Sensory neuron proteins interact with the intracellular domains of sodium channel NaV1.8

Molecular Brain Research 110 (2003) 298–304 www.elsevier.com / locate / molbrainres

Research report

Sensory neuron proteins interact with the intracellular domains of sodium channel Na V 1.8 Misbah Malik-Hall, W.-Y. Louisa Poon, Mark D. Baker, John N. Wood, Kenji Okuse* Department of Biology, University College London, Gower Street, London WC1 E 6 BT, UK Accepted 4 November 2002

Abstract Voltage-gated sodium channels initiate and propagate action potentials in excitable cells. The tetrodotoxin-resistant Na 1 channel (Na V 1.8 / SNS) is expressed in damage-sensing neurons (nociceptors) and plays an important role in pain pathways. Expression of high levels of functional Na V 1.8 in heterologous cells has proved problematic, even in the presence of known sodium channel accessory b-subunits. This suggests that other regulatory proteins are required for normal levels of Na V 1.8 expression. Here we report the use of a yeast two-hybrid system and a rat dorsal root ganglion cDNA library to identify 28 different clones encoding proteins which interact with intracellular domains of Na V 1.8. Many clones are expressed at high levels in small diameter DRG neurons as judged by in situ hybridization. Interacting proteins include cytoplasmic elements and linker proteins (e.g. b-actin and moesin), enzymes (e.g. inositol polyphosphate 5-phosphatase and TAO2 thousand and one protein kinase), channels and membrane-associated proteins (voltagedependent anion channel VDAC3V and tetraspanin), as well as motor proteins (dynein intermediate and light chain) and transcripts encoding previously undescribed proteins. Immunoprecipitation (pull-down) assays confirm that some of the proteins interact with, and may hence regulate, Na V 1.8 in vivo.  2002 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Sodium channels Keywords: Yeast two hybrid; Accessory subunits; Sensory neurons; Nociception and pain

1. Introduction Ten distinct pore-forming voltage-gated sodium channel a-subunits have been identified by molecular cloning [6]. The tetrodotoxin-resistant sodium channel Na V 1.8 / SNS is expressed predominantly in small diameter sensory neurons in the dorsal root and cranial sensory ganglia and plays an important role in pain pathways [1]. Four auxiliary b-subunits which associate with sodium channel a-subunits have been identified [7,8,10,14]. Co-expression of b-subunits with a-subunits such as Na V 1.2 in mammalian cell lines and Xenopus oocytes results in an increased level of expression of sodium currents. The voltage dependence of activation and inactivation also shifts to levels closer to the values exhibited by endogen*Corresponding author. Tel.: 144-207-679-7943. E-mail address: [email protected] (K. Okuse).

ous channels [7]. In the case of Na V 1.8, the b 1 subunit up-regulates expression in Xenopus oocytes [21]. b 3 also up-regulates functional Na V 1.8 expression in a mammalian cell line [18]. However, none of the b-subunits increase functional Na V 1.8 expression to endogenous levels seen in DRG, and the properties of the expressed a-subunits are aberrant in terms of their current–voltage relationship and kinetics. In contrast, microinjection of Na V 1.8 cDNA into the nuclei of DRG neurons of Na V 1.8 null mutant mice or superior cervical ganglia (SCG) neurons results in high level expression of sodium currents which show the same channel properties as those observed in wild-type DRG neurons [1,3]. This suggests that normal levels of Na V 1.8 sodium channel expression require the presence of, as yet, unidentified accessory subunits. Here we describe the use of the yeast two-hybrid system to identify proteins that interact with cytoplasmic regions of Na V 1.8. Using a combination of in situ hybridization

0169-328X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00661-7

M. Malik-Hall et al. / Molecular Brain Research 110 (2003) 298–304

and immuno-precipitation assays we show that clones are present within Na V 1.8 expressing neurons, and may physically associate with Na V 1.8.

2. Materials and methods

2.1. Yeast two-hybrid screening Polymerase chain reaction (PCR) primers were designed to amplify the intracellular segments of the a-subunit of Na V 1.8 from full length cDNA in pRK7 vector (17) to generate six baits corresponding to the following amino acid residues: N-terminal (bait I, amino acids 1 to 127), between D1 and D2 (bait II, amino acids 400 to 660), between D2 and D3 (bait III, amino acids 893 to 1148), between D3 and D4 (bait IV, amino acids 1420 to 1472), the C-terminal divided into two parts (bait Va, amino acids 1724–1844 and bait Vb, 1842–1956). Control experiments demonstrated that the long C-terminal bait V had intrinsic transcriptional activating effects, and this region of the sodium channel was therefore sub-cloned into two baits, where the endogenous activity was lost. EcoRI and NotI restriction sites were introduced into PCR products in the 59 and 39 direction, respectively. The five intracellular loops were cloned in frame into the pEG202 vector: Forward primers SNSYH-1 SNSYH-3 SNSYH-5 SNSYH-7 SNSYH-9 SNSYH-12 Reverse primers SNSYH-2 SNSYH-4 SNSYH-6 SNSYH-8 SNSYH-10

59-GCGAATTCATGGAGCTCCCCTTTG-39 ]]] 59-GCGAATTCGAAGAGCAGAGCCAGG-39 ]]] 59-GCGAATTCAGCGCGGACAACCTCAC-39 ]]] 59-GCGAATTCGACAACTTCAACCAACAG-39 ]]] 59-GCGAATTCGAGAACTTCAACGTAGCC-39 ]]] 59-GCGAATTCCCAATAGCCACCACCC-39 ]]] 59-TATAGCGGCCGCTTTGATGGCTGTTCTTC-39 59-TATAGCGGCCGCGAACAGCGCCATCTTG-39 59-TATAGCGGCCGCGCGGTAGCAGGTCTTG-39 59-TATAGCGGCCGCGTCAAACACGAAGCCTTG-39 59-TATAGCGGCCGCTCACTGAGGTCCAGG-39

SNSYH-11

299

59-TATAGCGGCCGCGGCTATTGGTTCATAG-39

EcoRI sites are underlined, NotI sites are boldfaced. PCR was used to amplify the inserts using the following primer sets: SNSYH-1 / SNSYH-2, SNSYH-3 / SNSYH-4, SNSYH-5 / SNSYH-6, SNSYH-7 / SNSYH-8, SNSYH-9 / SNSYH-10, SNSYH-12 / SNSYH-11 for bait I, bait II, bait III, bait IV, bait Va, and bait Vb, respectively (see Fig. 1). A cDNA library from postnatal day 1 (P1) dorsal root ganglia (DRG) was expressed as in-frame fusions with the Gal4 transcriptional activation domain [11,17]. Approximately 5310 6 yeast transformants for each bait were screened for both b-galactosidase activity and growth in the absence of leucine as described [5,17]. Positive library plasmids were rescued from yeast and electroporated into E. coli KC8 and selected on M9 agar plates lacking tryptophan. The entire inserts in the rescued library plasmids were sequenced. Although most of the clones were identified as full length cDNA, only partial sequences were isolated for some clones. In such cases, RT-PCR was performed to obtain full length cDNA using the cDNA library as template with specific primer pairs for the clones. In such a way, full length cDNA for clones II-6 and A-140 were obtained. Full length Papin (clone III-42) and Periaxin (clone IV-40) cDNA were also obtained from Yoshimi Takai (Osaka University) and Peter Brophy (University of Edinburgh).

2.2. In situ hybridization PCR fragments of unique regions of individual clones were subcloned into pGEM-T Easy (Promega), and DIGUTP labelled sense or antisense cRNA probes were generated using T7 RNA polymerase. The regions of clones used as probes are nucleotide positions 5–288 (clone I-1), 215 bp 39-UTR (II-4), 94 bp 39-UTR (II-5), 274 bp 39-UTR (II-6), 7857–8200 (III-42), and 378–541 (IV-7-40). Frozen DRG sections (10 mm thick) were fixed for 15 min in 4% paraformaldehyde on ice and were

Fig. 1. Structure of the Na V 1.8 a-subunit showing the four homologous domains, each of which is composed of six membrane spanning segments. The location of the six baits (baits I, II, III, IV, Va, and Vb) is indicated by arrows and the numbers correspond to the amino-acid location. Bait II has the five sites for PKA phosphorylation at serine residues.

300

M. Malik-Hall et al. / Molecular Brain Research 110 (2003) 298–304

acetylated in 0.1 M triethanolamine, 0.25% acetic anhydride for 10 min. Prehybridization was carried out in 50% formamide, 43 SSC, 100 mg / ml herring sperm DNA, 50 mg / ml tRNA, 23 Denhardt’s solution at room temperature for 1 h. Hybridization was carried out in the same buffer containing 50 ng / ml cRNA probe at 65 8C for 16 h. Sections were washed in 0.13 SSC at 72 8C and incubated with alkaline phosphatase conjugated anti-digoxygenin antibody.

2.3. Translocation assays A stably transformed CHO cell line (CHO-SNS22 cells) that expresses rat Na V 1.8 protein in the cytosol was transfected with expression plasmid pBS500-GFP encoding candidate clones using lipofectamine. Three days after transfection, the cells were fixed with 4% paraformaldehyde for 15 min on ice and subsequently incubated with anti-Na V 1.8 polyclonal antibody (SNS11) for 1 h at room temperature [16]. The cells were washed three times with PBS and incubated with rhodamine-labelled anti-rabbit IgG. The cells were mounted in CITIfluor and analysed with a confocal microscope [17].

2.4. Co-immunoprecipitation pull-down assays Using PCR with the forward primer Nco-HA (59TAACCATGGCCTACCCTTATGATGTG-39) and reverse primer BCO2-2 (59-AGACATCTAGACAACCTTGATTGGAG-39), rat sensory neuron cDNA library inserts in pJG4-5 vector were amplified with the restriction sites NcoI and XbaI introduced into the 59 and 39 end of the PCR products, respectively, to retain the HA-tag. The PCR products were then digested with NcoI and XbaI restriction enzymes and ligated into linearized pBS500. Because the antisera directed against Na V 1.8 recognise only the C-terminal, the intracellular loops were all fused to loop V to enable interactions to be visualised. Using recombinant PCR, intracellular loop IV (between D3 and D4 amino acids 1420–1472) was fused with the C-terminal of Na V 1.8 and then cloned into pBS500 vector that had been digested with NcoI and XbaI restriction enzymes. PCR primers were designed for the 39 end of the intracellular loops IV to bind to the 59 end of the amplified C-terminal region. PCR was carried out using SNSYH-7-2 (59-CACCATGGACAACTTCAACCAACAG-39) and SNSYH-8-2 (59-AAGTTCTCGTCAAACACGAAGCCTTG-39) primer pairs. C-terminal of Na V 1.8 was amplified by SNSYH-9-5 (59-TGTTTGACGAGAACTTCAACGTAGCC-39) and SNSYH-10-2 (59-GATCTAGATCACTGAGGTCCAGGG-39) to match the 39 end of the PCR products of intracellular loop IV. This fusion Na V 1.8 was used for co-immunoprecipitation against clones IV-40 and IV-7-40. The intracellular loops I, II, and III were also fused to the C-terminal domain of Na V 1.8 for the other clones.

COS cells were transiently co-transfected with HAtagged cDNA library inserts and Na V 1.8 recombinant plasmid DNA in the pBS500 expression vector by means of lipofection. Transfected cells were harvested after 3 days, washed with phosphate-buffered saline (PBS, pH 7.4) at 4 8C three times and then incubated in 1 ml of ice-cold RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 8.0) with 1 mg / ml each of aprotinin, leupeptin, and pepstatin and 0.01 mM PMSF (phenylmethylsulfonyl fluoride) at 4 8C for 1 h. The lysate was centrifuged at 10,000 g for 10 min at 4 8C. The supernatant was retained and centrifuged at 14,000 g for 30 min at 4 8C. The supernatant was then incubated with a 1:1000 dilution of anti-hemaglutinin or anti-Na V 1.8 antibody for 1 h at 4 8C. 100 ml of Protein-A Sepharose beads was added and left at 4 8C for 1 h. The mixture was centrifuged (10,000 g) for 10 min and the Sepharose beads were washed with lysis buffer three times. Sepharose beads were boiled in SDS–PAGE sample buffer. The mixture was spun briefly and 20 ml of supernatant was loaded onto an 8% SDS–PAGE gel. After electrophoresis, protein bands on the gel were transferred to a nitrocellulose membrane for Western blotting. The membrane was incubated with primary antibody, a 1:1000 dilution of anti-HA antibody, at room temperature for 1 h. After incubation, the membrane was washed in PBS containing 0.1% Tween-80 and incubated with a 1:1000 dilution of horseradish peroxidase conjugated secondary antibody, antimouse IgG, for 1 h at room temperature. The membrane was washed as described above. ECL Western blotting detection reagents (Amersham) were mixed in 1:1 ratio and applied onto the membrane for 1 min. The membrane was then exposed to BioMax (Kodak) film.

3. Results

3.1. Yeast two-hybrid screening The regions of Na V 1.8 used as baits are shown in Fig. 1. Using yeast two-hybrid screening, 28 positive clones were isolated from a DRG cDNA library, including p11 which was previously reported [17], as listed in Table 1. The accession numbers of clones identified by sequencing (or close homologues of identified clones) are shown in parentheses. In addition, the region of the sodium channel that the baits associate with is shown by the subscript of each clone number. Some of the clones were isolated only once as prey, while the others were isolated multiple times. The number of clones isolated for each prey are shown in Table 1.

3.2. In situ hybridization To determine whether the clones were expressed in Na V 1.8-positive small diameter neurons in DRG, in situ

M. Malik-Hall et al. / Molecular Brain Research 110 (2003) 298–304

301

Table 1 Identity of interacting clones, the number of copies cloned, their presence within DRG neurons as judged by in situ hybridization (clones against baits I–IV) and RT-PCR (baits Va and Vb), data for plasma membrane translocation analyzed by anti-Na V 1.8 antibody, and in vitro binding studied by GST pull-down assay (clone I-1, Ref. [17]) and co-immunoprecipitation assay (clones III-42, IV-40, IV-7-40) Bait

Clone identity

Copy number

mRNA in DRG

Translocation assay

In vitro binding

Bait I I-1

p11 Annexin II light chain (J03627)

5

1

1

1

Bait II II-4 II-5 II-6

Similar to human Huntintin-interacting protein (U79734) Similar to mouse Zyxin-related protein (AF097511) Follistatin-related protein (U06864)

3 1 2

1 1 1

2 ND 2

ND ND ND

2

1

ND

ND

III-42

Similar to mouse syntrophin-associated serine / threonine kinase (NM 019945) ] Papin (AF169411)

2

1

2

1

Bait IV IV-40 IV-73 IV-7-40

Periaxin (Z29649) Cdc37 (NM 053743) ] Tctex-1 Dynein light chain (AB010119)

3 2 9

1 1 1

2 2 2

1 ND 1

Similar to rat Inositol polyphosphate 5-phosphatase (AB032551) missing seven amino acids at position 35 Similar to rat VDAC3 V voltage-dependent anion channel (AF048830) having extra 10 amino acids at N-terminal Similar to mouse TM4SF (NM 019656) ] Similar to mouse Necdin (NM 010882) ] IC2 Dynein intermediate chain (U39044) Connexin 43 (NM 012567) ] Similar to mouse Pelota (AF148638) Moesin (AF004811) Alpha-tubulin (RNATUBZ) Similar to mouse tyrosine-rich heat shock protein (XM 110140) ] Similar to mouse Prnpb long incubation prion protein (U29187) TAO2 thousand and one protein kinase (AF140556) Similar to mouse 10 days embryo cDNA, clone 2610001E01 (AK011254)

2

1

2

ND

3

1

2

ND

2 2 3 2 1 2 3 3 1 2 1

ND 1 1 ND 1 1 1 1 ND 1 1

ND 2 ND ND ND 2 ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND

10 1 1 2

1 1 1 1

2 2 ND 2

ND ND ND ND

2 1

1 1

2 ND

ND ND

Bait III III-27

Va A-10 A-32 A-91 A-103 A-123 A-133 A-136 A-140 A-145 A-148 A-150 A-165 A-189 Vb B-4 B-13 B-15 B-18 B-20 B-25

Calmodulin III (NM 012518) ] Calmodulin II (NM 017326) ] Calmodulin I (AF178845) Similar to rat PKC zeta-interacting protein (Y08355) and rat oxidative stress induced protein (NM 011018) ] Beta-actin (NM 031144) ] Similar to human RAB3 GTPase-activating protein (XM 040048) ]

hybridization was performed on 2 week old rat DRG sections. All clones binding to baits I–IV were tested and all demonstrated strong staining in small diameter neurons, showing that these clones are expressed in neurons that are known to express endogenous Na V 1.8. Expression of mRNA in DRG for most of the clones binding to baits Va and Vb were tested by RT-PCR. The pattern of expression of mRNA encoding transcripts detected by in situ hybridization (for clones against baits I–IV) and RT-PCR (for clones against baits Va and Vb) are scored in Table 1. Fig. 2A shows the expression of mRNA encoding p11 (clone I-1) in small (red arrow) and large (yellow arrow) diameter sensory neurons, and Na V 1.8-like immunoreactivity can be

detected only in small diameter neurons (Fig. 2B). Antisense probes for Huntingtin-interacting protein (clone II4), Zyxin-related protein (II-5), Follistatin-related protein (II-6), Papin (III-42), and dynein light chain (IV-7-40) were detected both in small (red arrows) and large (yellow arrows) neurons (Fig. 2C–G), while sense probes for all of the clones did not show any positive staining (example for sense IV-7-40 probe shown in Fig. 2H).

3.3. Co-immunoprecipitation assays Using over-expression in heterologous expression systems, some clones were examined for direct interactions

302

M. Malik-Hall et al. / Molecular Brain Research 110 (2003) 298–304

the anti-Na V 1.8 antisera did not pull down HA-tagged clones in the absence of Na V 1.8 co-expression.

3.4. Translocation assays Previous studies have demonstrated that p11, an annexin light chain, can induce translocation of immunoreactive Na V 1.8 from cytosol to membrane of a permanentlytransfected cell line, CHO-SNS22. Using this same approach, clones were introduced into the cell line and translocation assessed as described [17]. p11 causes approximately a six-fold increase in membrane-associated immunoreactivity, whilst other clones showed no increased translocation over background levels (Table 1). An example of membrane translocation of Na V 1.8-like immunoreactivity in CHO-SNS22 cells transfected with full length p11 cDNA is shown in Fig. 4A as well as cells transfected with full length cDNA for clone IV-7-40 (Fig. 4B) as an example for negative data. Some clones could not be isolated in full length form, and these were not tested in the translocation assay (ND).

4. Discussion

Fig. 2. Antisense probes for clones I-1 (A), II-4 (C), II-5 (D), II-6 (E), III-42 (F), and IV-7-40 (G) demonstrate strong staining in both small (red arrow) and large (yellow arrow) diameter neurons, showing that mRNAs for these clones are expressed in nociceptive small diameter neurons that have endogenous Na V 1.8 revealed by immunohistochemistry using antiNa V 1.8 antibody SNS11 (B). Sense probes (H: example for clone IV-740) do not show any specific neuronal staining.

with fragments of Na V 1.8. For example, clones IV-40 and IV-7-40 fused at their N-terminals to HA epitopes were generated. These were co-transfected into COS cells with loop IV of Na V 1.8, previously fused to the immunoreactive C-terminal of Na V 1.8. Immunoprecipitation of cell lysates was carried out with either anti-HA or anti-Na V 1.8 antibodies, and the subsequent Western blots were probed with anti-HA antibodies. Anti-Na V 1.8 brought down HA-immunoreactive bands of appropriate sizes for both IV-40 (Fig. 3A) and IV7-40 (Fig. 3B), showing an interaction between co-expressed proteins. Controls demonstrated that

The yeast two-hybrid screen devised by Fields and Song is a high throughput genetic screen for interacting proteins [4]. This approach has already been used to study proteins that bind to sodium channels. For example, the TTXresistant sodium channel Na V 1.9 has been shown to interact with a growth factor, fibroblast growth factor homologous factor-1B (FHF1B), a member of the fibroblast growth factor family. FHF1B selectively interacts via its N-terminal 5–77 amino acid residues with the C-terminal region of Na V 1.9. The interaction is specific because the C-termini of two other sodium channels, Na V 1.7 and Na V 1.8, do not bind FHF1B [12]. Na V 1.9 has also been shown to interact with contactin, a cell adhesion molecule known to be involved in the quaternary complex of extracellular and intracellular proteins involved with the spatial pattern of expression of sodium channels [13]. Contactin (also known as F3 or F11) is a surface glycoprotein that has significant homology with the b2 subunit of voltage-gated Na 1 channels [9]. Contactin and Na 1 channels can be co-immunoprecipitated from brain homogenates. Contactin binds directly to Na V 1.9 and recruits tenascin to the protein complex in vitro. Na V 1.9 and contactin co-immunoprecipitate from dorsal root ganglia and transfected Chinese hamster ovary cells. Co-transfection of CHO cells with Na V 1.9 and contactin has been found to enhance the surface expression of Na V 1.9 [9]. An interaction trap analysis for proteins that regulate Na V 1.8 expression is of particular interest, because this channel is hard to express in heterologous systems, and appears to play a specialised role in pain pathways [1,3]. This paper describes a comprehensive analysis of binding

M. Malik-Hall et al. / Molecular Brain Research 110 (2003) 298–304

303

Fig. 3. Western blot obtained with expressed HA-tagged clones IV-40 and IV7-40 when the bait IV–V fragment of Na V 1.8 was co-expressed and precipitated with either anti-HA or anti-Na V 1.8 antisera, before visualisation with anti-HA antibodies. The HA-tagged clones IV-40 and IV7-40 bands are absent in the anti-Na V 1.8 antibody precipitates obtained from cells without Na V 1.8 co-expression. These two clones thus directly interact with Na V 1.8.

proteins for all the intracellular regions of the sensory neuron-specific channel Na V 1.8. We have accumulated evidence that the transcripts encoding these proteins are present in the same cell population that expresses the Na V 1.8 channel, and have shown that over-expression of some HA-epitope-tagged proteins results in association with domains of the voltage-gated sodium channel which they bind to in the yeast selection system. The functional consequences of the interactions may be assessed by examining the effect of over-expression of the clones on sodium channel density, as well as the consequences of antisense application to sensory neurons in culture. We have only completed an analysis for one clone (p11), which binds to the N-terminal intracellular domain of Na V 1.8 [17]. This protein has been found to be necessary, although not sufficient, for high level expression of Na V 1.8 in DRG sensory neurons [17]. Such analyses for all of the clones alone, or in combination, are desirable, although difficult to accomplish. The range of interacting protein subtypes associated with Na V 1.8 is remarkably varied. As well as cytoskeletal proteins and linker proteins, there are interactions with two components of the dynein motor protein that may be important in axonal transport [20]. There are also associa-

tions with other channel-like molecules such as tetraspanins and an anion channel. Some enzymes seem to associate with Na V 1.8 (e.g. inositol polyphosphate 59phosphatase), whilst other clones of unknown function show structural homology with known proteins (e.g. follistatin-related protein). There are also unknown proteins (e.g. A-189), as well as very large proteins (e.g. Papin) that are difficult to express at high levels. Finally, there are some proteins that have previously been shown to have a role in regulating sodium channel function. Calmodulin has been demonstrated to interact with the Cterminal regions of other sodium channels by yeast twohybrid screening, and evidence that the interaction of calmodulin with sodium channels has functional consequences has been obtained [2,15]. In summary, a range of defined proteins that show high affinity for the intracellular loops of Na V 1.8 have been identified using a genetic screen. Distribution studies and some biochemical analyses support the possibility of interactions between Na V 1.8 and these clones. The functional significance of the putative interactions described in this paper remains to be established. However, downregulating the expression of Na V 1.8, which contributes substantially to electrogenesis in C-type DRG neurons [19]

304

M. Malik-Hall et al. / Molecular Brain Research 110 (2003) 298–304

Fig. 4. (A) Na V 1.8-like immunoreactivity translocated to the plasma membrane in CHO-SNS22 cells transfected with full length p11 (clone I-1) cDNA. (B) Cells transfected with full length cDNA for tctex-1 (clone IV-7-40) shows cytosolic immunoreactive Na V 1.8 with no obvious membrane translocation.

and is important in pain pathways, has already been suggested as a potentially useful new analgesic drug strategy [1,17].

Acknowledgements We acknowledge generous support from the Medical Research Council and the Wellcome Trust, the kind gift of a DRG-derived cDNA library from Moses Chao (NYU), GFP expression and yeast vectors from Brian Sauer (NIH) and Alan Hall (LMCB London), cell lines from Mathew Lo (Wilmington) and helpful comments from K. Pojak.

References [1] A.N. Akopian, V. Souslova, S. England, K. Okuse, N. Ogata, J. Ure, A. Smith, B.J. Kerr, S.B. McMahon, S. Boyce, R. Hill, L.C. Stanfa, A.H. Dickenson, J.N. Wood, The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways, Nature Neurosci. 2 (1999) 541–548. [2] I. Deschenes, N. Neyroud, D. DiSilvestre, E. Marban, D.T. Yue, G.F. Tomaselli, Isoform-specific modulation of voltage-gated Na(1) channels by calmodulin, Circ. Res. 90 (4) (2002) E49–E57. [3] S. England, K. Okuse, N. Ogata, J.N. Wood, Heterologous expression of the sensory neurone-specific sodium channel (SNS) asubunit in rat sympathetic neurones, J. Physiol. 511P (1998) 124P.

[4] S. Fields, O. Song, A novel genetic system to detect protein–protein interactions, Nature 340 (1989) 245–246. [5] R.L. Finley Jr., R. Brent, Interaction trap cloning with yeast, in: D. Glover, B.D. Hames (Eds.), DNA Cloning-expression Systems: A Practical Approach, Oxford University Press, Oxford, UK, 1995, pp. 170–203. [6] A.L. Goldin, Resurgence of sodium channel research, Annu. Rev. Physiol. 63 (2001) 871–894. [7] L.L. Isom, D.S. Ragsdale, K.S. De Jongh, R.E. Westenbroek, B.F. Reber, T. Scheuer, W.A. Catterall, Structure and function of the b-2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif, Cell 83 (1995) 433–442. [8] L.L. Isom, K.S. De Jongh, D.E. Patton, B.F. Reber, J. Offord, H. Charbonneau, K. Walsh, A.L. Goldin, W.A. Catterall, Primary structure and functional expression of the beta-1 subunit of the rat brain sodium channel, Science 256 (1992) 839–842. [9] K. Kazarinova-Noyes, J.D. Malhotra, D.P. McEwen, L.N. Mattei, E.O. Berglund, B. Ranscht, S.R. Levinson, M. Schachner, P. Shrager, L.L. Isom, Z.C. Xiao, Contactin associates with Na 1 channels and increases their functional expression, J. Neurosci. 21 (19) (2001) 7517–7525. [10] K.A. Kazen-Gillespie, D.S. Ragsdale, M.R. D’Andrea, L.N. Mattei, K.E. Rogers, L.L. Isom, Cloning, localization, and functional expression of sodium channel b1A subunits, J. Biol. Chem. 275 (2000) 1079–1088. [11] H. Kong, J. Boulter, J.L. Weber, C. Lai, M.V. Chao, An evolutionarily conserved transmembrane protein that is a novel downstream target of neurotrophin and ephrin receptors, J. Neurosci. 21 (2001) 176–185. [12] C.-J. Liu, S.D. Dib-Hajj, S.G. Waxman, Fibroblast growth factor homologous factor 1B binds to the C terminus of the tetrodotoxinresistant sodium channel rNa v 1.9a (NaN), J. Biol. Chem. 276 (2001) 18925–18933. [13] C.-J. Liu, S. Dib-Hajj, J.A. Black, J. Greenwood, Z. Lian, S.G. Waxman, Direct interaction with contactin targets voltage-gated sodium channel Nav1.9 / NaN to the cell membrane, J. Biol. Chem. 276 (2001) 46553–46561. [14] K. Morgan, E.B. Stevens, B. Shah, P.J. Cox, A.K. Dixon, K. Lee, R.D. Pinnock, J. Hughes, P.J. Richardson, K. Mizuguchi, A.P. Jackson, b-3: An additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics, Proc. Natl. Acad. Sci. USA 97 (2000) 2308–2313. [15] M. Mori, T. Konno, T. Ozawa, M. Murata, K. Imoto, K. Nagayama, Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca 21 sensitivity?, Biochemistry 39 (2000) 1316–1323. [16] K. Okuse, S.R. Chaplan, S.B. McMahon, Z.D. Luo, N.A. Calcutt, B.P. Scott, A.N. Akopian, J.N. Wood, Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain, Mol. Cell. Neurosci. 10 (1997) 196–207. [17] K. Okuse, M. Malik-Hall, M.D. Baker, W.-Y.L. Poon, H. Kong, M.V. Chao, J.N. Wood, Annexin II light chain regulates sensory neuronspecific sodium channel expression, Nature 417 (6889) (2002) 653–656. [18] M.A. Powell, J.R. Glenney, Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src, Biochem. J. 247 (1987) 321–328. [19] M. Renganathan, T.R. Cummins, S.G. Waxman, Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in neurons, J. Neurophysiol. 86 (2) (2001) 629–640. [20] J.V. Shah, D.W. Cleveland, Slow axonal transport: fast motors in the slow lane, Curr. Opin. Cell Biol. 14 (1) (2002) 58–62. [21] K. Vijayaragavan, M.E. O’Leary, M. Chahine, Gating properties of Na(v)1.7 and Na(v)1.8 peripheral nerve sodium channels, J. Neurosci. 21 (2001) 7909–7918.