Journal: Journal of Traditional Chinese Medicine

Article Title: Astragaloside IV plays a role in reducing radiation-induced liver inflammation in mice by inhibiting thioredoxin-interacting protein/nod-like receptor protein 3 signaling pathway

doi: 10.19852/j.cnki.jtcm.2023.01.008

Figure Lengend Snippet: A: immunofluorescence expression of NLRP3 inflammasome in each group. A1: Control group; A2: DMSO group; A3: IR group; A4: IR+AS-IV-20 group; A5: IR+AS-IV-40 group; B1: the expressions of TXNIP, NLRP3, ASC, Caspase-1 and IL-1β in liver were measured by Western blot; B2: relative density of TXNIP; B3: relative density of NLRP3; B4: relative density of ASC. B5: relative density of Caspase-1; B6: relative density of IL-1β. DMSO: dimethyl sulfoxide; IR: irradiation; IR+AS-20: irradiation+AS-IV (20 mg/kg); IR + AS-40: irradiation +AS-IV (40 mg/kg); AS-IV: astragaloside IV; TXNIP: Thioredoxin-interacting protein; NLRP3: nod-like receptor protein 3; ASC: apoptosis-associated speck-like protein containing a CARD; Caspase-1: cysteinyl aspartate-specific proteinase 1; IL-1β: interleukin 1beta. aP < 0.001 vs Control group; bP < 0.001 vs IR group; cP < 0.05 vs IR + AS-20 group; dP < 0.01 vs IR group; eP < 0.01 vs IR + AS-20 group.

Article Snippet: After blocked in 5% nonfat dry milk for 1 h, membranes were washed with Tris-buffered saline with tween 20 (TBST) for 3 × 10 min. Then, membranes were incubated with the primary antibodies against NLRP3 (1:1000), ASC (1:1000; bs-6741R; Bioss, Beijing, China), cysteinyl aspartate-specific proteinase 1 (Caspase-1; 1:1000; bs-10442R; Bioss, Beijing, China), IL-1β (1:1000; bs-0812R; Bioss, Beijing, China), TXNIP (1:1000; ET1705-72, Huabio, Hangzhou, China), tumor necrosis factor alpha (TNF-α, 1:1000; bs-0078R; Bioss, Beijing, China), interleukin 6 (IL-6, 1:1000; bs-0782R; Bioss, Beijing, China)and GAPDH (1:1000; K200057M, Solarbio, Beijing, China) overnight at 4 ℃.

Techniques: Immunofluorescence, Expressing, Western Blot, Irradiation

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Nav1.7 immunolabeling (IL) of arterioles (Ar), arteriole-venule shunts (AVS) and associated innervation in normal human plantar glabrous skin with Alomone (A, B) or Yale (C) Nav1.7 antibodies (red). Co-labeling of innervation (arrows) as marked with anti-PGP 9.5 (PGP, green, A ) or smooth muscle cells in tunica media (tm) as marked with anti α-smooth muscle actin antibody (αSMA, green, B , C ). Nuclei are DAPI-labeled (blue). Left images (each panel) show only red fluorescence, middle images green; right images show triple-label combinations. Large white rectangles are 2X-enlargements of small rectangles. A-C . Nav1.7-IL is expressed on endothelial cells of tunica intima (red arrowheads) and tm smooth muscle cells as confirmed by double-labeling with anti-αSMA (B, C) . Nav1.7-IL is expressed on virtually all vascular innervation (arrows) in tunica adventitia (ta) as confirmed by anti-PGP 9.5 double-labeling ( A , yellow arrows). N=nerve. D-E . Nav1.7-IL on arteriole endothelial cells shown as 2X-enlargements of areas indicated by white rectangles in B , C . First images (each panel) show Nav1.7-IL on smooth muscle cells in tm and endothelial cells (red arrowheads). The second images show α-SMA co-labeling of only the smooth muscle cells of tm (green). The third images show merge of first and second images with DAPI (blue). Sections re-labeled with anti-PECAM (green) to show co-labeling with Nav1.7 on endothelial cells (yellow arrowheads, fourth and fifth images). F-G . Background Cy3 fluorescence is limited with no primary antibody in arteriole deep in dermis (F) , epidermis (Ep) and upper-dermis (UD) (G) . In F , broken line shows tm perimeter with dotted line around arteriole lumen. In G , broken line indicates basement membrane of epidermis and dotted line indicates boundary of dead and live superficial keratinocyte layers (stratum corneum, sc and stratum granulosum, sg, respectively). Stratum spinosum, ss; stratum basalis, sb; dermal papilla (dp). Scale bars=150μm (A); 100μm (B ,C, F, G) ; 50μm in D , E .

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Immunolabeling, Labeling, Fluorescence

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Digital fluorescence images of Nav1.7 immunolabeling (IL) of arterioles (Ar), arteriole-venule shunts (AVS) and associated innervation in normal human glabrous skin biopsies from the plantar foot (A,C,D) and palmar hand (B) . All sections are labeled with an Alomone (A,C) or Yale (B,D) rabbit anti-rat Nav1.7 antibody revealed by a donkey anti-rabbit Cy3-conjugated secondary antibody (red fluorescence). Secondary antibodies conjugated to Alexa 488 (green fluorescence) were used to assess co-labeling for peptidergic sensory innervation revealed with a sheep anti-human CGRP antibody (A,B) or noradrenergic sympathetic innervation revealed with a sheep anti-human NPY antibody (C,D) . Cell nuclei are labeled with DAPI (blue fluorescence). The left images in each panel show only the red fluorescence, the middle images only the green, and the right images the triple label combinations. Areas outlined in large white rectangles are 2X enlargement of the areas in the small rectangles. A-D . Nav1.7-IL is expressed on the endothelial cells of the tunica intima (red arrowheads) and on smooth muscle cells of the tunica media (tm). A , B . Peptidergic sensory innervation co-expresses Nav1.7-IL and CGRP-IL (yellow straight arrows). Other innervation labeled only with Nav1.7 (red curved arrows) is likely the noradrenergic sympathetic innervation that expresses NPY-IL as shown in C and D . C , D . Noradrenergic sympathetic innervation co-expresses Nav1.7-IL and NPY-IL (yellow curved arrows). Other innervation labeled with only Nav1.7 (red straight arrows) is likely the peptidergic sensory innervation that expresses CGRP-IL as shown in A and B . Scale Bar = 150 μm in A and B , 100 μm in C and D .

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Fluorescence, Immunolabeling, Labeling

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Nav1.7 immunolabeling (IL) of arterioles and associated innervation in normal human palmar glabrous skin biopsies, in alternating sections cut parallel to and through lumen (*) of branched arteriole (A-C) and parallel to arteriole, skimming the interface between tunica media (tm) and tunica adventitia (ta) (D,E) . All sections are labeled with Abcam anti-human Nav1.7 antibody (red). Secondary antibodies conjugated to Alexa 488 (green) were used to assess co-labeling for: smooth muscle cells revealed with mouse anti-α−smooth muscle actin antibody (αSMA, A ); peptidergic sensory innervation revealed with sheep anti-CGRP antibody (yellow straight arrows, B , D ); and noradrenergic sympathetic innervation revealed with sheep anti-NPY antibody (yellow curved arrows, C , E ): Nuclei are labeled with DAPI (blue). Left images in each panel show only the red fluorescence, middle images only green, and right images the triple-label combinations. Areas outlined in large white rectangles (A-C) are 2X enlargements of areas in small rectangles. A-E . A . Nav1.7-IL is expressed on endothelial cells of tunica intima (red arrowheads) and smooth muscle cells of tm as confirmed by double-labeling with anti-αSMA. Nav1.7-IL is expressed on innervation (arrows) in ta, near and at the border with tm. B , D . Peptidergic sensory innervation co-expresses Nav1.7-IL and CGRP-IL (yellow straight arrows). Other innervation labeled only with Nav1.7 (red curved arrows) is likely noradrenergic sympathetic innervation that expresses NPY-IL (C,E) . C , E . Noradrenergic sympathetic innervation co-expresses Nav1.7-IL and NPY-IL (yellow curved arrows). Other innervation labeled with only Nav1.7 (red straight arrows) is likely peptidergic sensory innervation that expresses CGRP-IL as shown in B and D . Scale bar = 100 μm in A-C , 50 μm in D and E .

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Immunolabeling, Labeling, Fluorescence

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Nav1.7 expression in smooth muscle cells of deep dermis arterioles within skin from lateral malleolus of three healthy subjects. Smooth muscle cells (arrowheads) of the arteriole tunica media exhibit robust Nav1.7 (red) immunolabeling (antibody Nav1.7 Y ), which is co-localized with alpha smooth muscle actin (green). Skin samples from 3 healthy subjects (Subject 1: A ; Subject 2: B , C ; Subject 3: D ) display similar patterns of Nav1.7 labeling in the smooth muscle cells of the dermal arterioles. Co-localization of Nav1.7 and alpha smooth muscle actin is yellow in the merged panels. E . Sections incubated without primary antibodies followed by secondary antibodies displayed background levels of immunofluorescence in skin vasculature.

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Expressing, Immunolabeling, Labeling, Incubation, Immunofluorescence

Journal: Molecular Pain

Article Title: Sodium channel Nav1.7 in vascular myocytes, endothelium, and innervating axons in human skin

doi: 10.1186/s12990-015-0024-3

Figure Lengend Snippet: Digital fluorescence images of Nav1.7 (red) and PGP 9.5 (green) immunolabeling (IL) in the epidermis (Ep) and upper dermis (UD) biopsies of normal human palmar glabrous skin ( A , Abcam anti-Nav1.7) and normal human plantar glabrous skin ( B , Alomone anti-Nav1.7). Stratum corneum, sc; stratum granulosum, sg; stratum spinosum (ss); stratum basalis (sb), dermal papilla (dp). Straight arrows indicate epidermal sensory endings, curved arrows indicate small nerves and individual axons or endings in the upper dermis. The areas enclosed in the large rectangles are 2X enlargements of those in the smaller rectangles. Of all the innervation revealed by anti-PGP 9.5, only some express Nav1.7-IL (yellow straight and curved arrows) whereas other only express PGP 9.5-IL (green straight and curved arrows). Aβ-fiber innervation of a Meissner corpuscle (MC) has little if any Nav1.7-IL. Kertinocytes especially in stratum granulosum label for Nav1.7 (arrowheads) which has a more membranous distribution with the Alomone anti-Nav1.7 antibody, but more diffuse labeling with the Abcam anti-Nav1.7 antibody. Scale bar = 100 μm.

Article Snippet: Three affinity purified antibodies generated to different amino acid sequences in rat and human Nav1.7 were utilized in these studies: rabbit polyclonal Nav1.7 Al : Alomone Labs, ASC-008, rat 446–460 aa sequence, 1:100; rabbit polyclonal Nav1.7 Y : Y083, [ ], rat 514–532 aa sequence, 1:250; and rabbit polyclonal Nav1.7 Ab : Abcam Inc, ab85167, human 1000–1100 aa sequence, 1:500.

Techniques: Fluorescence, Immunolabeling, Labeling

Journal: Basic Research in Cardiology

Article Title: Inhibition of Na V 1.8 prevents atrial arrhythmogenesis in human and mice

doi: 10.1007/s00395-020-0780-8

Figure Lengend Snippet: Expression of Na V 1.8 in the human atrium. Data are presented as scatter plot with mean ± SEM. P values were calculated using unpaired Student's t test. a Original Western blot for Na V 1.8 expression in atrial myocardium from patients with sinus rhythm compared to ventricular myocardium from non-failing donors (NF). b Normalized densitometry data comparing the protein expression of Na V 1.8 in human atria ( n = 6) and human ventricle ( n = 5). GAPDH was used as an internal loading control in all blots. c Normalized mRNA expression of Na V 1.8/GAPDH in human atrial myocardium ( n = 7 patients) compared to ventricular myocardium from healthy subjects ( n = 10 patients). d Original Western Blot for Na V 1.8 and Na V 1.5 protein in human atria from patients with sinus rhythm (SR) or atrial fibrillation (AF). e Normalized densitometry data from Western Blots using atrial myocardium from patients with SR or AF showing the protein expression of Na V 1.5 (SR: n = 14 patients, AF: n = 13) and ( f ) Na V 1.8 (SR: n = 14 patients, AF: n = 14). GAPDH was used as an internal loading control in all blots. g Normalized mRNA expression of Na V 1.5/GAPDH (SR: n = 8 patients, AF: n = 8) and Na V 1.5/GAPDH (SR: n = 8 patients, AF: n = 8) in human atrial myocardium from SR compared to AF

Article Snippet: Mouse monoclonal anti-Na V 1.8 antibodies (1:1,000, LSBio, LS-C109037), rabbit polyclonal anti-Na V 1.5 (1:2,000, Alomone labs, ASC-005), and mouse monoclonal anti-GAPDH (1:20,000, BIOTREND, BTMC-A473-9) were used.

Techniques: Expressing, Western Blot

Journal: Pain

Article Title: The role of Na v 1.7 in human nociceptors: insights from human induced pluripotent stem cell–derived sensory neurons of erythromelalgia patients

doi: 10.1097/j.pain.0000000000001511

Figure Lengend Snippet: Functional sensory neurons are generated from iPS cells. (A) Differentiation scheme of iPS cells into sensory neurons with dual-SMAD inhibition (LDN193189 and SB431542), VEGF/FGF/PDGF inhibition (SU5402), Notch inhibition (DAPT), and WNT activation (CHIR99021) for 10 days (d0-d10), followed by growth factor (NGF, BDNF, and GDNF)-driven neuron maturation for 8 weeks. On maturation day M35 and M55, neurons were used for analysis. (B) Representative phase-contrast and immunofluorescence images of iPS cell–derived neurons expressing peripherin (green) and TUJ-1 (red) of IEM 1. Scale bar 100 µm. (C and D) Representative immunofluorescence images of neurons from IEM 1 stained positive for Na V 1.8 (C) (see also Supplementary Fig. S2, available at http://links.lww.com/PAIN/A749 ) and TRPV1 (D) (both red) and TUJ-1 (green). Nuclei were counterstained with DAPI (blue). Scale bar 100 µm. (E) Representative calcium imaging recording performed on iPS cell–derived neurons from clone IEM 1. Neurons were stimulated with capsaicin (1 µM), AITC (100 µM), menthol (100 µM), pH 6.0, and KCl (60 mM) for 30 seconds each as indicated. Response profiles of 18 cells are overlaid. For quantification, see Supplementary Figure S3 (available at http://links.lww.com/PAIN/A749 ). (F) Representative recording of an IEM 1 neuron. Traces display the neuron's total (left) and TTX-r (right) currents, showing functional expression of TTX-r Na V channels. See also Table . (G) Representative recording of an IEM 1 neuron, showing functional expression of Na V 1.7. Total Na V current is shown before (black) and after (red) 20 minutes of application of the Na V 1.7-specific blocker ProTx-II (5 nM). Data shown in (B–E) were gathered on maturation day M35. IEM, inherited erythromelalgia; iPS cell, induced pluripotent stem cell.

Article Snippet: Induced pluripotent stem cell–derived neurons were stained with anti-peripherin (clone sc-7604; Santa Cruz Biotechnology), anti-β-III-tubulin (clone TUJ-1; R&D Systems), anti-Na V 1.8 (clone ASC-016, 1:1000, see Supplementary Fig. S2, available at http://links.lww.com/PAIN/A749 ), and anti-TRPV1 (clone ACC-030, 1:1000; all from Alomone Labs, Jerusalem, Israel).

Techniques: Functional Assay, Generated, Inhibition, Activation Assay, Immunofluorescence, Derivative Assay, Expressing, Staining, Imaging

Journal: Pain

Article Title: The role of Na v 1.7 in human nociceptors: insights from human induced pluripotent stem cell–derived sensory neurons of erythromelalgia patients

doi: 10.1097/j.pain.0000000000001511

Figure Lengend Snippet: The IEM mutation changes action potential characteristics. (A) Representative action potentials recorded in IEM 2 (left) and Ctrl 2 (right) nociceptors. Action potential thresholds for each particular neuron are indicated. Dotted line represents 0 mV. (B) The RMP was −47.7 ± 2.4 mV for IEM and −39.3 ± 3.3 for Ctrl nociceptors (n = 24 and 22; P = 0.05 unpaired t test: t = 2.07, df = 44). (C) The action potential threshold was significantly lowered in IEM nociceptors (−45.9 ± 0.6 mV in IEM and −40.1 ± 2.8 mV in Ctrl; n = 23 and 21; P = 0.02, Mann–Whitney test). (D) IEM nociceptors display a stronger hyperpolarization after each action potential (−61.2 ± 0.8 mV for IEM and −45.9 ± 3.7 mV for Ctrl; n = 23 and 21; P < 0.0001, Mann–Whitney test). (E) IEM action potentials had a mean amplitude of 120.9 ± 1.7 mV, compared with 103.1 ± 4.3 mV in Ctrl nociceptors (n = 23 and 21; P = 0.004, Mann–Whitney test). (F) Action potentials in IEM nociceptors had a mean half-width of 2.3 ± 0.6 ms, compared with 8.5 ± 3.8 ms in Ctrl nociceptors (n = 23 and 21; P = 0.02, Mann–Whitney test). (G) The time from pulse onset to the action potential peak is significantly shorter in IEM nociceptors (28.6 ± 2.4 ms, compared with 56.8 ± 11.8 ms in Ctrl; n = 23 and 21; P = 0.019, unpaired t test: t = 2.44, df = 42). (H) Nociceptors for IEM patients have a steeper maximum slope of the action potential upstroke (223.7 ± 13.2 V/s, compared with 136.3 ± 29 V/s in Ctrl; n = 23 and 21; P = 0.007, unpaired t test: t = 2.83, df = 42). (I) The slope of the subthreshold depolarization is not different between IEM and Ctrl nociceptors (1.5 ± 0.1 V/s for IEM vs 1.8 ± 0.3 V/s for Ctrl; n = 23 and 21; P = 0.8, Mann–Whitney test). (J) Action potential (AP) frequency induced by increasing stepwise current injections (n = 14 for IEM and n = 5 for Ctrl; note that some traces are overlaid). None of the measured Ctrl nociceptors fired more than 2 action potentials. The current injection protocol is identical to the one in Supplementary Fig. S5b (available at http://links.lww.com/PAIN/A749 ). Neurons were held at an RMP around −70 mV and depolarized by square current injections in steps of 0.5 × rheobase to evoke action potential firing. IEM, inherited erythromelalgia; RMP, resting membrane potential.

Article Snippet: Induced pluripotent stem cell–derived neurons were stained with anti-peripherin (clone sc-7604; Santa Cruz Biotechnology), anti-β-III-tubulin (clone TUJ-1; R&D Systems), anti-Na V 1.8 (clone ASC-016, 1:1000, see Supplementary Fig. S2, available at http://links.lww.com/PAIN/A749 ), and anti-TRPV1 (clone ACC-030, 1:1000; all from Alomone Labs, Jerusalem, Israel).

Techniques: Mutagenesis, MANN-WHITNEY, Injection, Membrane

Journal: Pain

Article Title: The role of Na v 1.7 in human nociceptors: insights from human induced pluripotent stem cell–derived sensory neurons of erythromelalgia patients

doi: 10.1097/j.pain.0000000000001511

Figure Lengend Snippet: Voltage-clamp protocol to measure TTX-s currents of iPS cell–derived nociceptors. (A–F) Example recordings from a Ctrl 2 nociceptor explain the protocols used in voltage-clamp recordings on iPS cell–derived cells. (A) Shown is the prepulse protocol, used to eliminate space clamp artifacts in this particular Ctrl 2 neuron. The protocol consisted of a short prepulse (generally 4-6 ms, −50 to −15 mV), followed by a repolarizing interpulse (generally 1 ms, −120 to −70 mV), followed by the regular testpulse (40 ms, −80 to +40 mV, 10 mV increments). Prepulse and interpulse voltage and duration were adjusted to each cell individually to eliminate artefacts (F) and obtain optimal voltage-clamp conditions. When tested in Na V 1.7-expressing HEK293 cells, such prepulse protocols did not affect voltage dependence of activation of Na V 1.7 and provided measurable current amplitudes during the testpulse (Supplementary Fig. S6, available at http://links.lww.com/PAIN/A749 ). (B) Total current measured in this particular neuron using the protocol shown in (A) before application of TTX. (C) TTX-resistant (TTX-r) current obtained from the same neuron after application of 500 nM TTX. (D) The TTX-sensitive (TTX-s) current component was calculated by subtracting the TTX-r current (C) from the total current (B). (E) Current–voltage (IV) relationships of the same neuron, obtained from the total current (black) shown in (B), the TTX-r current (blue) shown in (C), and the TTX-s current (red) shown in (D). The IV relationships confirm accurate voltage-clamp conditions, achieved by the prepulse protocol shown in (A). Incomplete inactivation of Na V s during the interpulse (here −70 mV) prevents current from reaching zero before the onset of the test pulse (see also B). (F) A regular voltage protocol without prepulse (40 ms, −80 to 40 mV, 10 mV increments) was run on the same neuron at the end of the recording after TTX had already been applied. The recorded TTX-r current shows bad voltage clamp and components from multiple cells, visible as delayed inward current peaks (red and blue with arrows). This confounds current–voltage analysis. iPS cell, induced pluripotent stem cell.

Article Snippet: Induced pluripotent stem cell–derived neurons were stained with anti-peripherin (clone sc-7604; Santa Cruz Biotechnology), anti-β-III-tubulin (clone TUJ-1; R&D Systems), anti-Na V 1.8 (clone ASC-016, 1:1000, see Supplementary Fig. S2, available at http://links.lww.com/PAIN/A749 ), and anti-TRPV1 (clone ACC-030, 1:1000; all from Alomone Labs, Jerusalem, Israel).

Techniques: Derivative Assay, Expressing, Activation Assay

Journal: Pain

Article Title: The role of Na v 1.7 in human nociceptors: insights from human induced pluripotent stem cell–derived sensory neurons of erythromelalgia patients

doi: 10.1097/j.pain.0000000000001511

Figure Lengend Snippet: Proposed distribution of Na V channels during action potential generation. Top: schematic representations of action potentials from wild-type (WT, left) or Na V 1.7/I848T (right) nociceptors. The individual phases of the action potential are shaded in different colors to match the assumed contribution of different Na V isoforms to each particular phase. Bottom: schematic activation curves for the different Na V channel categories, based on the data presented in this study. We propose that in the wild-type (left panel) Na V 1.1, 1.2, 1.3, 1.6, and 1.9 (purple) contribute mainly to subthreshold depolarizations because their voltage dependence is very hyperpolarized. Na V 1.7 (red) has a more depolarized activation curve and defines the action potential threshold but also contributes to the upstroke. Na V 1.8 (blue) with its very depolarized activation contributes mainly to the action potential upstroke. In patients carrying the IEM mutation Na V 1.7/I848T (right panel), activation of Na V 1.7 is crucially shifted towards negative potentials. This leads to more synchronized activation of Na V 1.7 and other TTX-s Na V s (note the vicinity of the red and purple activation curves). This synchronized activity leads to a lowering of the action potential threshold and to a speeding up of the upstroke. It is possible that Na V 1.2 and 1.3 now also contribute to the action potential upstroke in addition to Na V 1.7 and 1.8. This further enhances slope and amplitude of the action potential upstroke. Taken together, action potentials in IEM (I848T) patients are faster and larger and display particular changes, summarized on the right. Comparing the 2 graphs of activation curves (bottom panel), please note the shorter X-axis in the bottom right graph. We do not propose a shift of either the purple or the blue (Na V 1.8) activation curve. IEM, inherited erythromelalgia.

Article Snippet: Induced pluripotent stem cell–derived neurons were stained with anti-peripherin (clone sc-7604; Santa Cruz Biotechnology), anti-β-III-tubulin (clone TUJ-1; R&D Systems), anti-Na V 1.8 (clone ASC-016, 1:1000, see Supplementary Fig. S2, available at http://links.lww.com/PAIN/A749 ), and anti-TRPV1 (clone ACC-030, 1:1000; all from Alomone Labs, Jerusalem, Israel).

Techniques: Activation Assay, Mutagenesis, Activity Assay