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Molecular architecture of a sodium channel S6 helix: radial tuning of the voltage-gated sodium channel 1.7 activation gate.

ABSTRACT: BACKGROUND: In-frame deletion mutation (Del-L955) in NaV1.7 sodium channel from a kindred with erythromelalgia hyperpolarizes activation. RESULTS: Del-L955 twists the S6 helix, displacing the Phe960 activation gate. Replacement of Phe960 at the correct helical position depolarizes activation. CONCLUSION: Radial tuning of the activation gate is critical to the activation of NaV1.7 channel. SIGNIFICANCE: Structural modeling guided electrophysiology reveals the functional importance of radial tuning of the S6 segment. Voltage-gated sodium (NaV) channels are membrane proteins that consist of 24 transmembrane segments organized into four homologous domains and are essential for action potential generation and propagation. Although the S6 helices of NaV channels line the ion-conducting pore and participate in channel activation, their functional architecture is incompletely understood. Our recent studies show that a naturally occurring in-frame deletion mutation (Del-L955) of NaV1.7 channel, identified in individuals with a severe inherited pain syndrome (inherited erythromelalgia) causes a substantial hyperpolarizing shift of channel activation. Here we took advantage of this deletion mutation to understand the role of the S6 helix in the channel activation. Based on the recently published structure of a bacterial NaV channel (NaVAb), we modeled the WT and Del-L955 channel. Our structural model showed that Del-L955 twists the DII/S6 helix, shifting location and radial orientation of the activation gate residue (Phe(960)). Hypothesizing that these structural changes produce the shift of channel activation of Del-L955 channels, we restored a phenylalanine in wild-type orientation by mutating Ser(961) (Del-L955/S961F), correcting activation by ?10 mV. Correction of the displaced Phe(960) (F960S) together with introduction of the rescuing activation gate residue (S961F) produced an additional ?6-mV restoration of activation of the mutant channel. A simple point mutation in the absence of a twist (L955A) did not produce a radial shift and did not hyperpolarize activation. Our results demonstrate the functional importance of radial tuning of the sodium channel S6 helix for the channel activation.

SUBMITTER: Yang Y

PROVIDER: S-EPMC3650411 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Molecular architecture of a sodium channel S6 helix: radial tuning of the voltage-gated sodium channel 1.7 activation gate.

Yang Yang Y Yang Yang Y Estacion Mark M Dib-Hajj Sulayman D SD Waxman Stephen G SG

The Journal of biological chemistry 20130327 19

<h4>Background</h4>In-frame deletion mutation (Del-L955) in NaV1.7 sodium channel from a kindred with erythromelalgia hyperpolarizes activation.<h4>Results</h4>Del-L955 twists the S6 helix, displacing the Phe960 activation gate. Replacement of Phe960 at the correct helical position depolarizes activation.<h4>Conclusion</h4>Radial tuning of the activation gate is critical to the activation of NaV1.7 channel.<h4>Significance</h4>Structural modeling guided electrophysiology reveals the functional i ...[more]

PMID: 23536180

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Project description:BackgroundTemporomandibular disorders (TMDs) have the highest prevalence in women of reproductive age. The role of estrogen in TMDs and especially in TMDs related pain is not fully elucidated. Voltage-gated sodium channel 1.7 (Nav1.7) plays a prominent role in pain perception and Nav1.7 in trigeminal ganglion (TG) is involved in the hyperalgesia of inflamed Temporomandibular joint (TMJ). Whether estrogen could upregulate trigeminal ganglionic Nav1.7 expression to enhance hyperalgesia of inflamed TMJ remains to be explored.MethodsEstrous cycle and plasma levels of 17?-estradiol in female rats were evaluated with vaginal smear and enzyme linked immunosorbent assay, respectively. Female rats were ovariectomized and treated with 17?-estradiol at 0 ?g, 20 ?g and 80 ?g, respectively, for 10 days. TMJ inflammation was induced using complete Freund's adjuvant. Head withdrawal thresholds and food intake were measured to evaluate the TMJ nociceptive responses. The expression of Nav1.7 in TG was examined using real-time PCR and western blot. The activity of Nav1.7 promoter was examined using luciferase reporter assay. The locations of estrogen receptors (ER? and ER?), the G protein coupled estrogen receptor (GPR30), and Nav1.7 in TG were examined using immunohistofluorescence.ResultsUpregulation of Nav1.7 in TG and decrease in head withdrawal threshold were observed with the highest plasma 17?-estradiol in the proestrus of female rats. Ovariectomized rats treated with 80 ?g 17?-estradiol showed upregulation of Nav1.7 in TG and decrease in head withdrawal threshold as compared with that of the control or ovariectomized rats treated with 0 ?g or 20 ?g. Moreover, 17?-estradiol dose-dependently potentiated TMJ inflammation-induced upregulation of Nav1.7 in TG and also enhanced TMJ inflammation-induced decrease of head withdrawal threshold in ovariectomized rats. In addition, the estrogen receptor antagonist, ICI 182,780, partially blocked the 17?-estradiol effect on Nav1.7 expression and head withdrawal threshold in ovariectomized rats. ER? and ER?, but not GPR30, were mostly co-localized with Nav1.7 in neurons in TG. In the nerve growth factor-induced and ER?-transfected PC12 cells, 17?-estradiol dose-dependently enhanced Nav1.7 promoter activity, whereas mutations of the estrogen response element at -1269/-1282 and -1214/-1227 in the promoter completely abolished its effect on the promoter activity.ConclusionEstradiol could upregulate trigeminal ganglionic Nav1.7 expression to contribute to hyperalgesia of inflamed TMJ.

Project description:The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.

Dataset Information

Molecular architecture of a sodium channel S6 helix: radial tuning of the voltage-gated sodium channel 1.7 activation gate.

Publications

Molecular architecture of a sodium channel S6 helix: radial tuning of the voltage-gated sodium channel 1.7 activation gate.

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