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1.  A Novel Nav1.7 Mutation Producing Carbamazepine-Responsive Erythromelalgia 
Annals of neurology  2009;65(6):733-741.
Objective
Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Nav1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ).
Methods
We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Nav1.7 complementary DNA. Wild-type Nav1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents.
Results
Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Nav1.7 but does not affect these parameters in wild-type Nav1.7.
Interpretation
Our results demonstrate a normalizing effect of CBZ on mutant Nav1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Nav1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders.
doi:10.1002/ana.21678
PMCID: PMC4103031  PMID: 19557861
2.  Structural homology modeling and mutant cycle analysis predict pharmacoresponsiveness of a NaV1.7 mutant channel 
Nature communications  2012;3:1186.
The NaV1.7 voltage-gated sodium channel is critical for pain signaling in humans. Gain-of-function mutations are associated with several pain syndromes including inherited erythromelalgia (IEM). Most IEM patients with NaV1.7 mutations are resistant to pharmacotherapy, but carbamazepine (CBZ) normalizes activation of NaV1.7-V400M mutant channels from a family with CBZ-responsive IEM. Here we show that structural modeling and mutant cycle analysis predict pharmacoresponsiveness to CBZ of a NaV1.7 mutant channel that substitutes a residue 159 amino acids distant from V400M in the channel peptide. Structural modeling reveals that this IEM mutation (S241T) is only 2.4-angstrom (Å) apart from V400M in the folded NaV1.7 channel and mutant cycle analysis demonstrates that V400M is energetically coupled to S241T during channel activation. We further show that the atomic proximity and energetic coupling of V400M and S241T are paralleled by pharmacological coupling, as CBZ at therapeutic concentration (30 μM) causes a depolarizing shift of S214T mutant channel activation curve, similar to that previously reported for V400M mutant channel. This pharmacoresponsiveness of S241T to CBZ was further evident at a cellular level, where CBZ normalized the hyperexcitability of dorsal root ganglion (DRG) neurons expressing S241T mutant channel. We suggest that a similar approach might facilitate screening for amino acid variants of a variety of channels that confer enhanced pharmacoresponsiveness on the channel.
doi:10.1038/ncomms2184
PMCID: PMC3530897  PMID: 23149731
3.  Intra- and interfamily phenotypic diversity in pain syndromes associated with a gain-of-function variant of NaV1.7 
Molecular Pain  2011;7:92.
Background
Sodium channel NaV1.7 is preferentially expressed within dorsal root ganglia (DRG), trigeminal ganglia and sympathetic ganglion neurons and their fine-diamter axons, where it acts as a threshold channel, amplifying stimuli such as generator potentials in nociceptors. Gain-of-function mutations and variants (single amino acid substitutions) of NaV1.7 have been linked to three pain syndromes: Inherited Erythromelalgia (IEM), Paroxysmal Extreme Pain Disorder (PEPD), and Small Fiber Neuropathy (SFN). IEM is characterized clinically by burning pain and redness that is usually focused on the distal extremities, precipitated by mild warmth and relieved by cooling, and is caused by mutations that hyperpolarize activation, slow deactivation, and enhance the channel ramp response. PEPD is characterized by perirectal, periocular or perimandibular pain, often triggered by defecation or lower body stimulation, and is caused by mutations that severely impair fast-inactivation. SFN presents a clinical picture dominated by neuropathic pain and autonomic symptoms; gain-of-function variants have been reported to be present in approximately 30% of patients with biopsy-confirmed idiopathic SFN, and functional testing has shown altered fast-inactivation, slow-inactivation or resurgent current. In this paper we describe three patients who house the NaV1.7/I228M variant.
Methods
We have used clinical assessment of patients, quantitative sensory testing and skin biopsy to study these patients, including two siblings in one family, in whom genomic screening demonstrated the I228M NaV1.7 variant. Electrophysiology (voltage-clamp and current-clamp) was used to test functional effects of the variant channel.
Results
We report three different clinical presentations of the I228M NaV1.7 variant: presentation with severe facial pain, presentation with distal (feet, hands) pain, and presentation with scalp discomfort in three patients housing this NaV1.7 variant, two of which are from a single family. We also demonstrate that the NaV1.7/I228M variant impairs slow-inactivation, and produces hyperexcitability in both trigeminal ganglion and DRG neurons.
Conclusion
Our results demonstrate intra- and interfamily phenotypic diversity in pain syndromes produced by a gain-of-function variant of NaV1.7.
doi:10.1186/1744-8069-7-92
PMCID: PMC3248882  PMID: 22136189
4.  Effects of ranolazine on wild-type and mutant hNav1.7 channels and on DRG neuron excitability 
Molecular Pain  2010;6:35.
Background
A direct role of sodium channels in pain has recently been confirmed by establishing a monogenic link between SCN9A, the gene which encodes sodium channel Nav1.7, and pain disorders in humans, with gain-of-function mutations causing severe pain syndromes, and loss-of-function mutations causing congenital indifference to pain. Expression of sodium channel Nav1.8 in DRG neurons has also been shown to be essential for the manifestation of mutant Nav1.7-induced neuronal hyperexcitability. These findings have confirmed key roles of Nav1.7 and Nav1.8 in pain and identify these channels as novel targets for pain therapeutic development. Ranolazine preferentially blocks cardiac late sodium currents at concentrations that do not significantly reduce peak sodium current. Ranolazine also blocks wild-type Nav1.7 and Nav1.8 channels in a use-dependent manner. However, ranolazine's effects on gain-of-function mutations of Nav1.7 and on DRG neuron excitability have not been investigated. We used voltage- and current-clamp recordings to evaluate the hypothesis that ranolazine may be effective in regulating Nav1.7-induced DRG neuron hyperexcitability.
Results
We show that ranolazine produces comparable block of peak and ramp currents of wild-type Nav1.7 and mutant Nav1.7 channels linked to Inherited Erythromelalgia and Paroxysmal Extreme Pain Disorder. We also show that ranolazine, at a clinically-relevant concentration, blocks high-frequency firing of DRG neurons expressing wild-type but not mutant channels.
Conclusions
Our data suggest that ranalozine can attenuate hyperexcitability of DRG neurons over-expressing wild-type Nav1.7 channels, as occurs in acquired neuropathic and inflammatory pain, and thus merits further study as an alternative to existing non-selective sodium channel blockers.
doi:10.1186/1744-8069-6-35
PMCID: PMC2898769  PMID: 20529343
5.  Paroxysmal extreme pain disorder M1627K mutation in human Nav1.7 renders DRG neurons hyperexcitable 
Molecular Pain  2008;4:37.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1744-8069-4-37
PMCID: PMC2556659  PMID: 18803825
6.  Blockade of maitotoxin-induced oncotic cell death reveals zeiosis 
BMC Physiology  2002;2:2.
Background
Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca2+ influx via non-selective cation channels, 2) uptake of vital dyes via formation of large pores, and 3) release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC) in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells.
Results
Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 ≈ 1.9 and 0.66 μM, respectively) suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program.
Conclusions
Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms.
doi:10.1186/1472-6793-2-2
PMCID: PMC65053  PMID: 11825342
7.  Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells 
BMC Physiology  2001;1:2.
Background
Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca2+]i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells.
Results
Maitotoxin produced a concentration-dependent increase in [Ca2+]i followed by a biphasic uptake of ethidium. Comparison of ethidium (Mw 314 Da), YO-PRO-1 (Mw 375 Da), and POPO-3 (Mw 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation.
Conclusions
These results suggest that maitotoxin-induced Ca2+ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.
doi:10.1186/1472-6793-1-2
PMCID: PMC32181  PMID: 11231888

Results 1-7 (7)