Comprehensive Proteomic Profiling of Human Nav1.7-Interacting Proteins Reveals Conserved Regulatory Networks Involved in Nociceptive Signaling

Abstract

The voltage-gated sodium channel Nav1.7, encoded by the SCN9A gene, is critically involved in the initiation and propagation of nociceptive signals. While prior research has delineated the interactome of mouse Nav1.7 (mNav1.7), the molecular partners associated with its human homolog (hNav1.7) remain largely undefined. In this study, we employed tandem affinity purification (TAP) combined with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to systematically characterize the protein-protein interaction (PPI) network of hNav1.7 in stably transfected HEK293 cells. Functional expression of TAP-tagged hNav1.7 was confirmed by immunofluorescence, immunoblotting, and whole-cell patch-clamp electrophysiology. A total of 261 interacting proteins were identified, primarily localized to the plasma membrane and cytoplasm, and predominantly enriched in protein translation, folding, and trafficking pathways. Comparative proteomic analysis revealed conserved interactors shared between human and mouse Nav1.7, including translation elongation factors (Eef1a1, Eef2), chaperonin subunits (CCT2, CCT3, CCT5, CCT6A, CCT7), and members of the kinesin and Rab GTPase families. Knockdown of 2 conserved interactors, CCT5 and TMED10, significantly reduced hNav1.7 current density, confirming their functional relevance. These findings provide new insights into the proteomic architecture and regulatory mechanisms of hNav1.7, offering potential targets for modulating channel function in pain pathophysiology.

Keywords

Nav1.7 sodium channel protein-protein interaction tandem affinity purification mass spectrometry

Introduction

Pain is a complex physiological and pathological phenomenon affecting a substantial proportion of the global population, with prevalence increasing in parallel with aging demographics and rising psychosocial stressors. Epidemiological studies suggest that the number of individuals suffering from chronic pain now exceeds the combined prevalence of heart disease, cancer, and diabetes.¹ Despite the widespread use of pharmacologic, surgical, and physical therapies, pain relief remains inadequate in nearly 40% of patients, often due to limited efficacy and adverse effects, including addiction and respiratory depression.² Consequently, identifying new molecular targets for safe and effective analgesics remains a major research priority.

Voltage-gated sodium channels (Nav) are key mediators of action potential initiation and propagation in excitable tissues. Among these, Nav1.7, encoded by the SCN9A gene, plays a central role in human nociception.³ Gain-of-function mutations in SCN9A have been linked to severe pain disorders such as primary erythromelalgia,⁴ paroxysmal extreme pain disorder,⁵ and small fiber neuropathy,⁶ whereas loss-of-function mutations lead to congenital insensitivity to pain without affecting motor or cognitive function.⁷ Animal studies further support the crucial role of Nav1.7 in pain transmission, as conditional knockout of SCN9A in sensory neurons results in pronounced deficits in inflammatory and acute pain responses.^8-11

While selective Nav1.7 antagonists have been pursued for analgesic development, progress has been hampered by pharmacokinetic limitations, off-target effects, and the high structural homology among Nav isoforms.^12,13 An alternative therapeutic strategy involves modulating Nav1.7 indirectly by targeting proteins that regulate its expression, trafficking, and degradation. Indeed, several such regulators have been reported, including CRMP2,¹⁴ NEDD4-2,¹⁵ and FGF13.¹⁶ However, a comprehensive map of the Nav1.7 protein interaction network in human cells has yet to be established.

In a previous study, we systematically profiled the protein interactome of mouse Nav1.7 (mNav1.7) using an epitope-tagged knock-in model and affinity purification followed by mass spectrometry.¹⁷ In the present study, we extend this work to human Nav1.7 (hNav1.7), utilizing a TAP-tagged hNav1.7 construct stably expressed in HEK293 cells. Through tandem affinity purification (TAP), size-exclusion chromatography, and mass spectrometry, we identified and characterized the core interactome of hNav1.7. Moreover, by comparing our results with the previously established mNav1.7 interactome, we identified conserved protein partners likely to be essential for channel function. Functional validation via siRNA knockdown and patch-clamp electrophysiology further substantiated the role of selected interactors in modulating Nav1.7 activity. Our findings provide a foundation for understanding hNav1.7 regulation and may guide the development of new therapeutic strategies for pain management.

Materials and Methods

Bioinformatic Analysis of Gene Expression

Gene expression data for SCN9A and associated regulatory genes in human dorsal root ganglion (DRG) neurons and HEK293 cells were retrieved from BioGPS (http://biogps.org). Data were extracted from the Geneatlas U133A-gcrma and NCI60 on U133A-gcrma datasets. Expression values were normalized against GAPDH as an internal reference to compare relative mRNA expression levels across key genes involved in protein translation, trafficking, and membrane localization of Nav1.7.

Generation of TAP-Tagged hNav1.7 Stable Cell Line

A stably transfected HEK293 cell line expressing human Nav1.7 with a tandem affinity purification (TAP) tag was constructed.¹⁸ The TAP tag-a fusion of a histidine affinity tag (HAT) and 3 tandem FLAG epitopes-was cloned in-frame immediately upstream of the SCN9A stop codon. Plasmid constructs were transfected using Lipofectamine 2000, and stable clones were selected in G418-containing medium. Expression and membrane localization of the TAP-tagged hNav1.7 protein were verified by immunocytochemistry and western blotting.

Immunocytochemistry

Immunocytochemistry was performed according to previously described methods.¹⁷ HEK293 cells were cultured on poly-D-lysine-coated coverslips for 24 hours. Following permeabilization with cold methanol and fixation with acetone, cells were blocked with PBS containing 0.3% Triton X-100 and 10% goat serum. Primary anti-FLAG antibodies (1:500, Sigma, F1804) were applied overnight at 4°C. Cells were then incubated with Alexa Fluor 488-conjugated secondary antibodies (1:2000, Invitrogen) for 2 hours at room temperature. Nuclei were counterstained with DAPI and visualized using fluorescence microscopy.

Western Blot Analysis

Western blot analysis was performed according to our previously described procedures.¹⁷ Protein lysates were prepared in buffer containing 20 mM Tris, 100 mM NaCl, 1% n-dodecyl-β-D-maltoside (DDM), 0.2% CHS, and protease inhibitors (pH 7.4). Lysates were centrifuged at 20,000g for 10 minutes at 4°C. Protein concentrations were determined using the Pierce BCA assay, and 40 µg of protein per sample was resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk and probed with antibodies against FLAG, Nav1.7, CCT5, or TMED10. Horseradish peroxidase-conjugated secondary antibodies were used, and signals were detected via chemiluminescence.

RNA Interference

Small interfering RNAs (siRNAs) targeting human CCT5 (NM_012073) and TMED10 (NM_006827) were synthesized. The sequences used were: CCT5 siRNA: 5′-CCGAGUCCAUUGUUAAUGATT-3′ and 5′-UCAUUAACAAUGGACUCGGTT-3′; TMED10 siRNA: 5′-GGCGAUGUGACUAUAACAATT-3′ and 5′-UUGUUAUAGUCACAUCGCCTT-3′

A scrambled siRNA was used as a negative control. siRNAs were transfected into HEK293 cells using Lipofectamine 2000. Knockdown efficiency was confirmed by western blotting.

Electrophysiological Recordings

Whole-cell patch-clamp recordings were performed following our previously established protocol.¹⁷ Recordings were conducted using an Axopatch 200B amplifier and a Digidata 1322A digitizer. Microelectrodes (2.5-4 MΩ) were filled with internal solution containing 140 mM CsF, 10 mM NaCl, 1.1 mM EGTA, and 1 mM MgCl₂. The external solution contained 140 mM NaCl, 3 mM KCl, 1 mM CaCl₂, and 1 mM MgCl₂. Cells were held at −100 mV before stimulation. Current-voltage relationships were obtained, and voltage-dependent activation/inactivation parameters were fit using Boltzmann equations.¹⁹ Data were analyzed with pCLAMP and Origin 9.0 software.

Tandem Affinity Purification and Mass Spectrometry

Tandem affinity purification (TAP) and mass spectrometry (MS) were performed following our previously established protocol.¹⁷ Briefly, TAP-tagged hNav1.7 and its associated protein complexes were isolated using a sequential two-step affinity purification strategy. Cell lysates were first incubated with anti-FLAG resin (Sigma), followed by elution using 3 × FLAG peptide. The eluate was subjected to secondary purification with Ni-NTA agarose (Qiagen) and eluted with imidazole buffer. Purified complexes were concentrated using 100-kDa cutoff Centricon filters and resolved by size exclusion chromatography (SEC) on a Superose 6 column. Fractions were subjected to LC-MS/MS for protein identification.

Protein Annotation and Comparative Analysis

Identified proteins were analyzed using the PANTHER Classification System to determine subcellular localization and functional enrichment. Comparative analysis with previously published mNav1.7 interactors¹⁷ was conducted to identify conserved protein partners across species and tissues.

Statistical Analysis

Data are presented as mean ± standard deviation (SD). Group comparisons were performed using unpaired Student’s t-tests or one-way ANOVA where appropriate. A P-value < .05 was considered statistically significant.

Results

Comparable Expression of Nav1.7-Related Genes in Human DRG and HEK293 Cells

To evaluate the suitability of HEK293 cells as a surrogate model for studying human Nav1.7 (hNav1.7) protein-protein interactions, we first examined the expression profiles of Nav1.7 and its associated regulatory genes in human dorsal root ganglia (DRG) and HEK293 cells using publicly available transcriptomic datasets from BioGPS. Genes implicated in sodium channel α- and β-subunits, chaperonin-assisted folding, cytoskeletal transport, membrane anchoring (eg, ankyrins, contactins), and post-translational modifications (eg, phosphorylation, ubiquitination) were similarly expressed in both cell types (Figure 1A-F). These findings support the use of HEK293 cells as a feasible system for investigating Nav1.7 protein interactions in vitro.

Figure 1.

Comparative expression of Nav1.7-related genes in human dorsal root ganglia (DRG) and HEK293 cells. (A-F) Relative mRNA expression profiles of key genes associated with Nav1.7 biogenesis and regulation were obtained from publicly available BioGPS transcriptomic datasets (Geneatlas U133A-gcrma and NCI60 on U133A-gcrma). Expression values for each gene were normalized to GAPDH. Panels show representative groups of genes encoding (A) sodium channel α- and β-subunits, (B) cytoskeletal and membrane-anchoring proteins (eg, ANK3, CNTN1), (C) chaperonin subunits (eg, calnexin, HSP1A1), (D) regulatory molecules related to protein trafficking and localization, (E) kinesin family motor proteins (eg, KIF5B, KIF5C, KIF11), and (F) post-translational modification enzymes involved in phosphorylation and ubiquitination.

Generation and Characterization of TAP-Tagged hNav1.7 in HEK293 Cells

To facilitate biochemical purification, we established a stable HEK293 cell line expressing hNav1.7 fused with a C-terminal TAP tag composed of a polyhistidine affinity domain and 3 FLAG epitopes (Figure 2A). Immunofluorescence staining using an anti-FLAG antibody confirmed membrane localization of the TAP-hNav1.7 construct, whereas no signal was detected in parental HEK293 cells (Figure 2B). Western blot analysis further verified successful expression of the full-length TAP-tagged hNav1.7 protein (Figure 2C).

Figure 2.

Generation and functional characterization of TAP-tagged human Nav1.7 (hNav1.7) in HEK293 cells. (A) Schematic representation of the tandem affinity purification (TAP) construct, showing the C-terminal fusion of a histidine affinity tag (HAT) and 3 tandem FLAG epitopes to the hNav1.7 channel. (B) Immunocytochemistry of HEK293 cells stably expressing TAP-tagged hNav1.7 using anti-FLAG antibody (green) demonstrates predominant membrane localization, whereas no fluorescence signal was observed in untransfected control cells. Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm. (C) Western blot analysis confirming the expression of full-length TAP-tagged hNav1.7 (~260 kDa) using anti-FLAG and anti-Nav1.7 antibodies; no signal was detected in parental HEK293 lysates. (D-G) Representative whole-cell sodium current traces recorded from HEK293-hNav1.7 cells. (H and I) Current-voltage (I-V) relationships and steady-state activation/inactivation curves of TAP-hNav1.7. n = 6-10 cells per group.

To assess the electrophysiological functionality of the construct, whole-cell patch-clamp recordings were performed. Voltage-dependent activation and inactivation curves demonstrated that TAP-hNav1.7 retained characteristic sodium channel kinetics comparable to those previously reported (Figure 2D-I), indicating that the TAP tag did not impair channel activity.

Isolation and Purification of hNav1.7 Protein Complexes

TAP-tagged hNav1.7 protein complexes were solubilized using DDM-CHS detergent and subjected to a two-step purification protocol. Initial anti-FLAG affinity chromatography (SS-AP) was followed by Ni-NTA agarose-based purification exploiting the histidine tag (TAP; Figure 3A). Western blot analysis confirmed efficient sequential enrichment of TAP-hNav1.7 (Figure 3B). Eluted complexes were further purified using size-exclusion chromatography (SEC), and peak fractions were resolved by SDS-PAGE and visualized by Coomassie blue staining (Figure 3C). Selected fractions were subjected to LC-MS/MS analysis for proteomic profiling.

Figure 3.

Tandem affinity purification (TAP) and validation of hNav1.7 protein complexes. (A) Schematic workflow illustrating the sequential two-step purification strategy used to isolate TAP-tagged hNav1.7 and its associated protein complexes from stably transfected HEK293 cells. (B) Western blot validation of hNav1.7 enrichment at each purification step using anti-FLAG antibody, demonstrating efficient sequential recovery of the TAP-tagged channel. (Ci) Size-exclusion chromatography (SEC) elution profile of purified hNav1.7 protein complexes on a Superose 6 Increase column, showing a single major protein peak corresponding to the hNav1.7-containing fractions. (Cii) Coomassie blue-stained SDS-PAGE gel of SEC fractions, illustrating protein bands representing co-purified components of the hNav1.7 complex. (D) Co-immunoprecipitation (Co-IP) validation of selected interactors, confirming physical association of KIF11, CCT5, and TMED10 with hNav1.7. Detection was performed using specific antibodies against each candidate protein following anti-FLAG immunoprecipitation.

Identification and Annotation of hNav1.7-Interacting Proteins

A total of 261 proteins were identified as hNav1.7 interactors (Table 1). Among these, 3 conserved candidate proteins-KIF11, CCT5, and TMED10-were randomly selected for validation as representatives of distinct functional categories. Co-immunoprecipitation using anti-FLAG resin followed by western blotting with specific antibodies confirmed their physical interaction with hNav1.7 (Figure 3D).

Table 1.

Identified hNaV1.7-Associated Proteins.

Protein	Gene	Protein name	Score	Mass	Num. of matches	Num. of significant matches	Num. of sequences	Num. of significant sequences	emPAI
KIF11	KIF11	Kinesin-like protein KIF11	18 792	119 085	531	531	44	44	7.08
TBB5	TUBB	Tubulin beta chain	3927	49 639	111	111	13	13	4.12
TBB4B	TUBB4B	Tubulin beta-4B chain	3416	49 799	101	101	13	13	4.59
TBB2A	TUBB2A	Tubulin beta-2A chain	3216	49 875	99	99	10	10	3.2
ANM5	PRMT5	Protein arginine N-methyltransferase 5	3083	72 638	98	98	20	20	3.25
ALBU	ALB	Serum albumin	2763	69 321	91	91	16	16	2.22
TBB4A	TUBB4A	Tubulin beta-4A chain	2702	49 554	88	88	11	11	3.24
CALX	CANX	Calnexin	2450	67 526	62	62	15	15	2.33
TBA1B	TUBA1B	Tubulin alpha-1B chain	1862	50 120	40	40	9	9	1.35
TBA1A	TUBA1A	Tubulin alpha-1A chain	1740	50 104	37	37	9	9	1.36
MEP50	WDR77	Methylosome protein 50	1662	36 701	38	38	6	6	1.17
PIGS	PIGS	GPI transamidase component PIG-S	1562	61 617	48	48	12	12	1.74
K2C1	KRT1	Keratin, type II cytoskeletal 1	1539	65 999	47	47	13	13	1.56
TAB1	TAB1	TGF-beta-activated kinase 1 and MAP3K7-binding protein 1	1305	54 610	32	32	10	10	1.39
SCYL2	SCYL2	SCY1-like protein 2	1302	103 642	33	33	10	10	0.59
K1C10	KRT10	Keratin, type I cytoskeletal 10	1286	58 792	42	42	14	14	2.37
PIGT	PIGT	GPI transamidase component PIG-T	1150	65 658	44	44	15	15	1.97
KCTD5	KCTD5	BTB/POZ domain-containing protein KCTD5	1137	26 076	24	24	5	5	1.48
K22E	KRT2	Keratin, type II cytoskeletal 2 epidermal	978	65 393	27	27	10	10	1.08
KCD17	KCTD17	BTB/POZ domain-containing protein KCTD17	959	35 648	21	21	6	6	1.54
GPAA1	GPAA1	Glycosylphosphatidylinositol anchor attachment 1 protein	916	67 580	28	28	7	7	0.76
BIP	HSPA5	Endoplasmic reticulum chaperone BiP	837	72 288	23	23	12	12	1.21
MCM3	MCM3	DNA replication licensing factor MCM3	817	90 924	30	30	13	13	0.98
GPI8	PIGK	GPI-anchor transamidase	744	45 223	28	28	7	7	1.09
EF1A1	EEF1A1	Elongation factor 1-alpha 1	721	50 109	24	24	6	6	0.77
HSP7C	HSPA8	Heat shock cognate 71 kDa protein	656	70 854	21	21	8	8	0.72
RS3	RPS3	40S ribosomal protein S3	656	26 671	27	27	8	8	3.96
EF1D	EEF1D	Elongation factor 1-delta	643	31 103	13	13	5	5	1.15
TTC28	TTC28	Tetratricopeptide repeat protein 28	640	270 715	19	19	10	10	0.19
ACTB	ACTB	Actin, cytoplasmic 1	598	41 710	12	12	5	5	0.77
M3K7	MAP3K7	Mitogen-activated protein kinase kinase kinase 7	526	67 153	19	19	5	5	0.43
HS71A	HSPA1A	Heat shock 70 kDa protein 1A	519	70 009	23	23	9	9	0.85
EFTU	TUFM	Elongation factor Tu, mitochondrial	497	49 510	14	14	5	5	0.62
GOLP3	GOLPH3	Golgi phosphoprotein 3	488	33 790	11	11	6	6	1.33
MCM5	MCM5	DNA replication licensing factor MCM5	469	82 233	12	12	5	5	0.34
PRP8	PRPF8	Pre-mRNA-processing-splicing factor 8	460	273 427	25	25	16	16	0.32
E2AK3	EIF2AK3	Eukaryotic translation initiation factor 2-alpha kinase 3	390	125 137	12	12	6	6	0.26
SERPH	SERPINH1	Serpin H1	377	46 411	11	11	5	5	0.67
KPYM	PKM	Pyruvate kinase PKM	371	57 900	12	12	6	6	0.64
EF1G	EEF1G	Elongation factor 1-gamma	367	50 087	14	14	5	5	0.61
SF3B3	SF3B3	Splicing factor 3B subunit 3	358	135 492	13	13	8	8	0.33
RBBP7	RBBP7	Histone-binding protein RBBP7	347	47 790	12	12	6	6	0.82
RPN1	RPN1	Dolichyl-diphosphooligosaccharide–protein glycosyltransferase subunit 1	321	68 527	14	14	7	7	0.63
PRDX1	PRDX1	Peroxiredoxin-1	321	22 096	14	14	6	6	2.61
RS16	RPS16	40S ribosomal protein S16	317	16 435	9	9	3	3	1.36
U5S1	EFTUD2	116 kDa U5 small nuclear ribonucleoprotein component	310	109 366	14	14	11	11	0.62
VIME	VIM	Vimentin	300	53 619	11	11	5	5	0.56
RS18	RPS18	40S ribosomal protein S18	296	17 708	8	8	3	3	1.22
PSA4	PSMA4	Proteasome subunit alpha type-4	285	29 465	10	10	3	3	0.62
JAK1	JAK1	Tyrosine-protein kinase JAK1	282	133 191	13	13	9	9	0.38
STK38	STK38	Serine/threonine-protein kinase 38	281	54 155	10	10	5	5	0.55
OST48	DDOST	Dolichyl-diphosphooligosaccharide–protein glycosyltransferase 48 kDa subunit	281	50 769	13	13	5	5	0.6
TCPE	CCT5	T-complex protein 1 subunit epsilon	277	59 633	8	8	5	5	0.49
FLNA	FLNA	Filamin-A	276	280 564	11	11	8	8	0.15
HNRPK	HNRNPK	Heterogeneous nuclear ribonucleoprotein K	271	50 944	4	4	2	2	0.21
EF1B	EEF1B2	Elongation factor 1-beta	268	24 748	4	4	2	2	0.47
ICLN	CLNS1A	Methylosome subunit pICln	255	26 199	9	9	1	1	0.2
HS90B	HSP90AB1	Heat shock protein HSP 90-beta	255	83 212	9	9	5	5	0.33
CH60	HSPD1	60 kDa heat shock protein, mitochondrial	242	61 016	8	8	6	6	0.6
TMEDA	TMED10	Transmembrane emp24 domain-containing protein 10	237	24 960	4	4	3	3	0.77
RS27A	RPS27A	Ubiquitin-40S ribosomal protein S27a	230	17 953	6	6	2	2	0.69
RS9	RPS9	40S ribosomal protein S9	225	22 578	15	15	6	6	2.52
OCAD2	OCIAD2	OCIA domain-containing protein 2	220	16 943	4	4	2	2	0.74
KCTD2	KCTD2	BTB/POZ domain-containing protein KCTD2	217	28 509	6	6	3	3	0.65
LRC59	LRRC59	Leucine-rich repeat-containing protein 59	213	34 909	12	12	4	4	0.73
HNRH1	HNRNPH1	Heterogeneous nuclear ribonucleoprotein H	209	49 198	5	5	3	3	0.34
RS2	RPS2	40S ribosomal protein S2	193	31 305	10	10	5	5	1.13
TIF1B	TRIM28	Transcription intermediary factor 1-beta	183	88 493	6	6	3	3	0.18
TRAP1	TRAP1	Heat shock protein 75 kDa, mitochondrial	176	80 060	5	5	3	3	0.2
CREL1	CRELD1	Cysteine-rich with EGF-like domain protein 1	174	45 409	7	7	4	4	0.52
PIGU	PIGU	Phosphatidylinositol glycan anchor biosynthesis class U protein	168	50 019	2	2	1	1	0.1
PRP19	PRPF19	Pre-mRNA-processing factor 19	167	55 146	6	6	3	3	0.3
VAPA	VAPA	Vesicle-associated membrane protein-associated protein A	165	27 875	4	4	2	2	0.41
EF2	EEF2	Elongation factor 2	159	95 277	6	6	6	6	0.35
GRP75	HSPA9	Stress-70 protein, mitochondrial	155	73 635	3	3	1	1	0.07
ARHGA	ARHGEF10	Rho guanine nucleotide exchange factor 10	152	151 516	5	5	4	4	0.13
MCM7	MCM7	DNA replication licensing factor MCM7	145	81 257	5	5	5	5	0.34
RL18	RPL18	60S ribosomal protein L18	145	21 621	2	2	1	1	0.24
ASCC3	ASCC3	Activating signal cointegrator 1 complex subunit 3	139	251 301	6	6	4	4	0.08
HAT1	HAT1	Histone acetyltransferase type B catalytic subunit	138	49 481	3	3	2	2	0.21
U520	SNRNP200	U5 small nuclear ribonucleoprotein 200 kDa helicase	133	244 353	5	5	5	5	0.1
RL7	RPL7	60S ribosomal protein L7	126	29 207	6	6	4	4	0.92
ANM1	PRMT1	Protein arginine N-methyltransferase 1	125	42 434	7	7	4	4	0.57
RL13	RPL13	60S ribosomal protein L13	124	24 247	7	7	3	3	0.8
TERA	VCP	Transitional endoplasmic reticulum ATPase	123	89 266	7	7	7	7	0.45
1433B	YWHAB	14-3-3 protein beta/alpha	123	28 065	6	6	3	3	0.66
TCPB	CCT2	T-complex protein 1 subunit beta	121	57 452	5	5	3	3	0.28
RS8	RPS8	40S ribosomal protein S8	121	24 190	6	6	2	2	0.48
TMED5	TMED5	Transmembrane emp24 domain-containing protein 5	121	25 988	4	4	2	2	0.44
DHX15	DHX15	Pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15	118	90 875	5	5	4	4	0.23
RS6	RPS6	40S ribosomal protein S6	118	28 663	4	4	2	2	0.39
HNRPU	HNRNPU	Heterogeneous nuclear ribonucleoprotein U	117	90 528	6	6	4	4	0.24
K1C9	KRT9	Keratin, type I cytoskeletal 9	115	62 027	5	5	2	2	0.17
VDAC2	VDAC2	Voltage-dependent anion-selective channel protein 2	115	31 547	2	2	1	1	0.16
ADT2	SLC25A5	ADP/ATP translocase 2	112	32 831	5	5	2	2	0.34
HACD3	HACD3	Very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase 3	112	43 132	1	1	1	1	0.12
SF3B4	SF3B4	Splicing factor 3B subunit 4	111	44 357	3	3	2	2	0.24
TCPH	CCT7	T-complex protein 1 subunit eta	111	59 329	2	2	1	1	0.08
PRPS1	PRPS1	Ribose-phosphate pyrophosphokinase 1	109	34 812	3	3	2	2	0.31
DDX3X	DDX3X	ATP-dependent RNA helicase DDX3X	108	73 198	4	4	4	4

Gene ontology analysis using the PANTHER classification system revealed that the majority of hNav1.7 interactors were localized to the cytoplasm and plasma membrane (Figure 4A), and functionally annotated as components involved in translation, chaperone-mediated folding, protein modification, cytoskeletal transport, and signal transduction (Figure 4B).

Figure 4.

Comparative bioinformatic analysis of human and mouse Nav1.7 interactomes using the PANTHER classification system. (A and B) Gene Ontology (GO)-based categorization of human Nav1.7 (hNav1.7)-interacting proteins identified by tandem affinity purification and LC-MS/MS. (A) Subcellular localization analysis showing that the majority of hNav1.7 interactors are distributed in the cytoplasm and plasma membrane, consistent with the channel’s membrane-associated function. (B) Functional classification of hNav1.7 interactors, revealing enrichment in protein translation, folding, trafficking, and signal transduction pathways. (C and D) Corresponding PANTHER analysis of mouse Nav1.7 (mNav1.7) interactors. (C) Cellular localization pattern highlighting a similar cytoplasmic and membrane-associated distribution, with additional enrichment in synaptic components. (D) Functional classification of mNav1.7 interactors, revealing enrichment in protein modification, folding, trafficking, and translation.

Comparison with mNav1.7 Interactome Reveals Conserved Protein Families

To examine evolutionary conservation in Nav1.7 regulation, we compared the hNav1.7 interactome with our previously published mNav1.7 dataset. Most interactors in both datasets were localized to similar subcellular compartments, although a subset of mNav1.7 interactors was enriched in synaptic regions (Figure 4C). Functionally, both human and mouse interactomes were dominated by proteins involved in translation, trafficking, and membrane integration (Figure 4D).

Direct comparison revealed a conserved set of proteins shared between hNav1.7 and mNav1.7 interactomes. These include elongation factors EEF1A1 and EEF2, as well as T-complex protein subunits TCP1, CCT2, CCT3, CCT5, CCT6A, and CCT7. Notably, members of the kinesin motor protein family (eg, KIF5B, KIF5C, KIF11) and Rab GTPases (eg, Rab5a, Rab1a) were also represented across both species, highlighting their potential roles in Nav1.7 trafficking and vesicular transport (Table 2).

Table 2.

Comparison Between mNav1.7- and hNaV1.7-Interacted Proteins.

Translational protein

Homo

Eef1a1

Eef1b2

Eef1d

Eef2

Eftud2

Mrps23

Mrps34

Rpl11

Rpl13

Rpl14

Rpl18

Rpl18a

Rpl23

Rpl27

Rpl27a

Rpl6

Rpl7

Rplp0p6

Rps11

Rps15a

Rps16

Rps17

Rps18

Rps2

Rps25

Rps3

Rps3a

Rps4x

Rps5

Rps6

Rps8

Rps9

Tufm

Mouse

Eef1a1

Eef1a2

Eef2

Eif3a

Eif3b

Eif3c

Eif3d

Eif3e

Eif3i

Eif3l

Tufm

Chaperone

Homo

Canx

Cct2

Cct3

Cct5

Cct6a

Cct7

Clgn

Clip1

Hsp90ab1

Hspa1a

Hspa5

Hspa8

Hspa9

Npm1

Pdia6

Tcp1

Trap1

Mouse

Ahsa1

Calr

Cct2

Cct3

Cct4

Cct5

Cct6a

Cct7

Cct8

Dctn1

Hsp90aa1

Hsp90ab1

Hspa5

Hspa9

Hsph1

Ppia

Tcp1

Protein modifying enzyme

Homo

Akt1

Carm1

Eif2ak3

Hpr

Jak1

Map3k7

Ogt

Ppm1b

Prkdc

Prmt1

Prmt5

Psma4

Scyl2

Sec11a

Stk38

Stub1

Tab1

Trim21

Trim28

Ubr5

Usp7

Usp9x

Mouse

Ank3

Asrgl1

Map2

Map3k7

Ogt

Ppm1a

Ppp1cc

Ppp2cb

Psma1

Psma4

Psma5

Psma7

Psmb2

Psmb4

Psmb5

Psmc2

Psmc3

Psmc4

Psmc5

Psmd1

Psmd12

Psmd13

Psmd2

Psmd3

Psmd4

Stk38

Stk38l

Tab1

Tcp1

Uchl1

Uqcrc1

Uqcrc2

Usp15

Cytoskeletal protein

Homo

Actb

Actn1

Capza1

Cfl1

Dnah17

Kif11

Krt10

Krt9

Pls3

Shroom3

Sptan1

Tuba1a

Tuba1b

Tubb

Tubb2a

Tubb4a

Tubb4b

Vim

Mouse

Ablim1

Actbl2

Actr1a

Actr1b

Actr2

Capza1

Cfl1

Clasp2

Cttn

Dbn1

Dctn4

Dctn5

Des

Dsp

Dst

Dstn

Dync1h1

Dynll2

Epb41l1

Epb41l3

Kif5b

Kif5c

Klc1

Myo6

Prph

Tmod2

Tuba4a

Tubb2b

Tubb5

RNA metabolism protein

Homo

Cpsf1

Cpsf6

Ddb1

Ddx3x

Dhx15

E2f7

Hnrnph1

Hnrnpk

Larp1

Med14

Nop56

Nudt21

Pabpc1

Pcbp1

Pcf11

Pdcd11

Polr2a

Prpf19

Prpf31

Prpf6

Prpf8

Ptbp1

Sf3b1

Sf3b2

Sf3b3

Sf3b4

Sf3b6

Snrpd2

Syncrip

Tardbp

Mouse

Ddb1

Hnrnph1

Hnrnph2

Htatsf1

Sart3

Intercellular signal molecule

Homo

Gdf9

Tmpo

Mouse

Fgb

Fgg

Membrane traffic protein

Homo

Clstn3

Copa

Lman2l

Ociad2

Tmed10

Tmed2

Tmed5

Tmed9

Vapa

Mouse

Ankfy1

Ap2s1

Ap3b2

Bcap31

Dnm1

Napa

Napb

Nsf

Stx12

Syt2

Tmed10

Transfer/carrier protein

Homo

Alb

Lrp1

Slc25a5

Mouse

Fabp7

Hba

Transmembrane signal receptor

Homo

Adgre5

Or1l8

Pgrmc1

Pgrmc2

Mouse

Glud1

Tkt

Transporter

Homo

Abca8

Abcd3

Atp5f1a

Atp5mf

Ccar2

Clns1a

Mtch2

Pls3

Praf2

Slc25a3

Vcp

Vdac1

Mouse

Ap3d1

Aqp4

Atp1a1

Atp1a3

Atp2a2

Atp5a1

Atp5c1

Atp5d

Atp5i

Atp5j

Atp5j2

Atp5o

Atp6v1g2

Kpnb1

Scn3b

Sfxn3

Slc12a2

Slc12a5

Slc17a7

Slc1a3

Slc25a3

Slc2a1

Slc4a1

Slc4a8

Slc7a14

Slc7a5

Slc8a1

Usmg5

Vcp

Vdac1

Vdac3

Scaffold/adaptor protein

Homo

Ankrd28

Ascc3

Ivns1abp

Kctd17

Kctd2

Kctd5

Lrrc59

Lrrc8e

Snrnp200

Ttc28

Wdr87

Ywhab

Mouse

Akap12

Cyfip2

Kctd12

Mapk8ip3

Mpdz

Mpp2

Ppfia3

Ywhab

Ywhae

Ywhag

Ywhah

Ywhaq

Metabolite interconversion enzyme

Homo

Acot9

Acsm6

Cnp

Cox7a2

Ddost

Ganab

Hacd3

Hao1

Impdh2

Ldhb

Mt-Co2

Mthfd1

Nt5c2

Pfkfb3

Pkm

Prdx1

Prdx2

Prdx6

Prps1

Prpsap1

Prpsap2

Rpn1

Txn

Mouse

Acly

Aco2

Agpat3

Ampd2

Cox4i1

Cox5a

Crmp1

Dpysl2

Dpysl3

Dpysl4

Dpysl5

Echs1

Eno1

Glud1

Glul

Gpx4

Mccc2

Mthfd1l

Ndufa10

Ndufa7

Ndufa9

Ndufb10

Ndufb7

Ndufs7

Ndufs8

Ndufv3

Oxct1

Pccb

Pfkm

Pgam1

Pi4ka

Pkm

Prdx1

Prdx2

Sbf1

Synj1

Tkt

Calcium-binding protein

Homo

Anxa2

Cpne1

S100b

Mouse

Calb2

Calm1

Defense/immunity protein

Homo

C1qbp

Mouse

Ighg1

Ighm

Igsf8

Lsamp

Ntm

Gene-specific transcriptional regulator

Homo

Hdx

Mta1

Mouse

Erh

Protein-binding activity modulator

Homo

Arhgef10

Dock4

Gnal

Ppp2r1a

Prkag1

Rab5a

Serpina1

Serpinb10

Serpinh1

Srgap1

Mouse

Pebp1

Rab1a

Rp2

Others

Homo

Fasn

Flna

Krt1

Krt10

Krt2

Krt71

Krt9

Lmna

Ppp2r1a

Tln1

Mouse

Fasn

Flna

Krt2

Krt6a

Lmna

Ppp1cc

Ppp1r9a

Ppp2cb

Tln1

Tln2

In summary

Homo

Atp5f1a

Atp5mf

Canx

Capza1

Tcp1

Cct2

Cct3

Cct5

Cct6a

Mouse

Atp5a1

Atp5c1

Atp5d

Atp5i

Atp5j

Atp5j2

Atp5o

Calr

Capza1

Tcp1

Cct2

Cct3

Cct4

Cct5

Homo

Cct7

Cox7a2

Ddb1

Eef1a1

Eef1b2

Eef1d

Eef1g

Eef2

Fasn

Flna

Hnrnph1

Mouse

Cct6a

Cct7

Cct8

Cox4i1

Cox5a

Ddb1

Eef1a1

Eef1a2

Eef2

Fasn

Flna

Hnrnph1

Homo

Hnrnpk

Hnrnpm

Hnrnpu

Hsp90ab1

Hspa1a

Hspa5

Hspa8

Hspa9

Kctd2

Kctd5

Kif11

Krt1

Mouse

Hnrnph2

Hsp90aa1

Hsp90ab1

Hspa5

Hspa9

Kctd12

Kif5b

Kif5c

Homo

Krt10

Krt2

Krt71

Krt9

Lmna

Map3k7

Pkm

Ppm1b

Ppp2r1a

Prdx1

Prdx2

Mouse

Krt2

Krt6a

Lmna

Map3k7

Pkm

Ppm1a

Ppp1cc

Ppp1r9a

Ppp2cb

Prdx1

Prdx2

Psma1

Homo

Psma4

Rab5a

Slc25a3

Slc25a5

Stk38

Tab1

Tab2

Tab3

Tln1

Tmed10

Mouse

Psma4

Psma5

Psma7

Rab1A

Slc25a3

Stk38

Stk38l

Tab1

Tln1

Tln2

Tmed10

Homo

Tmed5

Tmed9

Tuba1a

Tuba1b

Tubb2a

Tubb4a

Tubb4b

Tubb5

Tufm

Usp7

Usp9x

Vcp

Vdac1

Vdac2

Mouse

Tuba4a

Tubb2b

Tubb5

Tufm

Usp15

Vcp

Vdac1

Vdac3

Homo

Ywhab

Mouse

Ywhab

Ywhae

Ywhag

Ywhah

Ywhaq

The bold and underlined entries denote a conserved subset of proteins that are shared between the human (hNav1.7) and mouse (mNav1.7) interactomes.

Functional Validation of Conserved Interactors CCT5 and TMED10

To investigate the functional relevance of conserved Nav1.7 interactors, we selected CCT5 and TMED10-two fully conserved proteins between human and mouse that were also validated by co-immunoprecipitation-for functional assays using siRNA knockdown in HEK293 cells. Western blot analysis confirmed efficient silencing of both targets (Figure 5A and B).

Figure 5.

Functional validation of conserved Nav1.7 interactors CCT5 and TMED10 in HEK293 cells. (A and B) Western blot analysis confirming efficient siRNA-mediated knockdown of CCT5 and TMED10 in HEK293-hNav1.7 cells. GAPDH was used as a loading control. n = 3 per group. (C-E) Representative whole-cell sodium current traces recorded from HEK293-hNav1.7 cells after transfection with scrambled control siRNA or target-specific siRNAs. (F and G) Averaged current–voltage (I-V) relationships and peak current density plots showing a significant reduction in sodium current amplitude following CCT5 or TMED10 knockdown compared with control. n = 6-14 cells per group.

Patch-clamp recordings revealed that CCT5 knockdown led to a ~50% reduction in hNav1.7 current density, while TMED10 knockdown resulted in a ~20% reduction (Figure 5C-G). These results suggest that both proteins play important roles in modulating Nav1.7 surface expression or channel activity, thereby validating their functional significance in the Nav1.7 regulatory network.

Discussion

In this study, we systematically mapped the protein-protein interaction (PPI) network of human Nav1.7 (hNav1.7) using tandem affinity purification (TAP) and LC-MS/MS in a stably transfected HEK293 cell line. We identified 261 Nav1.7-interacting proteins, the majority of which were functionally associated with protein synthesis, folding, intracellular transport, and membrane localization. Comparative analysis with our previous murine Nav1.7 (mNav1.7) interactome revealed conserved proteins and protein families across species and tissue types, including core chaperonins (eg, CCT5), trafficking mediators (eg, TMED10), translation elongation factors (EEF1A1, EEF2), kinesins, and Rab GTPases. Functional validation through RNAi knockdown demonstrated that depletion of CCT5 or TMED10 significantly reduced hNav1.7 current density, underscoring their regulatory roles in channel activity.

Despite considerable effort over the past 2 decades, pharmacological targeting of Nav1.7 for analgesia has yielded disappointing results, primarily due to structural similarity among sodium channel isoforms, poor CNS penetration, and adverse off-target effects. Alternative strategies have therefore shifted toward targeting regulatory mechanisms of Nav1.7 biogenesis, trafficking, and degradation. This study adds to a growing body of evidence suggesting that modulating accessory proteins and cofactors may represent a viable approach for tuning Nav1.7 function in a tissue-specific and isoform-selective manner.

One of the key findings from our cross-species comparison was the identification of conserved regulatory nodes in the Nav1.7 interactome. CCT5 has been implicated in chronic pain and is closely associated with the membrane trafficking of ion channels.^20,21 Our data indicate that CCT5 knockdown significantly reduced hNav1.7 current density, indicating that CCT5 contributes to the functional regulation of hNav1.7. Similarly, TMED10, a member of the p24 cargo receptor family, has previously been implicated in ER-Golgi vesicular transport.²² Its partial knockdown led to reduced Nav1.7 current density, suggesting an involvement in trafficking or membrane insertion of the channel.

Interestingly, we observed that while the identities of some individual interactors differed between human and mouse, their functional categories and protein family affiliations were highly conserved. For instance, KIF11 (human) and KIF5C (mouse), both members of the kinesin motor protein family,²³ were associated with Nav1.7 in their respective species. Likewise, Rab5a (human) and Rab1a (mouse), members of the Rab GTPase family,²⁴ were also identified as interactors. These results suggest that Nav1.7 function may be regulated by tissue- or species-specific paralogs of conserved protein families, a concept consistent with differential gene expression profiles across cell types.

Nevertheless, we acknowledge several limitations. First, although HEK293 cells provide a convenient and tractable system for biochemical purification, they do not recapitulate the full complexity of human sensory neurons. Some Nav1.7 interactors identified in neural tissues may not be represented in this model. Second, while 2 interactors (CCT5 and TMED10) were validated functionally, further work is required to define the mechanistic pathways through which these and other proteins influence Nav1.7 biology. Third, post-translational modifications and dynamic signaling events likely shape the Nav1.7 interactome under physiological and pathological conditions, which were not captured in the current static proteomic map.

Conclusion

This study presents the first comprehensive proteomic analysis of the human Nav1.7 interactome. Using a TAP-tagged expression system and high-resolution LC-MS/MS, we identified 261 hNav1.7-associated proteins, several of which were functionally conserved with mouse Nav1.7 interactors. Knockdown of 2 conserved proteins, CCT5 and TMED10, led to reduced Nav1.7 current density, confirming their involvement in channel regulation. These findings enhance our understanding of Nav1.7 biology and establish a framework for future exploration of indirect therapeutic strategies targeting Nav1.7 regulatory networks in pain disorders.

Footnotes

Author Contributions

X.L.Z. and J.Z. designed the experiments, performed the experiments, analyzed the data, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the National Natural Science Foundation of China (82001173, 82471279), Zhejiang Postdoctoral Science Foundation (ZJ2024076).

ORCID iD

Xuelong Zhou

Ethical Considerations

Not applicable.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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