 5171 micromanipulator (Eppendorf, Madison, WI), using custom-designed software (Ikeda 2004Go; Ikeda and Jeong 2004Go). Nodose ganglion neurons were treated with 50 ng/ml mouse 7S nerve growth factor (NGF; EMD Chemicals, La Jolla, CA) for 2 days prior to imaging. Plasmids were stored at –20°C as 0.3–1 µg/µl stock solution in TE buffer (10 mM Tris and 1 mM EDTA, pH 8). cDNA was injected at a pipette concentration of 0.1–0.2 µg/µl. When FP-fusion constructs were not used, neurons were coinjected with EGFP cDNA (pEGFP–N1; 5 ng/µl) to facilitate the identification of neurons receiving a successful intranuclear injection.

Cell culture, transfection, and imaging

HEK 293 and HeLa cells were cultured in MEM supplemented with 10% fetal calf serum under an atmosphere containing 5% CO2. The cells were transfected with the NaV1.8 cDNA clones as follows. A mixture of 1 µg of NaV1.8 (including FP-fusion constructs) in 150 µl of Opti-MEM (Invitrogen) and 10 µl of fully deacylated polyethyleneimine (Thomas et al. 2005Go) was added to achieve a final N:P ratio of 10. The mixture was incubated for 20 min at room temperature to allow complex formation. The transfection mixture was then applied to 24-well tissue-culture plates containing HEK 293 or HeLa cells at 50% confluence. After 24-h incubation, the cells were replated on plastic (Corning) or glass-bottom (MatTek, Ashland, MA) 35-mm tissue-culture dishes. HeLa cells were imaged on an Olympus IX-71 inverted fluorescence microscope equipped with a x60 1.45 NA (numerical aperture) objective. Images were captured using a cooled 12-bit charge-coupled device camera (Retiga EXi; Qimaging, Surrey, BC, Canada) and acquired using custom software written in IgorPro (WaveMetrics, Lake Oswego, OR). Wide-field fluorescence images were processed in IgorPro and ImageJ (W. S. Rasband, U.S. National Institutes of Health, http://rsb.info.nih.gov/ij/). Plasmids encoding ECFP targeted to either the endoplasmic reticulum (pECFP–ER) or Golgi apparatus (pECFP–Golgi) were obtained from Clontech. SCG and nodose ganglion neurons were imaged with an Achroplan IR x40 0.80 NA water-immersion objective mounted on a Zeiss Axioplan 2 microscope (Carl Zeiss, Jena, Germany) equipped with a Ti-sapphire laser (Chameleon, Coherent, Santa Clara, CA) for two-photon imaging. Excitation was at 970 nm in two-photon mode with a 500- to 550-nm emission filter. Zeiss software release 3.2 was used for image acquisition. Postprocessing of confocal images, including stack z-projection (average) and median filtering (1-pixel radius), was accomplished with ImageJ.

Electrophysiology

Rat SCG neurons were voltage-clamped using the whole cell patch-clamp technique with an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Electrodes were made from borosilicate glass capillaries (G85165T-4; Warner Instruments, Hamden, CT), coated with Sylgard (Dow Corning, Midland, MI) and fire polished to final resistances of 2 M{Omega} when filled with internal solutions. Mean uncompensated series resistance was about 5 M{Omega} and capacitance and series resistance were electronically compensated by ≥80%. For a representative series of recordings (n = 30), the estimated mean maximum (for the largest current recorded) voltage error was 3.8 ± 0.4 mV. Since no ion substitution experiments were performed, liquid junction potentials were not compensated for. Custom-designed software (S5) was used for voltage protocol generation and data acquisition on a Macintosh G4 computer (Apple Computer, Cupertino, CA) equipped with an ITC-18 data acquisition interface (InstruTECH, Port Washington, NY). Current traces were filtered at 5 kHz (–3 dB) using a four-pole low-pass Bessel filter and digitized at 20 kHz with the 16-bit A/D converter in the ITC-18 data acquisition interface. All experiments were carried out at room temperature (22–26°C).

Solutions and chemicals

For recording K+ currents, the external solution consisted of (in mM): 150 NaCl, 5.4 KCl, 5 MgCl2, 10 HEPES, and 15 D-glucose. The pipette solution contained (in mM): 140 KCl, 10 HEPES, 0.1 EGTA, 4.0 MgCl2, 4.0 Na2ATP, and 0.1 Na2GTP (pH adjusted to 7.2 with KOH). Osmolalities of the bath and pipette solutions were adjusted with sucrose to 300 and 285 mOsm/kg, respectively. Na currents were recorded with an external solution containing (in mM): 100 Na gluconate, 20 TEA-Cl, 10 HEPES, 2 MnCl2, 15 glucose, 40 sucrose, and 0.003 TTX. The internal solution contained (in mM): 120 N-methyl-D-glucamine, 20 TEA-OH, 11 EGTA, 10 HEPES, 10 sucrose, 1 CaCl2, 4 Na2ATP, 0.3 Na2GTP, and 14 Tris-creatine phosphate, pH 7.2, with methanesulfonic acid. Osmolalities of the bath and pipette solutions were adjusted with sucrose to 325 and 300 mOsm/kg, respectively.

Data analysis and statistics

Currents were analyzed using IgorPro software (WaveMetrics). Current amplitudes were measured from the peak of the current. Current traces were corrected during analysis for linear leak current based on the current amplitude at –80 mV and the assumption of a nonspecific ion conductance (i.e., reversal potential of 0 mV). Summary data are expressed as means ± SE. Statistical comparisons among groups were determined by one-way ANOVA followed by Student–Newman–Keuls post hoc test using GraphPad Prism software (GraphPad Software, San Diego, CA). P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Whole cell patch-clamp recordings were performed on acutely isolated adult mouse DRG neurons that natively express TTX-resistant INa arising from NaV1.8 (and NaV1.9). Rat SCG neurons, HEK 293, and HeLa cells were used as expression hosts for either wild-type or FP-tagged NaV1.8 to allow comparison of the functional expression and biophysical properties of heterologous and native channels. HeLa and HEK 293 cells transfected with 1 µg of NaV1.8 cDNA for 24 h consistently displayed very small inward currents. In solutions supportive of Na+ currents, depolarizing current pulses usually elicited no measurable inward current. In >20 cells, the largest inward current recorded was <50 pA. To ensure that the cells were transfection competent, HEK 293 cells were transfected using the same procedure with 250 ng of KV4.2 cDNA. In solutions supportive of K+ currents, depolarizing current pulses elicited very large transient outward currents, whereas cells transfected using EGFP alone displayed only a small background current of 390 ± 130 pA (n = 6) at +40 mV (Schofield and Ricci 2005Go). A typical K+ current trace elicited by a test pulse to +20 mV following a 1-s conditioning potential (–120 mV) is shown in Fig. 1B. The current–voltage (I–V) curves shown in Fig. 1C demonstrate the limited usefulness of HEK 293 and HeLa cells as a heterologous expression system for the study of NaV1.8 channel modulation or channel structure function studies.


Figure 1
View larger version (8K):
[in this window]
[in a new window]

  		FIG. 1. NaV1.8 expresses poorly in HEK 293 cells. A: tetrodotoxin (TTX)-resistant Na+ current (INa) elicited by a step to +10 mV from a holding potential of –80 mV in a HEK 293 cell transfected 24 h previously with a transfection mixture containing 1 µg NaV1.8, 250 ng pEGFP-N1 in polyethyleneimine (N:P ratio = 10). B: KV4.2 channel current elicited by a step to +40 mV from a conditioning potential of –120 mV in a HEK 293 cell transfected 24 h previously with a transfection mixture containing 250 ng KV4.2 and 250 ng of pEGFP in polyethyleneimine (N:P ratio = 10). C: current–voltage (I–V) relationships for the NaV1.8 and KV4.2 channel currents from the cells shown in A and B.

 
Conversely, NaV1.8 channels showed robust functional expression in a neuronal background, adult rat SCG neurons. Figure 2 compares TTX-resistant INa recorded from mouse DRG neurons, which natively express NaV1.8 channels, and SCG neurons injected 24 h previously with 100 ng/µl NaV1.8 cDNA and 5 ng/µl pEGFP–N1 (injected neurons were identified from EGFP fluorescence). Figure 2A shows superimposed inward currents elicited from an adult mouse DRG neuron by depolarizing steps from a holding potential of –80 mV. Currents began to activate at a potential of –45 mV, reached peak amplitude near –20 mV, and then declined approaching an extrapolated reversal potential near +40 mV. The deviation from the reversal potential predicted by the Nernst equation possibly arises from contaminating outward currents or nonlinearity arising from Goldman-type rectification. The extrapolated reversal potential of Na1.8 channel currents recorded from injected SCG neurons was hyperpolarized compared with DRG neurons perhaps due to imperfect current isolation. Although the TTX-resistant INa of DRG neurons consisted of both NaV1.8- and NaV1.9-containing Na+ channels, the holding potential utilized (–80 mV) primarily favors the NaV1.8 component (Coste et al. 2004Go; Cummins et al. 1999Go). This is evident from both the lack of current at potentials negative to –45 mV and the relatively minor component of INa that fails to inactivate during the test pulse. Figure 2B illustrates TTX-resistant INa traces recorded from a fluorescent SCG neuron (i.e., previously injected with NaV1.8 cDNA) that display a current trajectory and amplitude similar to those recorded from DRG neurons. It should be noted that rat SCG neurons possess a robust native INa that likely arises from NaV1.7-containing Na+ channels. However, this rapidly inactivating INa component is completely suppressed by TTX (Schofield and Ikeda 1988Go) and thus does not contribute to the current illustrated. Figure 2C depicts averaged I–V relationships comparing the native TTX-resistant INa recorded from mouse DRG neurons and the current produced by intranuclear injection of NaV1.8 cDNA in SCG neurons. Although the I–V relationships were superficially similar, expression of NaV1.8-containing channels in SCG neurons did not completely recapitulate the sensory neuron TTX-resistant INa phenotype because the I–V relationship for the heterologous NaV1.8 channels was shifted about 10 mV toward more depolarized potentials. In terms of absolute expression levels, the slope of the I–V curve at depolarized potentials was similar for both conditions, suggesting a comparable maximal conductance—a parameter analyzed in greater detail in the following text.


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		FIG. 2. Heterologously expressed NaV1.8 INa is similar to the TTX-resistant INa of mouse dorsal root ganglion (DRG) neurons. A: representative family of TTX-resistant INa recorded from an acutely isolated mouse DRG neuron. INa were elicited in the presence of 1 µM TTX with the voltage protocol illustrated. Internal and external solutions were designed to isolate INa from other potentially contaminating currents. B: representative family of TTX-resistant INa recorded from a superior cervical ganglion (SCG) neuron injected 24 h previously with a mixture containing 100 ng/µl NaV1.8 cDNA and 5 ng/µl pEGFP-N1. Currents were elicited as described in A. C: mean current–voltage (I–V) curves for natively expressed mouse DRG TTX-resistant INa (open circles) and NaV1.8 channel currents from injected SCG neurons (filled circles). Each point represents the means ± SE of 20 and 10 cells, respectively.

 
Since NaV1.8 channels exhibit robust functional expression in a neuronal background, the negligible currents observed in HEK 293 and HeLa cells transfected with NaV1.8 cDNA suggest that clonal cells are deficient in a factor(s) facilitating functional expression. For example, defects in trafficking may interfere with attaining functional expression levels comparable to those observed in sensory neurons. As a first step toward investigating these questions, three FP-tagged NaV1.8 constructs (Fig. 3A) were developed to investigate trafficking in HeLa cells and SCG neurons. An electrophysiological characterization of these constructs, after expression by nuclear injection in SCG neurons, revealed that the N- and C-terminally tagged NaV1.8 constructs produced similar appearing INa when compared with the untagged NaV1.8. On the other hand, insertion of Venus into the C-terminus at residue 1739 (taking advantage of a unique Eco NI site), a site just beyond helix IV-S6 in the predicted topology map, resulted in very small noninactivating currents following nuclear injection in SCG neurons (data not shown). Figure 3B compares activation curves of native TTX-resistant INa and heterologously expressed NaV1.8 channels by converting peak INa to conductance using the chord conductance equation

Formula
where gNa is the Na+ conductance, INa is the peak Na+ current, Vm is the step potential, and VNa is the INa reversal potential. The solid lines in Fig. 3B represent nonlinear regression fits of a modified Boltzmann equation to the data

Formula
where gmax is the maximal Na+ conductance, Vh is the half-activation potential, and k is a slope factor.


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 3. Activation of NaV1.8 currents expressed in rat SCG neurons and native TTX-resistant INa of mouse DRG neurons. A: schematic of the NaV1.8 channel to show the placement of enhanced green fluorescent protein (EGFP) (triangle) and Venus (star and square) fluorescent protein tags. B: plots of normalized conductance (means ± SE) vs. step potential for the native TTX-resistant INa (open circles) recorded from acutely isolated mouse DRG neurons, wild-type NaV1.8 (filled circles), NaV1.8 tagged with Venus at the C-terminus (filled squares), and NaV1.8 tagged with EGFP at the N-terminus (filled triangles). The solid lines represent nonlinear least-squares fits of a modified Boltzmann equation to the data. The bar graph (inset) shows the mean maximal conductance of the native TTX-resistant INa (open bar) and the heterologously expressed NaV1.8 (filled bars) INa. The numbers in parentheses indicate the number of neurons recorded from.

 
Native TTX-resistant INa began to activate near –45 mV, reached half activation at –27 ± 1 mV, and had a slope of 2.7 ± 2 mV (n = 20). All of the heterologously expressed NaV1.8 channels began to activate near –45 mV but Vh was shifted about 10 mV toward depolarized potentials when compared with the native channels. Fitted activation curve parameters are summarized in Table 1 and show that both Vh and voltage dependence (as indicated by k) of the heterologously expressed NaV1.8 channels (untagged and tagged) differed significantly from the natively expressed channels of mouse DRG neurons. The nonnormalized maximum conductance (gmax) for the native and heterologously expressed INa did not differ significantly (Fig. 3B, inset, P > 0.05), suggesting that the number of functional channels per neuron was similar, given assumptions of similar single-channel conductance and probability of opening. This comparison was not meant to establish identity among the various conditions but to illustrate that heterologous expression of NaV1.8 in SCG neurons produced functional expression comparable to native levels.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Activation and inactivation parameters of native and cloned TTX-resistant Na+ channels

 
To compare steady-state inactivation characteristics, mouse DRG neurons and SCG neurons heterologously expressing untagged and FP-tagged NaV1.8 channels were subjected to a voltage protocol in which 0.5-s conditioning pulses to progressively more depolarized potentials were followed by a constant test pulse to +10 mV (Fig. 4A, right). Examples of families of INa traces evoked with the inactivation voltage protocol for native TTX-resistant INa (Fig. 4A) and heterologously expressed NaV1.8 channels (Fig. 4B) are illustrated. The current traces show that the channels encoded by the NaV1.8 cDNA followed an inactivation trajectory similar to the TTX-resistant INa of mouse DRG neurons. Moreover, fusion of a fluorescent protein to either the C- or N-terminus had little overt effect on inactivation kinetics. Figure 4C depicts normalized inactivation curves for native TTX-resistant and heterologously expressed NaV1.8 channels. The solid lines represent nonlinear least-square fits of a modified Boltzmann equation (see previous text; k is negative for inactivation curves) to the averaged data after the subtraction of a small noninactivating component (means ranged from 5 to 11% and were not significantly different among the groups). The native TTX-resistant INa began to inactivate at potentials positive to –60 mV and reached a constant level of inactivation near –20 mV. Expression of untagged NaV1.8 cDNA resulted in currents that inactivated at potentials more positive than –80 mV and reached a steady-state level of inactivation at potentials near 0 mV. Fusion of fluorescent proteins to either the N- or the C-terminus shifted inactivation curves to more positive potentials when compared with the unlabeled channel. A summary of the inactivation parameters is shown in Table 1. Voltage dependencies of the inactivation curves for the heterologously expressed channels were all significantly less (and indicated by a greater |k| value) when compared with the native TTX-resistant currents. On the other hand, only the Vh of EGFP–NaV1.8 INa was significantly different from the native current.


Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 4. Voltage dependence of NaV1.8 channel inactivation is reduced compared with native TTX-resistant INa. A: family of native TTX-resistant INa traces recorded from a mouse DRG neuron using an inactivation voltage protocol (right). The conditioning voltages are shown under the current records. B: INa traces recorded from SCG neurons heterologously expressing wild-type and FP-tagged NaV1.8 channels. Inactivation was produced with the voltage protocol shown in A. C: Superimposed inactivation curves for native, wild-type and fluorescent protein (FP)–tagged NaV1.8 channel currents. Solid lines represent the best fit of a modified Boltzmann equation after subtraction of a small, noninactivating component (5–11%). Data were normalized to the fitted maximum current; bars are SE.

 
Imaging experiments were performed to determine the subcellular localization of heterologously expressed NaV1.8 channels that were fused with fluorescent proteins. SCG neurons were imaged using two-photon microscopy because the thickness of the neuronal somata results in substantial out-of-focus light with conventional wide-field fluorescence microscopy. Representative live cell images are shown in Fig. 5 (left column). For the N- and C-terminal fusion constructs, granular fluorescence was seen throughout the cytoplasm with clear exclusion from the nucleus. Surprisingly, there was no overt concentration of fluorescence near the plasma membrane ("rim fluorescence") despite the fact that both constructs produced INa amplitudes similar to those recorded in DRG neurons. Thus the majority of expressed channels were not trafficked to the plasma membrane and likely retained on internal membranes. The exclusion of fluorescence from the nucleus indicates that "free" fluorescent protein, as might occur from aberrant translational start, was not produced in significant quantities. The fluorescence pattern of NaV1.8–(1739)Venus, a construct that produced minimal INa, was similar to the functionally expressing NaV1.8 constructs with the exception of intensely fluorescent "plaques" found near the plasma membrane. This feature was common in the brighter neurons and was never seen with the N- and C-terminal NaV1.8 fusion constructs. It was unclear whether this peculiar fluorescence pattern represents targeting to a specific subcellular compartment or a reaction to a misfolded protein. HeLa cells were transfected with EGFP–NaV1.8, NaV1.8–(1739)Venus, or NaV1.8–Venus along with ECFP fusion proteins targeted to either the endoplasmic reticulum (ER; calreticulin fused to the 5' end and the ER retention signal, KDEL, fused to the 3' end) or the Golgi apparatus (N-terminal 81 amino acids of the human β-1,4-galactosyltransferase fused to the 5' end). Following incubation for about 24 h, HeLa cells were imaged with conventional wide-field fluorescence microscopy using a high NA objective and filter sets optimized for the FP under study. All of the NaV1.8 constructs displayed exclusion from the nucleus and a fine lace-like pattern indicative of ER localization. This impression was supported by obvious coincidence with the fluorescence of ECFP–ER. The ECFP–Golgi construct largely localized to intense puncta adjacent to the nucleus. There was some coincidence of NaV1.8 with ECFP–Golgi fluorescence but clearly the former was found outside of this distribution. To ascertain expression patterns in neurons that natively express NaV1.8 (Ikeda and Schofield 1987Go; Kwong et al. 2008Go), cDNA encoding EGFP–NaV1.8 and NaV1.8–Venus were injected into adult rat nodose ganglion neurons (Fig. 6). A lower concentration of cDNA (20 ng/µl) was injected to decrease expression levels and neurons were maintained in tissue culture for 48 h in the presence of NGF to encourage neurite outgrowth. Two-photon confocal imaging of the living neurons revealed fluorescence in the cell body and neurites. Both constructs were at least partially excluded from the nucleus with course granular fluorescence present throughout the somata. EGFP–NaV1.8 fluorescence was present in perinuclear structures. Both EGFP–NaV1.8 and NaV1.8–Venus fluorescence appeared slightly enriched near the plasma membrane (Fig. 6, right panels) but this was neither a robust nor a consistent finding. Brighter neurons (data not shown) displayed less granular fluorescence in the cytoplasm and plasma membrane enrichment was not evident.


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 5. Fluorescent images of FP-tagged NaV1.8 channels expressed in SCG neurons or HeLa cells. Left: representative 2-photon confocal image slices at the level of the nucleus from SCG neurons expressing either EGFP-NaV1.8, NaV1.8–Venus, or NaV1.8–(1739)Venus cDNA. All neurons were injected with 100 ng/µl FP-tagged NaV1.8 cDNA 24 h prior to imaging. Center: representative wide field fluorescence images of HeLa cells cotransfected with the NaV1.8 FP-tagged channel cDNAs and an ECFP fusion protein targeted to the endoplasmic reticulum. Right: representative wide field fluorescence images of HeLa cells cotransfected with the NaV1.8 FP-tagged channel cDNAs and an ECFP fusion protein targeted to the Golgi apparatus. The cells are outlined in the images showing the Golgi apparatus fluorescence since the fluorescence was highly localized and the cell outlines were indistinct. The grayscale was inverted for better visualization of the internal structures. Scale bars represents 10 µm.

 

Figure 6
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 6. Fluorescent images of FP-tagged NaV1.8 channels expressed in nodose ganglion neurons. Representative 2-photon confocal images of rat nodose ganglion neurons expressing either EGFP–NaV1.8 (top) or NaV1.8–(1739)Venus (bottom) cDNA. All neurons were injected with 20 ng/µl FP-tagged NaV1.8 cDNA 48 h prior to imaging. Left: Z-projection (average) of an image stack sampled at 1 µm through the entire depth of the neuron. Right: a representative single image sampled at the level of the nucleus. Images were processed using a median filter (1-pixel radius) to reduce noise in the image. Scale bar represents 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Proteins that are expressed solely in primary sensory neurons represent important therapeutic targets for the development of novel analgesic compounds because a restricted expression pattern predicts minimal CNS effects if a protein-specific ligand can be developed. NaV1.8, a product of the Scn10a gene, is arguably the most sensory neuron-specific ion channel identified thus far. Under nonpathological conditions, only small and medium-size primary sensory neurons of the DRG and cranial sensory ganglia have been found to contain functional channels. Accordingly, a key question concerns the mechanism(s) controlling the exquisite tissue specificity of NaV1.8 expression. In general terms, specificity appears to occur at two levels. First, transcription is tightly controlled through as yet uncharacterized promoter elements that restrict NaV1.8 mRNA to a subset of primary sensory neurons. Second, following translation, functional expression of NaV1.8 channels, at numbers comparable to those of DRG neurons, occurs only in neurons or cell lines derived from neuronal elements. We investigated the second phenomenon by injecting mouse NaV1.8 cDNA into the nuclei of sympathetic neurons and characterizing the properties and trafficking of the expressed Na+ channel.

The source of our NaV1.8 cDNA was a mouse ortholog of NaV1.8 (#AY538273) generated using the PCR and cDNA reverse transcribed from mouse DRG neuron mRNA. Unexpectedly, the sequence of the open reading frame differed at 170 positions from the sequence, #Y09108 (Souslova et al. 1997Go), used in the design of our oligonucleotide primers. About 50% of the base changes did not alter the predicted amino acid sequence. The source of these discrepancies remains unclear, although a BLAT search (Kent 2002Go) of the mouse genomic database produced an exact match with our cDNA sequence, whereas a sequence matching #Y09108 was not found. Thus it seems unlikely that a NaV1.8 pseudogene or extensive RNA editing accounts for the discrepancies.

Injection of mouse NaV1.8 cDNA into sympathetic neurons produced TTX-resistant INa similar in magnitude and characteristics to the natively expressed TTX-resistant INa recorded from mouse DRG neurons, compared with feeble expression in clonal cell lines using conventional transfection techniques. The weak expression of NaV1.8 cDNA in Xenopus oocytes (Akopian et al. 1996Go) and clonal cell lines (Okuse et al. 2002Go), as opposed to robust heterologous expression in sympathetic neurons (England et al. 1998Go) and DRG neurons from knockout mice (Akopian et al. 1999; Liu et al. 2005Go), has been noted previously. However, detailed characterization of INa expressed from NaV1.8 cDNA in neurons has not been published. Our experiments suggest that the numbers of NaV1.8 channels (per cell) heterologously expressed in SCG neurons attain levels similar to those in mouse DRG neurons. This interpretation is predicated on assumptions of similar single-channel conductance and maximum probability of opening at depolarized potentials. It is also assumed that the majority of TTX-resistant INa measured in mouse DRG neurons under the described experimental conditions arises from NaV1.8-containing channels. Although inclusion of TTX in the external recording solution blocks the majority of Na+ channels, many sensory neurons possess, in addition to NaV1.8, channels comprised of NaV1.9 (Amaya et al. 2000Go; Benn et al. 2001Go; Dib-Hajj et al. 1998Go) and possibly a minor component arising from NaV1.5 (Renganathan et al. 2002Go). However, the similarity in INa trajectory when compared with the heterologous NaV1.8 channels, single-component activation and inactivation curves, and holding potential of –80 mV (which serves to inactivate the majority of NaV1.9-containing channels) all argue for a relatively well isolated NaV1.8-mediated current component.

Comparison of the activation and inactivation properties of heterologously expressed NaV1.8, including FP-fusion constructs, revealed similarity to the TTX-resistant INa recorded from mouse DRG neurons. However, the activation Vh was consistently shifted to a more depolarized potential, whereas only minor differences in inactivation Vh were noted. In addition, the voltage dependence of the heterologous channels for both activation and inactivation was shallower. Thus the sympathetic neuron environment does not perfectly recapitulate the phenotype of the natively expressed channel. Because of differences in experimental conditions, especially recording solutions (e.g., internal and external Na+ concentrations, ions used to block Ca2+ channels, presence of F– in the internal solution, etc.), direct comparisons of biophysical parameters of INa among published studies are of limited utility. However, in studies that directly compared heterologously expressed NaV1.8 and TTX-resistant INa of sensory neurons (John et al. 2004Go; Zhao et al. 2007Go) the finding of a more depolarized activation Vh for expressed NaV1.8 was a consistent observation. Given the diversity of the expression hosts (sympathetic neurons, ND7-23 neuroblastoma, tsA201 cells) used in these studies and ours, it appears that sensory neurons have a specific factor that modifies the biophysical characteristics of NaV1.8-containing channels. Although a sensory neuron–specific interacting protein is a likely candidate, one cannot rule out posttranslational modifications (e.g., phosphorylation) or posttranscriptional processing as potential mechanisms. It should also be noted that the native INa was recorded from acutely dissociated neurons, whereas recordings from the heterologous NaV1.8 channels required waiting overnight for protein expression to occur. Therefore the differences noted might also arise from time-dependent changes that occur while the neurons were in culture.

A number of potential proteins interact with NaV1.8 (Malik-Hall et al. 2003Go) and some have been shown to influence the functional expression level. Both Na+ channel β-subunits (Liu et al. 2006Go; Shah et al. 2000Go) and the annexin II light chain/p11 (Okuse et al. 2002Go) increased NaV1.8-mediated currents when coexpressed. Conversely, the clathrin-associated protein-1A (Liu et al. 2005Go) reduced NaV1.8 current when coexpressed. It is thus possible that the presence of a number of proteins, when summed together, accounts for native levels of expression in sensory neurons. Recently, it was shown that pretreatment with the local anesthetic lidocaine dramatically increased the surface expression of NaV1.8 in tsA201 cells by releasing the channel from the endoplasmic reticulum (Zhao et al. 2007Go). Although the precise mechanism for this effect is unclear, the authors propose a chemical chaperoning function for lidocaine that might indicate an endogenous molecule found in sensory neurons serves a similar function. In all of the studies, including this one, it should be noted that multiple (and usually unknown) copies of a NaV1.8 transcription unit containing powerful viral promoters was used to drive expression. Thus achieving channel densities with heterologous expression comparable to those found in sensory neurons may not equate to equivalent expression efficiency. Thus it seems likely that a key component regulating surface expression of NaV1.8, much as Ca2+ channel β-subunits dramatically increase Ca2+ channel {alpha}-subunit plasma membrane insertion (Bichet et al. 2000Go), remains to be discovered.

To facilitate visualization of channel trafficking, fluorescent proteins were fused to the termini and at an internal site of NaV1.8. Expression of the C- and N-terminal fusion constructs in SCG neurons resulted in functional expression of Na+ channels with characteristics similar to channels arising from the untagged cDNA. Somewhat unexpectedly, fluorescence was found throughout the cytoplasm in a granular pattern with little evidence of concentration at the plasma membrane. Because functional expression levels were comparable to those of mouse DRG neurons, the great majority of channels reside within the neuron, presumably on internal membranes. We initially assumed this pattern resulted from an artifactual pulse of synchronized translation arising from intranuclear cDNA injection. However, immunostaining of natively expressed NaV1.8-containing channels in neurons reveals a very similar pattern (Amaya et al. 2000Go; Benn et al. 2001Go). Thus whether large numbers of sequestered NaV1.8-containing channels represent a physiological condition remains an unanswered question. Expression of FP-tagged constructs in HeLa cells revealed a fluorescence pattern consistent with proteins retained in the ER—an observation confirmed by coincidence with an ER-targeted fluorescent protein. This result confirms earlier observations in clonal cells (Zhao et al. 2007Go) and explains the minimal functional expression seen in these cells. Insertion of FP into the interior of NaV1.8 resulted in minimal functional expression and a peculiar fluorescence pattern in neurons. The result underscores the difficulty of inserting residues into internal sites that might result in disruption of secondary structure and/or protein folding. Finally, expression of the two FP-tagged NaV1.8 constructs in primary sensory neurons revealed fluorescence patterns that were superficially similar to those seen in sympathetic neurons. Imaging conditions were slightly different for sensory neurons, e.g., lower expression levels, culture media containing NGF, and 24 h longer in culture, thus precluding direct comparisons. Because even the functional FP-fusion constructs provided limited information in regard to plasma membrane targeting, a construct expressing an epitope that is accessible from the exterior of the neuron would be of great value for trafficking studies. Experiments are currently under way to develop such a tool.

In summary, intranuclear injection of mouse NaV1.8 cDNA into adult rat SCG neurons produces expression of TTX-resistant Na+ channels comparable to those seen in DRG neurons. The biophysical properties were similar, but not identical to, natively expressed NaV1.8-containing channels. NaV1.8 channels tagged on either N- or C-terminus with fluorescent proteins were functional and produced TTX-resistant INa equal to or greater than that of untagged proteins. The FP-tagged channels were localized in the ER of HeLa cells, thereby explaining the minimal functional expression observed in these cells.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by the intramural program of the National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Drs. Steven S. Vogel and Christopher Thaler for help with the confocal imaging, Dr. Ruquia Ahmed-Schofield for the fully deacylated polyethyleneimine, and M. King for invaluable contributions toward generating the mouse NaV1.8 cDNA.

Present address of G. G. Schofield: Center for Scientific Review, NIH, Rockledge II, Room 4187, 6701 Rockledge Drive, MSC 7850, Bethesda, MD 20892-7850.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. R. Ikeda, Section on Transmitter Signaling, Laboratory of Molecular Physiology, NIH/NIAAA, 5625 Fishers Lane, MSC 9411, Bethesda, MD 20892-9411 (E-mail: sikeda{at}mail.nih.gov)


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Akopian AN, Sivilotti L, Wood JN. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379: 257–262, 1996.[CrossRef][Medline]

Amaya F, Decosterd I, Samad TA, Plumpton C, Tate S, Mannion RJ, Costigan M, Woolf CJ. Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2. Mol Cell Neurosci 15: 331–342, 2000.[CrossRef][Web of Science][Medline]

Benn SC, Costigan M, Tate S, Fitzgerald M, Woolf CJ. Developmental expression of the TTX-resistant voltage-gated sodium channels NaV1.8 (SNS) and NaV1.9 (SNS2) in primary sensory neurons. J Neurosci 21: 6077–6085, 2001.[Abstract/Free Full Text]

Bichet D, Cornet V, Geib S, Carlier E, Volsen S, Hoshi T, Mori Y, De Waard M. The I–II loop of the Ca2+ channel {alpha}1 subunit contains an endoplasmic reticulum retention signal antagonized by the β subunit. Neuron 25: 177–190, 2000.[CrossRef][Web of Science][Medline]

Black JA, Dib-Hajj S, Baker D, Newcombe J, Cuzner ML, Waxman SG. Sensory neuron-specific sodium channel SNS is abnormally expressed in the brains of mice with experimental allergic encephalomyelitis and humans with multiple sclerosis. Proc Natl Acad Sci USA 97: 11598–11602, 2000.[Abstract/Free Full Text]

Black JA, Dib-Hajj S, McNabola K, Jeste S, Rizzo MA, Kocsis JD, Waxman SG. Spinal sensory neurons express multiple sodium channel alpha-subunit mRNAs. Brain Res Mol Brain Res 43: 117–131, 1996.[Medline]

Blum R, Kafitz KW, Konnerth A. Neurotrophin-evoked depolarization requires the sodium channel NaV1.9. Nature 419: 687–693, 2002.[CrossRef][Medline]

Catterall WA, Goldin AL, Waxman SG. International Union of Pharmacology. XLVII. Nomenclature and structure–function relationships of voltage-gated sodium channels. Pharmacol Rev 57: 397–409, 2005.[Abstract/Free Full Text]

Coste B, Osorio N, Padilla F, Crest M, Delmas P. Gating and modulation of presumptive NaV1.9 channels in enteric and spinal sensory neurons. Mol Cell Neurosci 26: 123–134, 2004.[CrossRef][Web of Science][Medline]

Cummins TR, Dib-Hajj SD, Black JA, Akopian AN, Wood JN, Waxman SG. A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons. J Neurosci 19: RC43, 1999.[Abstract/Free Full Text]

Cummins TR, Dib-Hajj SD, Waxman SG. Electrophysiological properties of mutant NaV1.7 sodium channels in a painful inherited neuropathy. J Neurosci 24: 8232–8236, 2004.[Abstract/Free Full Text]

Cummins TR, Sheets PL, Waxman SG. The roles of sodium channels in nociception: implications for mechanisms of pain. Pain 131: 243–257, 2007.[CrossRef][Web of Science][Medline]

Dib-Hajj SD, Tyrrell L, Black JA, Waxman SG. NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy. Proc Natl Acad Sci USA 95: 8963–8968, 1998.[Abstract/Free Full Text]

Elliott AA, Elliott JR. Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia. J Physiol 463: 39–56, 1993.[Abstract/Free Full Text]

England S, Okuse K, Ogata N, Wood JN. Heterologous expression of the sensory neurone-specific sodium channel (SNS) {alpha}-subunit in rat sympathetic neurones (Letter). J Physiol 511: 124P, 1998.

Felts PA, Yokoyama S, Dib-Hajj S, Black JA, Waxman SG. Sodium channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patterns in developing rat nervous system. Brain Res Mol Brain Res 45: 71–82, 1997.[Medline]

Gellens ME, George AL Jr, Chen LQ, Chahine M, Horn R, Barchi RL, Kallen RG. Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel. Proc Natl Acad Sci USA 89: 554–558, 1992.[Abstract/Free Full Text]

George AL Jr, Komisarof J, Kallen RG, Barchi RL. Primary structure of the adult human skeletal muscle voltage-dependent sodium channel. Ann Neurol 31: 131–137, 1992.[CrossRef][Web of Science][Medline]

Goldin AL, Snutch T, Lubbert H, Dowsett A, Marshall J, Auld V, Downey W, Fritz LC, Lester HA, Dunn R, Catterall WA, Davidson N. Messenger RNA coding for only the {alpha} subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes. Proc Natl Acad Sci USA 83: 7503–7507, 1986.[Abstract/Free Full Text]

Ikeda SR. Expression of G-protein signaling components in adult mammalian neurons by microinjection. Methods Mol Biol 259: 167–181, 2004.[Medline]

Ikeda SR, Jeong SW. Use of RGS-insensitive Galpha subunits to study endogenous RGS protein action on G-protein modulation of N-type calcium channels in sympathetic neurons. Methods Enzymol 389: 170–189, 2004.[Web of Science][Medline]

Ikeda SR, Schofield GG. Tetrodotoxin-resistant sodium current of rat nodose neurones, monovalent cation selectivity and divalent cation block. J Physiol 389: 255–270, 1987.[Abstract/Free Full Text]

Ikeda SR, Schofield GG, Weight FF. Na+ and Ca2+ currents of acutely isolated adult rat nodose ganglion cells. J Neurophysiol 55: 527–539, 1986.[Abstract/Free Full Text]

Isom LL, De Jongh KS, Patton DE, Reber BF, Offord J, Charbonneau H, Walsh K, Goldin AL, Catterall WA. Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel. Science 256: 839–842,1992.[Abstract/Free Full Text]

Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, Marron B, Atkinson R, Thomas J, Liu D, Krambis M, Liu Y, McGaraughty S, Chu K, Roeloffs R, Zhong C, Mikusa JP, Hernandez G, Gauvin D, Wade C, Zhu C, Pai M, Scanio M, Shi L, Drizin I, Gregg R, Matulenko M, Hakeem A, Gross M, Johnson M, Marsh K, Wagoner PK, Sullivan JP, Faltynek CR, Krafte DS. A-803467, a potent and selective NaV1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci USA 104: 8520–8526, 2007.[Abstract/Free Full Text]

Jeong SY, Goto J, Hashida H, Suzuki T, Ogata K, Masuda N, Hirai M, Isahara K, Uchiyama Y, Kanazawa I. Identification of a novel human voltage-gated sodium channel alpha subunit gene, SCN12A. Biochem Biophys Res Commun 267: 262–270, 2000.[CrossRef][Web of Science][Medline]

John VH, Main MJ, Powell AJ, Gladwell ZM, Hick C, Sidhu HS, Clare JJ, Tate S, Trezise DJ. Heterologous expression and functional analysis of rat NaV1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23. Neuropharmacology 46: 425–438, 2004.[CrossRef][Web of Science][Medline]

Kayano T, Noda M, Flockerzi V, Takahashi H, Numa S. Primary structure of rat brain sodium channel III deduced from the cDNA sequence. FEBS Lett 228: 187–194, 1988.[CrossRef][Web of Science][Medline]

Kent WJ. BLAT—the BLAST-like alignment tool. Genome Res 12: 656–664, 2002.[Abstract/Free Full Text]

Kostyuk PG, Veselovsky NS, Tsyndrenko AY. Ionic currents in the somatic membrane of rat dorsal root ganglion neurons—I. Sodium currents. Neuroscience 6: 2423–2430, 1981.[CrossRef][Web of Science][Medline]

Kwong K, Carr MJ, Gibbard A, Savage TJ, Singh K, Jing J, Meeker S, Undem BJ. Voltage-gated sodium channels in nociceptive versus non-nociceptive nodose vagal sensory neurons innervating guinea pig lungs. J Physiol (January 10, 2008). 10.1113/jphysiol.2007.146365.[Abstract/Free Full Text]

Liu C, Cummins TR, Tyrrell L, Black JA, Waxman SG, Dib-Hajj SD. CAP-1A is a novel linker that binds clathrin and the voltage-gated sodium channel NaV1.8. Mol Cell Neurosci 28: 636–649, 2005.[CrossRef][Web of Science][Medline]

Liu CJ, Priest BT, Bugianesi RM, Dulski PM, Felix JP, Dick IE, Brochu RM, Knaus H-G, Middleton RE, Kaczorowski GJ, Slaughter RS, Garcia ML, Köhler MG. A high-capacity membrane potential FRET-based assay for NaV1.8 channels. Assay Drug Dev Technol 4: 37–48, 2006.[CrossRef][Web of Science][Medline]

Lu CM, Han J, Rado TA, Brown GB. Differential expression of two sodium channel subtypes in human brain. FEBS Lett 303: 53–58, 1992.[CrossRef][Web of Science][Medline]

Malik-Hall M, Poon W-YL, Baker MD, Wood JN, Okuse K. Sensory neuron proteins interact with the intracellular domains of sodium channel NaV1.8. Brain Res Mol Brain Res 110: 298–304, 2003.[Medline]

Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20: 87–90, 2002.[CrossRef][Web of Science][Medline]

Okuse K, Malik-Hall M, Baker MD, Poon W-YL, Kong H, Chao MV, Wood JN. Annexin II light chain regulates sensory neuron-specific sodium channel expression. Nature 417: 653–656, 2002.[CrossRef][Medline]

Renganathan M, Dib-Hajj S, Waxman SG. NaV1.5 underlies the "third TTX-R sodium current" in rat small DRG neurons. Brain Res Mol Brain Res 106: 70–82, 2002.[Medline]

Sangameswaran L, Fish LM, Koch BD, Rabert DK, Delgado SG, Ilnicka M, Jakeman LB, Novakovic S, Wong K, Sze P, Tzoumaka E, Stewart GR, Herman RC, Chan H, Eglen RM, Hunter JC. A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat and human dorsal root ganglia. J Biol Chem 272: 14805–14809, 1997.[Abstract/Free Full Text]

Schofield GG, Ikeda SR. Sodium and calcium currents of acutely isolated adult rat superior cervical ganglion neurons. Pfluegers Arch 411: 481–490, 1988.[CrossRef][Web of Science][Medline]

Schofield GG, Ricci A. Electrophysiological characterization of C-terminal KV4 channel fusion proteins. Pfluegers Arch 450: 245–254, 2005.[CrossRef][Web of Science][Medline]

Shah BS, Stevens EB, Gonzalez MI, Bramwell S, Pinnock RD, Lee K, Dixon AK. β3, a novel auxiliary subunit for the voltage-gated sodium channel, is expressed preferentially in sensory neurons and is upregulated in the chronic constriction injury model of neuropathic pain. Eur J Neurosci 12: 3985–3990, 2000.[CrossRef][Web of Science][Medline]

Souslova VA, Fox M, Wood JN, Akopian AN. Cloning and characterization of a mouse sensory neuron tetrodotoxin-resistant voltage-gated sodium channel gene, Scn10a. Genomics 41: 201–209, 1997.[CrossRef][Web of Science][Medline]

Tate S, Benn S, Hick C, Trezise D, John V, Mannion RJ, Costigan M, Plumpton C, Grose D, Gladwell Z, Kendall G, Dale K, Bountra C, Woolf CJ. Two sodium channels contribute to the TTX-R sodium current in primary sensory neurons. Nat Neurosci 1: 653–655, 1998.[CrossRef][Web of Science][Medline]

Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM. Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci USA 102: 5679–5684, 2005.[Abstract/Free Full Text]

Toledo-Aral JJ, Moss BL, He ZJ, Koszowski AG, Whisenand T, Levinson SR, Wolf JJ, Silos-Santiago I, Halegoua S, Mandel G. Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons. Proc Natl Acad Sci USA 94: 1527–1532, 1997.[Abstract/Free Full Text]

Yoshida S, Matsuda Y, Samejima A. Tetrodotoxin-resistant sodium and calcium components of action potentials in dorsal root ganglion cells of the adult mouse. J Neurophysiol 41: 1096–1106, 1978.[Free Full Text]

Zhao J, Ziane R, Chatelier A, O'Leary ME, Chahine M. Lidocaine promotes the trafficking and functional expression of NaV1.8 sodium channels in mammalian cells. J Neurophysiol 98: 467–477, 2007.[Abstract/Free Full Text]

Zimmermann K, Leffler A, Babes A, Cendan CM, Carr RW, Kobayashi J, Nau C, Wood JN, Reeh PW. Sensory neuron sodium channel NaV1.8 is essential for pain at low temperatures. Nature 447: 855–858, 2007.[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
99/4/1917    most recent
01170.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schofield, G. G.
Right arrow Articles by Ikeda, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schofield, G. G.
Right arrow Articles by Ikeda, S. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the The American Physiological Society.