- Open Access
Paroxysmal extreme pain disorder M1627K mutation in human Nav1.7 renders DRG neurons hyperexcitable
© Dib-Hajj et al; licensee BioMed Central Ltd. 2008
- Received: 24 June 2008
- Accepted: 19 September 2008
- Published: 19 September 2008
Paroxysmal extreme pain disorder (PEPD) is an autosomal dominant painful neuropathy with many, but not all, cases linked to gain-of-function mutations in SCN9A which encodes voltage-gated sodium channel Nav1.7. Severe pain episodes and skin flushing start in infancy and are induced by perianal probing or bowl movement, and pain progresses to ocular and mandibular areas with age. Carbamazepine has been effective in relieving symptoms, while other drugs including other anti-epileptics are less effective.
Sequencing of SCN9A coding exons from an English patient, diagnosed with PEPD, has identified a methionine 1627 to lysine (M1627K) substitution in the linker joining segments S4 and S5 in domain IV. We confirm that M1627K depolarizes the voltage-dependence of fast-inactivation without substantially altering activation or slow-inactivation, and inactivates from the open state with slower kinetics. We show here that M1627K does not alter development of closed-state inactivation, and that M1627K channels recover from fast-inactivation faster than wild type channels, and produce larger currents in response to a slow ramp stimulus. Using current-clamp recordings, we also show that the M1627K mutant channel reduces the threshold for single action potentials in DRG neurons and increases the number of action potentials in response to graded stimuli.
M1627K mutation was previously identified in a sporadic case of PEPD from France, and we now report it in an English family. We confirm the initial characterization of mutant M1627K effect on fast-inactivation of Nav1.7 and extend the analysis to other gating properties of the channel. We also show that M1627K mutant channels render DRG neurons hyperexcitable. Our new data provide a link between altered channel biophysics and pain in PEPD patients.
- Dorsal Root Ganglion
- Dorsal Root Ganglion Neuron
- Mutant Channel
- Single Action Potential
Recent genetic studies have identified sodium channel Nav1.7 as a major contributor to pain [1, 2]. Nav1.7 is predominantly expressed in dorsal root ganglion (DRG) and sympathetic ganglion neurons [3–6], and specifically in most functionally-identified DRG nociceptive neurons . Global knock-out of Nav1.7 in mice is neonatal lethal while nociceptor-specific knock-out results in elevated mechanical and thermal pain thresholds and an attenuated pain response . Surprisingly, congenital loss of Nav1.7 in humans is not associated with cognitive or motor deficits, but causes complete indifference to pain [9–11]. In contrast, one set of gain-of-function mutations of Nav1.7 in inherited erythromelalgia (IEM) leads to severe episodes of pain, mostly in the feet and hands [12–18], and a different set of mutations results in paroxysmal extreme pain disorder (PEPD) . Because some cases of PEPD do not carry mutations in Nav1.7 , it is important to genetically profile new PEPD cases to determine the molecular basis of the disease which may influence possible therapy options.
Severe pain in PEPD patients accompanied by redness in the lower body can start in infancy, is induced by bowel movement or probing of perianal areas, and can be accompanied by tonic nonepileptic seizures, syncopes, bradycardia and occasionally asystole . The cause for the seizures and cardiac symptoms is not well understood. Pain progresses with age to affect ocular and maxillary/mandibular areas and is triggered by cold, eating or emotional state . Pain episodes can last seconds to minutes (and hours in extreme cases), and gradually subside.
Whole-cell voltage-clamp studies have shown that all of the IEM mutations lower the voltage threshold for Nav1.7 activation, with most mutations increasing the ramp response of the channel [13, 16, 17, 21–25]. In contrast, PEPD mutations in Nav1.7 impair fast-inactivation and can result in a persistent current . The M1627K mutation, which is located in domain IV S4–S5 linker (DIV/S4–S5) and first identified in a sporadic case of PEPD from France, has been shown to cause a significant shift in the voltage-dependence of steady-state fast-inactivation of mutant channels but does not produce a persistent current . The effects of M1627K on other properties that influence channel function, for example slow-inactivation, recovery from fast-inactivation, and responses to ramp stimuli, have not been reported. Also, while it is predicted that impaired fast-inactivation could be linked to repetitive firing in hyperexcitable DRG neurons , the effects of the PEPD mutation on DRG neuron excitability have not been experimentally demonstrated.
We report here the identification of M1627K mutation in a new family with PEPD. We confirm that the M1627K mutation causes a large depolarized shift in fast-inactivation without altering channel activation, and show that mutant channels inactivate from the open state with slower kinetics, but without altering the rate for development of closed-state inactivation. We demonstrate that M1627K channels recover from fast-inactivation faster than wild type (WT) channels, and produce larger currents in response to a ramp stimulus. We also show, for the first time, that a PEPD mutation reduces the threshold for single action potentials and increases the number of action potentials in DRG neurons in response to graded stimuli, providing a link between altered channel biophysics and the pain that occurs in PEPD.
An English family with a history of PEPD in two generations was evaluated for a linkage with mutations in SCN9A. The proband is a 36 year old Caucasian female who presented to her physician with a lifelong history of episodic erythema and painful burning sensations from the waist downward. The episodes of erythema and extreme pain last for approximately 20 minutes. Her symptoms began in infancy and occurred several times a year, with pain triggered by bowel movement, passage of flatus or any painful stimulus in the lower half of her body. The patient experienced two attacks during each of her two deliveries, each of which occurred by Caesarian section under general anesthesia. Treatment with carbamazepine was initiated in infancy and currently has been successful in reducing her attacks to approximately one per year. As a teenager, the patient tried to reduce the daily dose; however, at 400 mg of carbamazepine per day she experienced an increase in the frequency of her attacks. Her symptoms dramatically improved upon returning to the original dose of 600 mg per day. Based on the clinical evaluation, the patient was diagnosed with PEPD.
The proband's father and sister have a similar presentation of the disease since infancy. The father is currently not treated with any medication and feels that the frequency of attacks has increased with age. The proband's sister has been treated effectively with carbamazepine since childhood, but requires a lower dose of 200 mg/day. Prior to starting carbamazepine at one year of age, she had frequent episodes of pain and erythema from the waist downward. She self-reports that her symptoms have improved with age, and currently she has suffered just two attacks in the last five years. Unlike the proband, she can recall three attacks in her arms triggered by minor hand injuries. She also had two attacks during Caesarian section under general anesthesia.
Identification of M1627K mutation
Voltage-clamp electrophysiology: activation and deactivation
Voltage-clamp electrophysiology: inactivation
We next examined the effects of the M1627K mutation on inactivation properties. The voltage-dependence of steady-state fast-inactivation was dramatically shifted in the depolarizing direction by the M1627K mutation (Figure 3C). The midpoint of fast-inactivation (V1/2, measured with 500 ms pre-pulses) was -75.3 ± 1.7 mV for WT (n = 11) and -56.0 ± 1.4 mV for M1627K (n = 15) channels. The slope of the steady-state inactivation relationship was 6.2 ± 0.1 mV/e-fold for WT channels and 9.3 ± 0.6 mV/e-fold for M1627K channels. Differences in the V1/2 and slope of fast-inactivation fit were significant (p < 0.001). However, the fraction of current remaining available after the -10 mV inactivation prepulse was not significantly different (p > 0.05) between WT (1.3 ± 0.3%) and M1627K (1.9 ± 0.5%) channels.
By contrast, the voltage-dependence of slow-inactivation of hNav1.7R currents was only slightly altered by the M1627K mutation (Figure 3D). Ten second pre-pulses, followed by 100 ms recovery pulses to -120 mV to allow recovery from fast-inactivation, preceded the test pulse (to 0 mV for 20 ms) to determine the fraction of current available. The M1627K mutation reduced the fraction of slow-inactivated mutant channels that occurred at -80, -70 and -60 mV, which might contribute to increased channel availability at normal resting membrane potentials.
Voltage-clamp electrophysiology: recovery from fast-inactivation
The time course for recovery from fast-inactivation (repriming) of WT and M1627K channels was measured at recovery voltages ranging from -140 to -60 mV. Fast-inactivation was induced with 20 ms inactivating prepulses to -20 mV. The time course for recovery from inactivation for both WT and M1627K currents could be fitted with single exponential functions. Recovery from inactivation was significantly faster for M1627K channels than for WT channels (Figure 5B). For example, the time constant for recovery of WT channels (τ = 92 ± 11 ms, n = 7) was almost 4-fold larger at -70 mV than the corresponding time constant for M1627K channels (τ = 26 ± 3 ms, n = 7).
Voltage-clamp electrophysiology: response to a ramp stimulus
Current Clamp electrophysiology
Previous studies have not examined the effect of PEPD mutations on DRG neuron excitability . We assessed the effect of the M1627K mutation on DRG neuron excitability by recording in current-clamp mode from DRG neurons transfected with GFP and either WT or the M1627K mutant construct. The GFP-positive cells that were selected for recording were similar for the two groups in cell size, as measured either by apparent cell body diameter (24.1 ± 0.6 μm for M1627K vs. 25.5 ± 0.5 μm for WT) or by measured whole-cell capacitance (23.4 ± 1.5 pF for M1627K vs. 23.6 ± 1.4 pF for WT). The expression of the M1627K mutant channels caused a significant reduction in the threshold to generate action potentials. DRG neurons (n = 28) that express WT channels required on average about 300 pA to reach threshold (292 ± 37 pA), whereas DRG neurons that express the M1627K mutant channel (n = 31) only required, on average, about half the amount of current to reach threshold (154 ± 20 pA; p < 0.005). One possibility for the reduction in threshold is an increase in input resistance that would give a larger depolarization to a fixed stimulus current. However, the difference of the input resistance between the two groups of transfected cells did not reach statistical significance (M1627K: 795 ± 105 MΩ; WT: 662 ± 67 MΩ; p = 0.3). A similar small, but non-significant, difference had been reported for the A863P IEM mutant .
No significant difference in resting membrane potential (RMP) was observed between the two groups of cells (M1627K: -60.1 ± 1.2 mV; WT: -60.1 ± 1.6 mV; p = 0.97). Additionally, the amplitude of action potential elicited at threshold showed no significant differences between M1627K transfected DRG neurons (n = 31) compared to WT transfected DRG neurons (n = 28) for peak (M1627K: 60.3 ± 2.2 mV, WT: 54.8 ± 2.9 mV, Mann-Whitney rank sum test, p = 0.24) or for after-hyperpolarization (M1627K: -64.2 ± 1.3 mV, WT: -59.2 ± 1.8 mV, t-test, p = 0.07).
In this study, we identified a mutation, M1627K, in Nav1.7 from a previously unreported family with PEPD. Life-long symptoms in two members of the family are controlled by the sodium channel blocker carbamazepine. We investigated the functional effect of the M1627K substitution on Nav1.7, and confirm that it causes a large depolarizing shift in the voltage-dependence of steady-state fast-inactivation, with no effect on channel activation. Using current clamp, we show that M1627K mutant channels lower threshold for single action potentials and increase the number of action potentials in response to graded suprathreshold stimuli in small DRG neurons, most of which are nociceptors. Thus we show, for the first time, that a PEPD mutation produces nociceptor hyperexcitability.
Missense mutations in Nav1.7 have been linked to two types of inherited painful neuropathies, early-onset IEM [12–18], and PEPD . While missense mutations in Nav1.7 have been found in most families with IEM and PEPD in whom the gene was sequenced, both IEM and PEPD may be genetically heterogenous because mutations in the coding exons of SCN9A were not found in 5 cases of PEPD , or in cases of familial early-onset  and adult-onset IEM , suggesting that other target genes or mutations in non-coding regions of SCN9A might underlie these cases. Mutations in the coding exons of sensory neuron-specific sodium channels Nav1.8 and Nav1.9 have been ruled out as causative in these cases of inherited erythromelalgia . We present in this study a previously unreported familial case of PEPD from the UK with the mutation M1627K, in the DIV/S4–S5 linker, which is also present in a sporadic case of a male patient from France [19, 20].
The patients in our study have responded favorably to treatment with carbamazepine, similar to those reported previously . Carbamazepine targets voltage-gated sodium channels  and potently inhibits TTX-S channels in DRG neurons , including those specifically produced by wild-type Nav1.7 channels . Although we did not examine the sensitivity of M1672K channels to carbamazepine in this study, carbamazepine has been shown to block persistent currents generated by I1461T and T1464I PEPD Nav1.7 mutant channels , and it is likely that inhibition of M1672K channel activity contributes to the therapeutic action of carbamazepine in the two patients examined in our study.
Our observations of a 19 mV depolarizing shift in the voltage-dependence of fast-inactivation for M1627K confirm the linkage of impaired fast-inactivation of Nav1.7 and PEPD reported by Fertleman et al . We report here that recovery from fast-inactivation was accelerated in the M1627K, consistent with a destabilized inactivation state of the channel. We have also observed that M1627K display an increased ramp current, similar to IEM mutations [13, 16, 17, 21–25]. M1627K displayed a trend toward a small (< 3 mV) hyperpolarizing shift in activation, but this was not statistically significant. Depolarizing shifts in fast-inactivation have also been observed in some [13, 16, 17] but not all [21–25] mutations that cause IEM. However, the depolarizing shifts in fast-inactivation associated to date with IEM (= 10 mV) are smaller than the shifts in PEPD mutations ( and this study). It is intriguing that the IEM mutation A863P with a +10 mV depolarizing shift in fast-inactivation [13, 16, 17] does not yield symptoms of PEPD, suggesting that other factors, perhaps genetic makeup or bigger shifts in the voltage-dependence of fast-inactivation may contribute to clinical manifestations of the disease.
Gating properties of Nav1.7, for example slow recovery from fast-inactivation and an ability to respond to ramp stimuli [28, 35], suggest that, normally, it may act as a "threshold" channel which boosts subthreshold stimuli, and thus sets the gain in nociceptors [36, 37]. Therefore, it is not surprising that mutations lowering the voltage-threshold for channel activation as in IEM lead to DRG neuron firing in response to a weaker stimulus that may normally be innocuous. Mutations that impair fast-inactivation as in PEPD allow more current to pass through the mutant channel, and thus induce stronger depolarization that brings the DRG neuron closer to the voltage-threshold for all-or-none action potential firing. By analogy to mutations in the cardiac channel Nav1.5 which cause arrhythmias and in neuronal channel Nav1.1 which causes epilepsy , the M1627K PEPD mutation which impairs fast-inactivation of Nav1.7 would be expected to increase repetitive firing, leading to hyperexcitablity of DRG neurons.
Indeed, we now show in this study that a PEPD mutation in human Nav1.7 channels in DRG neurons renders these cells hyperexcitable. Current clamp recordings showed a lower threshold for single action potentials, and an increased firing rate in response to suprathreshold stimuli, but did not show a change in resting membrane potential for DRG neurons expressing M1627K channels. However, Harty et al [13, 16, 17] have shown that depolarization of RMP produced by an IEM mutation (A863P) contributes to, but is not solely responsible for, the increase in DRG neuron hyperexcitability produced by that mutation. Thus, impaired fast-inactivation, accelerated repriming, and the enhanced response to slow depolarizations may all have contributed to the hyperexcitability of DRG neurons expressing M1627K.
The DIV/S4–5 linker is highly conserved in length and sequence (Figure 1C) among all sodium channels described to date, suggesting an important role in the normal functioning of the channel. Increasing the length of DIV/S4–S5 linker in Nav1.4 channels renders the mutant channels non-functional . Importantly, mutations in this linker in several channels underlie pathological conditions [19, 38–44]. Interestingly, substitution of the first methionine (Ma) in this linker (Figure 1C) with a positively charged residue (M1627K) in Nav1.7 causes PEPD ( and this study), while substitution with a hydrophobic residue isoleucine (M1476I) in Nav1.4 causes cold-induced myotonia . Subsitution of the second methionine (Mb) with a positively charged residue (M1652R) in Nav1.5 causes LQT-3 syndrome . All three disorders are linked to hyperexcitability of the cell in which they are expressed, irrespective if it is a neuron or a myocyte. The similarity of the outcome suggests a common mechanism of action, consistent with a conserved function of this linker in channel gating.
Site-directed mutagenesis studies have suggested that the DIV/S4–5 linker contributes to the receptor for the fast-inactivation tripeptide IFM in loop 3 (L3) which links DIII and DIV [45–47]. Structural studies have shown that this linker can acquire an α-helical structure  with several residues including the MaMb (Figure 1C) forming a hydrophobic cluster that is important for inactivation, but indicate that these residues do not interact directly with the IFM motif [47, 49, 50]. Taken together, these studies suggest a model of two antiparallel α-helices ; this structure positions MaMb to interact with Y1470Y1471 (numbers according to Nav1.7) in L3. Substitution of the residues that correspond to Ma ( and this study) or Mb  or Y1470Y1471 residues destabilizes the inactivated state of the channel and yields similar gating changes in several channels. Interestingly, phosphorylation by Fyn kinase of Y1495 which is predicted to interact with Ma in Nav1.5 (equivalent to Y1471 in Nav1.7), produces a significant depolarizing shift in the voltage-dependence but no effect on the rate of steady-state fast-inactivation . Thus the introduction of a charged residue at either of these two sites destabilizes this interaction and leads to impaired binding of the inactivation gate with its receptor.
In summary, our results show that a PEPD mutation produces hyperexcitability in DRG neurons. Our findings also confirm the impairment of fast-inactivation previously associated with PEPD mutations, but show that, in addition, a PEPD mutation can enhance the response of the Nav1.7 channel to small, slow depolarizations and accelerate repriming. These data contribute to a better understanding of the pathophysiology of pain in patients with PEPD and provide additional support for efforts to develop Nav1.7-specific therapeutics for treatment of neuropathic pain.
The proband (Figure 1) is a 36 year old female with a history of erythema and burning pain in the lower parts of the body. Family consent was obtained according to an approved institutional review board protocol and blood samples were then withdrawn and analyzed for mutations in SCN9A.
Genomic DNA (gDNA) was purified from venous blood. Human Caucasian variation panel DNA (25 males, 25 females; The Coriell Institute, Camden, NJ) was used as a normal population control. Coding exons and flanking intronic sequences were amplified and sequenced as described previously . Briefly, PCR amplification was carried out using 150 ng gDNA, 1 μM primers and Expand Long Template polymerase (Roche, Indianapolis, IN) in a 50 μl reaction volume for 35 cycles (95°C × 30 s, 55°C × 30 s and 72°C × 1 min.). Short exons were amplified using two primers, whereas exon 26 required four sets of primers to cover its entirety. Genomic sequences were compared to the reference Nav1.7 cDNA  to identify sequence variation. Sequencing was performed at the Howard Hughes Medical Institute/Keck Biotechnology Center at Yale University. Sequence analysis used BLAST (National Library of Medicine) and Lasergene (DNAStar, Madison, WI).
The plasmid carrying the TTX-resistant (TTX-R) version of human Nav1.7 cDNA (hNav1.7R) was previously described . The M1627K mutation was introduced into hNav1.7R using QuickChange XL II site-directed mutagenesis (Stratagene, La Jolla, CA). Transfected HEK 293 cells, grown under standard culture conditions (5% CO2, 37°C) in Dulbeccos's Modified Eagle's Medium supplemented with 10% fetal bovine serum, were treated with G418 for several weeks, and stable cell lines that express the mutant channel were selected.
Whole-cell patch-clamp recordings were conducted at room temperature (~21°C) using an EPC-10 amplifier and the Pulse program (v 8.5, HEKA Electronic, Germany). Fire-polished electrodes (0.8–1.5 MΩ) were fabricated from 1.7-mm VWR capillary glass using a Sutter P-97 puller (Novato, CA). Average access resistance was 2.1 ± 0.1 MΩ (mean ± SE, n = 27). Voltage errors were minimized using ~40–75% series resistance compensation to achieve identical (1.5 ± 0.2 mV) voltage error after series resistance compensation for the two groups. Capacitance artifacts were canceled using computer-controlled circuitry of the patch clamp amplifier and linear leak subtraction was used for all voltage clamp recordings. Recordings were always started 3.5–4 minutes after establishing the whole-cell configuration. Membrane currents were filtered at 5 kHz and sampled at 20 kHz. The pipette solution contained (in mM): 140 CsF, 1 EGTA, 10 NaCl and 10 HEPES (pH 7.3). The standard bathing solution was (in mM) 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.3). Data were analyzed using Pulsefit (HEKA Electronic, Germany) and Origin (Microcal Software, Northampton, MA) software. Unless otherwise noted, statistical significance was determined (p < 0.05) using an unpaired t-test. Results are presented as mean ± SEM and error bars in the figures represent standard errors (SE).
Transfection of DRG neurons
The protocol for the care and sacrifice of rats used in the study was approved by the Veterans Administration CT Healthcare system IACUC. DRG tissue from 1- to 5-day old Sprague Dawley rats were harvested and dissociated using a protocol that was adapted from Rizzo et al . Briefly, dissected ganglia were placed in ice cold oxygenated complete saline solution (CSS), which contained (in mM) 137 NaCl, 5.3 KCl, 1 MgCl2, 25 sorbitol, 3 CaCl2, 10 N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES); pH 7.2. They were then transferred to an oxygenated, 37°C CSS solution containing 1.5 mg/ml Collagenase A (Roche Applied Science, Indianapolis, IN) and 0.6 mM EDTA and incubated with gentle agitation at 37°C for 20 min. This solution was then exchanged with an oxygenated, 37°C CSS solution containing 1.5 mg/ml Collagenase D (Roche Applied Science, Indianapolis, IN), 0.6 mM EDTA and 30 U/ml papain (Worthington Biochemical, Lakewood, NJ) and incubated with gentle agitation at 37°C for 20 min. The solution was then aspirated and the ganglia triturated in DRG media [(DMEM/Fl2 (1:1) with 100 U/ml penicillin, 0.1 mg/ml streptomycin (Invitrogen, Carlsbad, CA) and 10% fetal calf serum (Hyclone, Logan, UT)], which contained 1.5 mg/ml bovine serum albumin (Sigma-Aldrich, St. Louis, MO) and 1.5 mg/ml trypsin inhibitor (Roche Applied Science, Indianapolis, IN).
Either WT hNav1.7R or M1627K mutant channels were transiently transfected into the DRG neurons, along with enhanced-GFP, by electroporation with a Nucleofector II (Amaxa, Gaithersburg, MD) using Rat Neuron Nucleofector Solution and program G-013. The ratio of sodium channel to GFP constructs was 5:1. Immediately after transfection, the cells were allowed to recover for 5 minutes in Ca2+- and Mg2+-free culture medium (DMEM + 10% FBS). The cell suspension was then diluted with DRG media containing 1.5 mg/ml bovine serum albumin and 1.5 mg/ml trypsin inhibitor, 80 μl was plated on 12 mm circular poly-D-lysine/laminin pre-coated coverslips (BD Biosciences, Bedford, MA) and the cells incubated at 37°C in 5% CO2 for 30 min. DRG media (1 ml/well), supplemented with 50 ng/ml each of mNGF (Alomone Labs, Jerusalem, Israel) and GDNF (Peprotec, Rocky Hill, NJ), was then added and the cells maintained at 37°C in a 5% CO2 incubator for 18–48 hr before recording.
Small (20–30 μm diameter) GFP-labeled DRG neurons were used for current-clamp recording. Neurons with round cell body morphology and clear processes were selected for analysis and recordings were performed between 20- and 50-hours post-transfection. Pipette resistance was 1–3 MΩ when filled with the pipette solution which contained (in mM): 140 KCl, 0.5 EGTA, 5 HEPES, and 3 Mg-ATP, pH 7.3 with KOH (adjusted to 315 mOsm with dextrose). The extracellular solution contained the following (in mM): 140 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, pH 7.3 with NaOH (adjusted to 320 mOsm with dextrose). Formation of a GΩ seal and the transition to whole-cell configuration was performed in voltage-clamp mode before proceeding to the current-clamp recording mode. Recordings were obtained using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) connected to a Digidata 1422 interface controlled by Clampex software (Molecular Devices). Cells with resting membrane potentials (RMP) more negative than -40 mV that were stable (<10% variation during the first 5 minutes) were included for data analysis. Input resistance was determined by fitting the slope of a line fit to hyperpolarizing voltage responses to current steps of -5 pA to -25 pA in 5 pA increments. Threshold was determined by the first action potential elicited by a series of depolarizing current injections that increased in 5 pA increments. Action potentials were counted by detecting membrane potential transients that exceeded the threshold value of 0 mV during 1-sec long depolarizing current injections. Data are expressed as means ± SEM. Student's t-test was used to assess the significance (p < 0.05) of differences between parameters measured from DRG neurons transfected with WT or M1627K mutant channels. Where indicated, the Mann-Whitney rank sum test was applied because of non-normal data distributions. Statistical analysis was performed using Sigmaplot software (Systat Software, San Jose, CA).
We thank Dr. Fuki Hisama for helpful discussions, and Emmanuella Eastman and Bart Toftness for excellent technical assistance. This work was supported by the Medical Research Service and Rehabilitation Research Service, Dept. of Veterans Affairs and by a grant from the Erythromelalgia Association (SDH and SGW); TRC was supported by research grant NS053422 from the National Institute of Health. The Center for Neuroscience and Regeneration Research is a Collaboration of the Paralyzed Veterans of America, and the United Spinal Association with Yale University.
- Dib-Hajj SD, Cummins TR, Black JA, Waxman SG: From genes to pain: Nav1.7 and human pain disorders. Trends Neurosci 2007, 30(11):555–563. 10.1016/j.tins.2007.08.004View ArticlePubMedGoogle Scholar
- Waxman SG: Channel, neuronal and clinical function in sodium channelopathies: from genotype to phenotype. Nat Neurosci 2007, 10(4):405–409. 10.1038/nn1857View ArticlePubMedGoogle Scholar
- 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. Mol Brain Res 1996, 43(1–2):117–131. 10.1016/S0169-328X(96)00163-5View ArticlePubMedGoogle Scholar
- 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. Mol Brain Res 1997, 45(1):71–82. 10.1016/S0169-328X(96)00241-0View ArticlePubMedGoogle Scholar
- Sangameswaran L, Fish LM, Koch BD, Rabert DK, Delgado SG, Ilnicka M, Jakeman LB, Novakovic S, Wong K, Sze P, et al.: A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat and human dorsal root ganglia. J Biol Chem 1997, 272(23):14805–14809. 10.1074/jbc.272.23.14805View ArticlePubMedGoogle Scholar
- Toledo-Aral JJ, Moss BL, He ZJ, Koszowski AG, Whisenand T, Levinson SR, Wolf JJ, Silossantiago I, Halegoua S, Mandel G: Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons. Proc Natl Acad Sci (USA) 1997, 94(4):1527–1532. 10.1073/pnas.94.4.1527View ArticleGoogle Scholar
- Djouhri L, Newton R, Levinson SR, Berry CM, Carruthers B, Lawson SN: Sensory and electrophysiological properties of guinea-pig sensory neurones expressing Nav1.7 (PN1) Na+channel alpha-subunit protein. J Physiol (Lond) 2003, 546(Pt 2):565–576. 10.1113/jphysiol.2002.026559View ArticleGoogle Scholar
- Nassar MA, Stirling LC, Forlani G, Baker MD, Matthews EA, Dickenson AH, Wood JN: Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc Natl Acad Sci USA 2004, 101(34):12706–12711. 10.1073/pnas.0404915101PubMed CentralView ArticlePubMedGoogle Scholar
- Ahmad S, Dahllund L, Eriksson AB, Hellgren D, Karlsson U, Lund PE, Meijer IA, Meury L, Mills T, Moody A, et al.: A stop codon mutation in SCN9A causes lack of pain sensation. Hum Mol Genet 2007, 16(17):2114–2121. 10.1093/hmg/ddm160View ArticlePubMedGoogle Scholar
- Cox JJ, Reimann F, Nicholas AK, Thornton G, Roberts E, Springell K, Karbani G, Jafri H, Mannan J, Raashid Y, et al.: An SCN9A channelopathy causes congenital inability to experience pain. Nature 2006, 444(7121):894–898. 10.1038/nature05413View ArticlePubMedGoogle Scholar
- Goldberg Y, Macfarlane J, Macdonald M, Thompson J, Dube MP, Mattice M, Fraser R, Young C, Hossain S, Pape T, et al.: Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations. Clin Genet 2007, 71(4):311–319. 10.1111/j.1399-0004.2007.00790.xView ArticlePubMedGoogle Scholar
- Yang Y, Wang Y, Li S, Xu Z, Li H, Ma L, Fan J, Bu D, Liu B, Fan Z, et al.: Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia. J Med Genet 2004, 41(3):171–174. 10.1136/jmg.2003.012153PubMed CentralView ArticlePubMedGoogle Scholar
- Dib-Hajj SD, Rush AM, Cummins TR, Hisama FM, Novella S, Tyrrell L, Marshall L, Waxman SG: Gain-of-function mutation in Nav1.7 in familial erythromelalgia induces bursting of sensory neurons. Brain 2005, 128(Pt 8):1847–1854. 10.1093/brain/awh514View ArticlePubMedGoogle Scholar
- Drenth JP, Te Morsche RH, Guillet G, Taieb A, Kirby RL, Jansen JB: SCN9A mutations define primary erythermalgia as a neuropathic disorder of voltage gated sodium channels. J Invest Dermatol 2005, 124(6):1333–1338. 10.1111/j.0022-202X.2005.23737.xView ArticlePubMedGoogle Scholar
- Michiels JJ, te Morsche RH, Jansen JB, Drenth JP: Autosomal dominant erythermalgia associated with a novel mutation in the voltage-gated sodium channel alpha subunit Nav1.7. Arch Neurol 2005, 62(10):1587–1590. 10.1001/archneur.62.10.1587View ArticlePubMedGoogle Scholar
- Han C, Rush AM, Dib-Hajj SD, Li S, Xu Z, Wang Y, Tyrrell L, Wang X, Yang Y, Waxman SG: Sporadic onset of erythermalgia: a gain-of-function mutation in Nav1.7. Ann Neurol 2006, 59: 553–558. 10.1002/ana.20776View ArticlePubMedGoogle Scholar
- Harty TP, Dib-Hajj SD, Tyrrell L, Blackman R, Hisama FM, Rose JB, Waxman SG: NaV1.7 mutant A863P in erythromelalgia: effects of altered activation and steady-state inactivation on excitability of nociceptive dorsal root ganglion neurons. J Neurosci 2006, 26(48):12566–12575. 10.1523/JNEUROSCI.3424-06.2006View ArticlePubMedGoogle Scholar
- Lee MJ, Yu HS, Hsieh ST, Stephenson DA, Lu CJ, Yang CC: Characterization of a familial case with primary erythromelalgia from Taiwan. J Neurol 2007, 254(2):210–214. 10.1007/s00415-006-0328-3View ArticlePubMedGoogle Scholar
- Fertleman CR, Baker MD, Parker KA, Moffatt S, Elmslie FV, Abrahamsen B, Ostman J, Klugbauer N, Wood JN, Gardiner RM, et al.: SCN9A mutations in paroxysmal extreme pain disorder: allelic variants underlie distinct channel defects and phenotypes. Neuron 2006, 52(5):767–774. 10.1016/j.neuron.2006.10.006View ArticlePubMedGoogle Scholar
- Fertleman CR, Ferrie CD, Aicardi J, Bednarek NA, Eeg-Olofsson O, Elmslie FV, Griesemer DA, Goutieres F, Kirkpatrick M, Malmros IN, et al.: Paroxysmal extreme pain disorder (previously familial rectal pain syndrome). Neurology 2007, 69(6):586–595. 10.1212/01.wnl.0000268065.16865.5fView ArticlePubMedGoogle Scholar
- Choi JS, Dib-Hajj SD, Waxman SG: Inherited erythermalgia. Limb pain from an S4 charge-neutral Na channelopathy. Neurology 2006, 67(9):1563–1567. 10.1212/01.wnl.0000231514.33603.1eView ArticlePubMedGoogle Scholar
- Cummins TR, Dib-Hajj SD, Waxman SG: Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy. J Neurosci 2004, 24(38):8232–8236. 10.1523/JNEUROSCI.2695-04.2004View ArticlePubMedGoogle Scholar
- Lampert A, Dib-Hajj SD, Tyrrell L, Waxman SG: Size matters: Erythromelalgia mutation S241T in Nav1.7 alters channel gating. J Biol Chem 2006, 281(47):36029–36035. 10.1074/jbc.M607637200View ArticlePubMedGoogle Scholar
- Sheets PL, Jackson Ii JO, Waxman SG, Dib-Hajj S, Cummins TR: A Nav1.7 channel mutation associated with hereditary erythromelalgia contributes to neuronal hyperexcitability and displays reduced lidocaine sensitivity. J Physiol (Lond) 2007, 581: 1019–1031. 10.1113/jphysiol.2006.127027View ArticleGoogle Scholar
- Cheng X, Dib-Hajj SD, Tyrrell L, Waxman SG: Mutation I136V alters electrophysiological properties of the NaV1.7 channel in a family with onset of erythromelalgia in the second decade. Mol Pain 2008, 4(1):1. 10.1186/1744-8069-4-1PubMed CentralView ArticlePubMedGoogle Scholar
- Catterall WA, Yu FH: Painful channels. Neuron 2006, 52(5):743–744. 10.1016/j.neuron.2006.11.017View ArticlePubMedGoogle Scholar
- Klugbauer N, Lacinova L, Flockerzi V, Hofmann F: Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells. EMBO J 1995, 14(6):1084–1090.PubMed CentralPubMedGoogle Scholar
- Cummins TR, Howe JR, Waxman SG: Slow closed-state inactivation: a novel mechanism underlying ramp currents in cells expressing the hNE/PN1 sodium channel. J Neurosci 1998, 18(23):9607–9619.PubMedGoogle Scholar
- Jarecki BW, Sheets PL, Jackson Ii JO, Cummins TR: Paroxysmal Extreme Pain Disorder mutations within the D3/S4–S5 linker of Nav1.7 cause moderate destabilization of fast-inactivation. J Physiol (Lond) 2008, 586(Pt 17):4137–4153. 10.1113/jphysiol.2008.154906View ArticleGoogle Scholar
- Drenth JP, Te Morsche RH, Mansour S, Mortimer PS: Primary erythermalgia as a sodium channelopathy: screening for SCN9A mutations: exclusion of a causal role of SCN10A and SCN11A. Arch Dermatol 2008, 144(3):320–324. 10.1001/archderm.144.3.320View ArticlePubMedGoogle Scholar
- Burns TM, Te Morsche RH, Jansen JB, JP HD: Genetic heterogeneity and exclusion of a modifying locus at 2q in a family with autosomal dominant primary erythermalgia. Br J Dermatol 2005, 153(1):174–177. 10.1111/j.1365-2133.2005.06441.xView ArticlePubMedGoogle Scholar
- Ragsdale DS, Avoli M: Sodium channels as molecular targets for antiepileptic drugs. Brain Res Rev 1998, 26(1):16–28. 10.1016/S0165-0173(97)00054-4View ArticlePubMedGoogle Scholar
- Rush AM, Elliott JR: Phenytoin and Carbamazepine – Differential Inhibition Of Sodium Currents In Small Cells From Adult Rat Dorsal Root Ganglia. Neuroscience Letters 1997, 226(2):95–98. 10.1016/S0304-3940(97)00258-9View ArticlePubMedGoogle Scholar
- Sheets PL, Heers C, Stoehr T, Cummins TR: Differential block of sensory neuronal voltage-gated sodium channels by lacosamide [(2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide], lidocaine, and carbamazepine. J Pharmacol Exp Ther 2008, 326(1):89–99. 10.1124/jpet.107.133413View ArticlePubMedGoogle Scholar
- Herzog RI, Cummins TR, Ghassemi F, Dib-Hajj SD, Waxman SG: Distinct repriming and closed-state inactivation kinetics of Nav1.6 and Nav1.7 sodium channels in mouse spinal sensory neurons. J Physiol (Lond) 2003, 551(Pt 3):741–750. 10.1113/jphysiol.2003.047357View ArticleGoogle Scholar
- Rush AM, Cummins TR, Waxman SG: Multiple sodium channels and their roles in electrogenesis within dorsal root ganglion neurons. J Physiol (Lond) 2007, 579(Pt 1):1–14. 10.1113/jphysiol.2006.121483View ArticleGoogle Scholar
- Cummins TR, Sheets PL, Waxman SG: The roles of sodium channels in nociception: Implications for mechanisms of pain. Pain 2007.Google Scholar
- Mitrovic N, Lerche H, Heine R, Fleischhauer R, Pika-Hartlaub U, Hartlaub U, George AL Jr, Lehmann-Horn F: Role in fast inactivation of conserved amino acids in the IV/S4–S5 loop of the human muscle Na+ channel. Neuroscience Letters 1996, 214(1):9–12.PubMedGoogle Scholar
- Claes L, Ceulemans B, Audenaert D, Smets K, Lofgren A, Del-Favero J, Ala-Mello S, Basel-Vanagaite L, Plecko B, Raskin S, et al.: De novo SCN1A mutations are a major cause of severe myoclonic epilepsy of infancy. Hum Mutat 2003, 21(6):615–621. 10.1002/humu.10217View ArticlePubMedGoogle Scholar
- Nabbout R, Gennaro E, Dalla Bernardina B, Dulac O, Madia F, Bertini E, Capovilla G, Chiron C, Cristofori G, Elia M, et al.: Spectrum of SCN1A mutations in severe myoclonic epilepsy of infancy. Neurology 2003, 60(12):1961–1967.View ArticlePubMedGoogle Scholar
- Fleischhauer R, Mitrovic N, Deymeer F, Lehmann-Horn F, Lerche H: Effects of temperature and mexiletine on the F1473S Na+ channel mutation causing paramyotonia congenita. Pflugers Arch 1998, 436(5):757–765. 10.1007/s004240050699View ArticlePubMedGoogle Scholar
- Rossignol E, Mathieu J, Thiffault I, Tetreault M, Dicaire MJ, Chrestian N, Dupre N, Puymirat J, Brais B: A novel founder SCN4A mutation causes painful cold-induced myotonia in French-Canadians. Neurology 2007, 69(20):1937–1941. 10.1212/01.wnl.0000290831.08585.2cView ArticlePubMedGoogle Scholar
- Schoser BG, Schroder JM, Grimm T, Sternberg D, Kress W: A large german kindred with cold-aggravated myotonia and a heterozygous A1481D mutation in the SCN4A gene. Muscle Nerve 2007, 35: 599–606. 10.1002/mus.20733View ArticlePubMedGoogle Scholar
- Ruan Y, Liu N, Bloise R, Napolitano C, Priori SG: Gating properties of SCN5A mutations and the response to mexiletine in long-QT syndrome type 3 patients. Circulation 2007, 116(10):1137–1144. 10.1161/CIRCULATIONAHA.107.707877View ArticlePubMedGoogle Scholar
- Smith MR, Goldin AL: Interaction between the sodium channel inactivation linker and domain III S4–S5. Biophys J 1997, 73(4):1885–1895.PubMed CentralView ArticlePubMedGoogle Scholar
- McPhee JC, Ragsdale DS, Scheuer T, Catterall WA: A critical role for the S4–S5 intracellular loop in domain IV of the sodium channel alpha-subunit in fast inactivation. J Biol Chem 1998, 273(2):1121–1129. 10.1074/jbc.273.2.1121View ArticlePubMedGoogle Scholar
- Lerche H, Peter W, Fleischhauer R, Pikahartlaub U, Malina T, Mitrovic N, Lehmann-horn F: Role in fast inactivation of the IV/S4–S5 loop of the human muscle Na+ channel probed by cysteine mutagenesis. J Physiol (Lond) 1997, 505(2):345–352. 10.1111/j.1469-7793.1997.345bb.xView ArticleGoogle Scholar
- Miyamoto K, Nakagawa T, Kuroda Y: Solution structures of the cytoplasmic linkers between segments S4 and S5 (S4–S5) in domains III and IV of human brain sodium channels in SDS micelles. J Pept Res 2001, 58(3):193–203. 10.1034/j.1399-3011.2001.00912.xView ArticlePubMedGoogle Scholar
- Kellenberger S, Scheuer T, Catterall WA: Movement of the Na+ channel inactivation gate during inactivation. J Biol Chem 1996, 271(48):30971–30979. 10.1074/jbc.271.48.30971View ArticlePubMedGoogle Scholar
- Filatov GN, Nguyen TP, Kraner SD, Barchi RL: Inactivation and secondary structure in the D4/S4–5 region of the SkM1 sodium channel. J Gen Physiol 1998, 111(6):703–715. 10.1085/jgp.111.6.703PubMed CentralView ArticlePubMedGoogle Scholar
- Ahern CA, Zhang JF, Wookalis MJ, Horn R: Modulation of the cardiac sodium channel NaV1.5 by Fyn, a Src family tyrosine kinase. Circ Res 2005, 96: 991–998. 10.1161/01.RES.0000166324.00524.ddView ArticlePubMedGoogle Scholar
- Rizzo MA, Kocsis JD, Waxman SG: Slow sodium conductances of dorsal root ganglion neurons: intraneuronal homogeneity and interneuronal heterogeneity. J Neurophysiol 1994, 72(6):2796–2815.PubMed CentralPubMedGoogle Scholar
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