Title page
Copyright page
Dedication
Foreword
Preface
Acknowledgments
I Dissecting God’s Megaphone
1. 1 Dissecting God’s Megaphone: The Search for a Pain Gene
2. 2 Sherrington’s Enchanted Loom and Huxley’s Science Fiction
II Chasing Men on Fire: The Search
III Beyond the Search: Expanding Horizons
IV Muting God’s Megaphone: From the Squid toward the Clinic
Glossary
Index

List of Tables

Table 2.1 Peripheral Sodium Channels
Table 1 Patient Baseline Characteristics
Table 2 Intensity of Pain (NRS: 0–10) before Surgery and after 1, 3, and 6 Months
Table 1 Clinical Description of Patients with Small Nerve Fiber Neuropathy and SCN9A Mutations
Table 2 SFN Symptoms Inventory Questionnaire Findings in Patients with SCN9A Novel Mutations
Table 1 Clinical Phenotype of IEM Subjects

List of Illustrations

Figure 1.1 Drawing, entitled A Constant Battle, submitted to The Erythromelalgia Association for their 2012 art contest, depicting the pain of erythromelalgia. Reproduced with permission of Jennifer Beech and The Erythromelalgia Association.
Figure 2.1 Nerve impulse (action potential) from a dorsal root ganglion (DRG) neuron, shown in green. Until it is stimulated, the neuron is quiescent and sits at resting membrane potential (RMP) with the inside of the cell negative by about –60 millivolts with respect to the outside. When the cell is depolarized by a sufficient amount, it reaches threshold, and, at that point, there is an explosive, nearly simultaneous activation of many sodium channels, producing a pulse-like depolarization of the cell membrane which actually crosses 0 millivolts, so that the inside of the cell is briefly positive before the cell repolarizes and returns to resting potential. The action potential, which always has the same configuration and time course in any given cell, lasts about 1 millisecond. Na_V1.7 sodium channels play a particularly important role in DRG neurons. They act within the subthreshold domain, below threshold, to amplify small depolarizing stimuli (blue). Acting in this way, Na_V1.7 channels determine the sensitivity, or “set the gain,” on DRG neurons. Modified from Rush et al. (2007).
Figure 2.2 Dorsal root ganglion (DRG) neurons, with cell bodies within the dorsal root ganglia, extend an axon from the body surface and organs, all the way into the spinal cord. Sodium channels within the cell membrane of DRG neurons enable them to produce action potentials (APs). Pain-signaling DRG neurons are excited by dangerous levels of pressure, heat, cold, acidity (pH), or irritating chemicals and, in response, send action potentials to the spinal cord, which relays them to the brain. Multiple types of sodium channels, shown in orange, red, and green, participate in this signaling. Na_V1.7 channels (green) play a particularly crucial role, amplifying small stimuli in the periphery and thereby setting the gain on DRG neurons, and facilitating impulse transmission close to the spinal cord. Modified from Waxman and Zamponi (2014).
Figure 2.3 Inappropriate repetitive firing of action potentials, recorded with a microelectrode from a single axon within the sciatic nerve of a rat that had received a nerve injury one year previously. The aberrant repetitive action potentials sit upon an abnormal depolarization of the axon membrane which suggests abnormal sodium channel activity. From Kocsis and Waxman (1983).
Figure 2.4 Microelectrode recording from a single axon within the sural nerve of a patient with a painful peripheral neuropathy. The nerve was biopsied for diagnostic evaluation. Aberrant repetitive action potentials can be seen, arising from an abnormal depolarization suggesting abnormal sodium channel activity within the axon membrane. From Kocsis and Waxman (1987).
Figure 2.5 Atomic-level model of the Na_V1.7 sodium channel. The green, salmon, purple, and blue spirals show the course of the channel protein as it weaves in and out of the cell membrane within four different parts (domains) of the Na_V1.7 channel. Single amino acids can be seen in red and gold. The top diagram shows a side view of the channel, as seen by an observer within the membrane. The bottom diagram shows the channel as seen from within the cell, looking out. Just above the yellow amino acid, the pore in the center of the channel can be seen. From Yang et al. (2012).
Figure 3.1 A drawing depicting the pain of erythromelalgia, entitled “Chained to Fire,” prepared by Bailey Deacon when she was fourteen years old, and submitted to an art contest sponsored by The Erythromelalgia Association in 2012. As in many patients with erythromelalgia, the pain is most severe in the feet. Reproduced courtesy of Bailey Deacon, Todd Deacon, and The Erythromelalgia Association.
Figure 1 The I848T and L858H mutations of hNa_V1.7 alter activation and deactivation. (A) Current traces recorded from representative HEK293 cells expressing either wild-type hNa_V1.7 or mutant channels, I848T or L858H. Cells were held at −100 mV, and currents were elicited with 50 msec test pulses to potentials ranging from −80 to 40 mV. (B) Normalized peak current–voltage relationship for wild-type (filled squares; n = 29), I848T (open circles; n = 27), and L858H (open triangles; n = 27) channels. (C) Representative tail currents of WT, I848T, and L858H channels. Cells were held at −100 mV and depolarized to −20 mV for 0.5 msec, followed by a repolarization to −50 mV to elicit tail currents. (D) Time constants for tail current deactivation at repolarization potentials ranging from −40 to −100 mV for wild-type (filled squares; n = 7), I848T (open circles; n = 7), and L858H (open triangles; n = 7) hNa_V1.7 channels. Time constants were obtained with single exponential fits to the deactivation phase of the currents. Error bars represent SE.
Figure 2 The I848T and L858H mutations differentially alter inactivation of hNa_V1.7. (A) Fast inactivation kinetics as a function of voltage for wild-type (filled squares; n = 8), I848T (open circles; n = 8), and L858H (open triangles; n = 8) hNa_V1.7 channels. Currents elicited as described in figure 1A were fit with Hodgkin–Huxley type m³h model to estimate the inactivation time constants. (B) Comparison of steady-state fast inactivation for wild-type (filled squares; n = 20), I848T (open circles; n = 19), and L858H (open triangles; n = 17) hNa_V1.7 channels. Currents were elicited with test pulses to 0 mV after 500 msec inactivating prepulses. (C) Comparison of steady-state slow inactivation for wild-type hNa_V1.7 (filled squares; n = 9), I848T (open circles; n = 8), and L858H (open triangles; n = 9) hNa_V1.7 channels. Slow inactivation was induced with 30 sec prepulses, followed by 100 msec pulses to −120 mV to allow recovery from fast inactivation. A test pulse to 0 mV for 20 msec was used to determine the fraction of current available. Error bars represent SE.
Figure 3 The I848T and L858H mutations enhance ramp currents of hNa_V1.7. Representative ramp currents elicited with 500 msec ramp depolarizations from −100 to 0 mV from HEK293 cells expressing wild-type, I848T, and L858H channels.
Figure 1 Family pedigree. Circles denote females; squares denote males. The proband is shown by an arrow. Blackened symbols indicate subjects affected with erythromelalgia. (+) denotes subjects heterozygous for the T4393G mutation; (–) denotes subjects without the mutation.
Figure 2 F1449 (arrow) is conserved within loop 3 in all known sodium channels. The lower line delineates the sequence of transmembrane segment S6 of domain III and the N-terminal half of L3 for Na_V1.1–Na_V1.9, together with the F1449V mutation (Na_V1.7 m). The asterisk denotes the position of V1293 which is replaced with isoleucine (I) in the skeletal muscle disorder paramyotonia congenita (Green et al., 1998). The fast inactivation tripeptide IFM is underlined.
Figure 3 The F1449V mutation alters activation but not deactivation of hNa_V1.7. (A) Current traces recorded from representative HEK293 cells expressing either wild-type (left) or F1449V (right) channels. Cells were held at –100 mV and currents elicited with 50 ms test pulses to −80 to +40 mV. (B) Normalized peak current–voltage relationship for wild-type (filled squares, n = 11) and F1449V (open circles, n = 12) channels. (C) Time constants for tail current deactivation at repolarization potentials from −40 to −100 mV for wild-type (filled squares, n = 8) and F1449V (open circles, n = 7) channels. Time constants were obtained with single exponential fits. Error bars show standard errors.
Figure 4 The F1449V mutation differentially alters fast and slow inactivation of hNa_V1.7. (A) Comparison of steady-state fast inactivation for wild-type (filled squares, n = 13) and F1449V (open circles, n = 14) channels. Currents were elicited with test pulses to 0 mV following 500 ms inactivating pre-pulses. The voltage dependence of activation, derived by fitting Boltzmann functions to the data shown in figure 3, is shown for wild-type (closed triangles) and F1449V (open triangles). (B) Comparison of steady-state slow inactivation for wild-type (filled squares, n = 4) and F1449V (open circles, n = 4) channels. Slow inactivation was induced with 30 s pre-pulses, followed by 100 ms pulses to −120 mV to allow recovery from fast inactivation. A 20 ms test pulse to 0 mV was used to determine the fraction of current available. (C) Fast inactivation kinetics as a function of voltage for wild-type (filled squares, n = 7) and F1449V (open circles, n = 8) channels. The decay phases of currents elicited as described in figure 3A were fitted with single exponentials to estimate open state inactivation time constants. (D) Time constants for development of closed state inactivation were estimated from single exponential fits to time courses measured at inactivation potentials from −90 to −50 mV for wild-type (filled squares, n = 6) and F1449V channels (open circles, right; n = 9), by holding cells at −100 mV, pre-pulsing to the inactivation potential for increasing durations, then stepping to 0 mV to determine the fraction of current inactivated during the pre-pulse. Development of closed state inactivation for F1449V currents is faster than for wild-type currents.
Figure 5 Recovery from inactivation kinetics is faster for F1449V mutant channels than for wild-type channels. (A) Time constants for recovery from inactivation of wild-type (filled squares, n = 8) and F1449V (open circles, n = 8) currents are shown as a function of voltage. Time constants were estimated from single exponential fits to time courses measured at recovery potentials from −140 to −60 mV, by pre-pulsing the cell to +20 mV for 20 ms to inactivate all of the current, then stepping back to the recovery potential for increasing recovery durations prior to the test pulse to 0 mV. The maximum pulse rate was 0.5 Hz. (B) Representative ramp currents, elicited with 500 ms ramp depolarizations from −100 to 0 mV for wild-type and mutant F1449V. The increase in ramp current amplitude, seen for the previously described I848T mutation (Cummins et al., 2004), is not observed.
Figure 6 F1449V, expressed in small DRG neurons, lowers the current threshold for action potential generation and repetitive firing. Action potentials were evoked using depolarizing current injections from a membrane potential of –60 mV. (A) Representative traces from a cell expressing wild-type Na_V1.7, showing subthreshold responses to 50–65 pA current injections and subsequent all-or-none action potentials evoked by injections of 130 pA (current threshold for this neuron) and 155 pA. (B) In contrast, in a cell expressing F1449V, action potentials were evoked by a 60 pA current injection, demonstrating a lower current threshold for action potential generation. The voltage for takeoff of the all-or-none action potential (dotted line) was similar for the neurons in (A) and (B). (C and D) The frequency of firing at graded stimulus intensities was higher for cells expressing F1449V mutant channels than with wild-type channels. (C) The firing of a neuron expressing hNa_V1.7 (same neuron as in A), which responded to a 950 ms stimulation of 150 pA with two action potentials. In contrast, D shows that, in a neuron expressing the mutant channel F1449V (same cell as in B), an identical 150 pA depolarizing stimulus evoked high frequency firing. (E) There is a significant (*P < 0.05) reduction in current threshold in cells expressing F1449V (n = 19) compared with cells expressing wild-type Na_V1.7 (n = 16). (F) There is a significant increase in the frequency of firing in response to 100 and 150 pA stimuli (950 ms) following expression of F1449V (n = 12), in comparison with wild-type Na_V1.7 (n = 11, 9) (**P < 0.01; *P < 0.05).
Figure 1 Domain structure of Na_V1.7 and locations of characterized mutations in Na_V1.7-related pain disorders. (a) The sodium channel α-subunit is a long polypeptide that folds into four homologous domains (DI–DIV), each of which consists of six transmembrane segments (S1–S6). The four domains are joined by three loops (L1–L3). Within each domain, S1–S4 comprise the voltage-sensing domain (VSD; S4, depicted in green, characteristically contains positively charged arginine and lysine residues), and S5–S6 and their extracellular linker comprise the pore module (PM). The linear schematic of the full-length channel shows the locations of amino acids affected by the gain-of-function SCN9A mutations that are linked to inherited erythromelalgia (IEM; red symbols), paroxysmal extreme pain disorder (PEPD; grey symbols), and small-fibre neuropathy (SFN; yellow symbols). (b) View of the folded Na_V1.7 from the intracellular side of the membrane based on the recently determined crystal structure of a bacterial sodium channel.³ The structure shows the central ion-conducting PM and four peripheral VSDs. Conformational changes in the VSDs in response to membrane depolarization are transmitted to the PMs through the S4–S5 linkers (identified by arrows through the helices). Mutations that seem distant from each other on the linear model can in fact be in close proximity to each other in the more biologically relevant folded structure. *The patient with this mutation showed symptoms common to IEM and SFN. ^‡The patient carrying this mutation showed symptoms and channel properties common to both IEM and PEPD. ^§This substitution is encoded by a polymorphism that was present in approximately 30% of ethnically matched Caucasian individuals of European descent in a control population.⁸¹ Part a is modified, with permission, from ref. 37 © (2007) Elsevier Science.
Figure 2 Pain signal transmission from peripheral terminals of DRG neurons that form synapses onto second-order neurons within the spinal cord. Dorsal root ganglion (DRG) neurons can be broadly classified into three types based on their soma size and the state of myelination of their axons: large diameter with heavily myelinated and rapidly conducting axons (Aβ-fibres; not shown here for simplicity); medium diameter with thinly myelinated and intermediate conducting axons (Aδ-fibres; cyan); and small diameter with unmyelinated and slowly conducting axons (C-fibres; red). Five voltage-gated sodium channels are reported to be expressed in DRG neurons,¹³ with Na_V1.7 expressed in the majority of small unmyelinated neurons and in a notable population of medium and large diameter myelinated neurons (see middle panel; Na_V1.7 expression is shown in red in this and other panels). Signals originating from the periphery are initiated by external stimuli (for example, thermal, mechanical or chemical stimuli) or injury- and inflammation-induced mediators (for example, cytokines or trophic factors), and are transduced by specific G protein-coupled receptors or acid- and ligand-gated ion channels at peripheral termini. Membrane depolarizations evoked by the graded receptor potential are integrated by voltage-gated sodium channels; when a threshold is reached, an action potential is initiated at these terminals and centrally propagated. Na_V1.7 extends to the peripheral ends of these terminals (left panel) where it amplifies small depolarizing inputs. Although Na_V1.7 is considered a peripheral sodium channel because it is expressed in peripheral neurons, it is present in central axonal projections of DRG neurons and their presynaptic terminals within the dorsal horn (right panel) where it may facilitate impulse invasion or evoked release of neurotransmitters that may include substance P, calcitonin-gene related peptide and glutamate.
Figure 3 Biophysical properties of wild-type and mutant Na_V1.7 channels. (a) Inherited erythromelalgia (IEM)-related SCN9A mutations shift the activation of Na_V1.7 in a hyperpolarized direction, allowing the mutant channels to open in response to a weaker depolarization than open wild-type (WT) channels. A comparison of the activation of WT and P1308L mutant Na_V1.7 channels⁷³ shows that the latter exhibits a hyperpolarizing shift (–9.6 mV) in activation. (b) Activation of Na_V1.7 boosts small, slow depolarizations, producing ramp currents. The ramp currents produced by the IEM I136V mutant Na_V1.7 channel,¹⁴⁰ normalized to maximal peak currents elicited by step depolarizations, are markedly increased compared with the ramp currents for WT Na_V1.7 channels. (c) The slow-inactivated state of Na_V1.7 makes these channels unavailable for further opening after they have been activated by sustained (>10 s) membrane depolarization. Mutations in SCN9A that impair slow inactivation (such as N395K and I739V) increase the firing rate of dorsal root ganglion (DRG) neurons.⁶⁷^,¹⁴¹ Error bars represent standard error of the mean. (d) Fast inactivation is a process that transiently makes Na_V1.7 unavailable for further opening after it has been activated by relatively short (100–500 ms) depolarizations. A hallmark of paroxysmal extreme pain disorder (PEPD)-related SCN9A mutations is that they cause a depolarizing shift in fast inactivation that results in fewer inactivated channels at any given potential, and resistance of a subpopulation of channels to inactivation. The PEPD G1607R mutant Na_V1.7 channel⁷⁰ shows a –30 mV depolarizing shift in fast inactivation, and the presence of a subpopulation of channels that resist inactivation (represented by orange shading in the graph). Error bars represent standard error of the mean. (e) Normalized current traces for WT and G1607R Na_V1.7 evoked by a depolarizing pulse to 0 mV show the transient current (I_Na-trans) and that the mutant channels retain a persistent current (I_Na-per) at the end of a 100 ms depolarizing pulse (represented by orange shading in the graph). (f) Resurgent currents (I_Na-res) are triggered by repolarization following a strong depolarization, and support burst firing. Note the increase in resurgent current recorded from DRG neurons expressing the M932L/V991L Na_V1.7 variant from a patient with small-fibre neuropathy.³⁹ Impaired fast- and slow-inactivation and resurgent currents are manifested by PEPD and SFN channel variants, as indicated in the main text, and the panels in this figure should be regarded as examples of these changes. Part a is modified from ref. 73. Part b is modified from ref. 140. Part c is modified, with permission, from ref. 67 © (2007) The Physiological Society. Parts d and e are modified, with permission, from ref. 70 © (2011) Macmillan Publishers Limited. All rights reserved. Part f is modified, with permission, from ref. 39 © (2012) American Neurological Association.
Figure 4 The F1449V mutation in Na_V1.7 makes DRG neurons hyperexcitable. (a,b) Representative traces from small (<30 μm) dorsal root ganglion (DRG) neurons expressing wild-type (WT) Na_V1.7 or Na_V1.7 with the F1449V mutation (the variant linked to inherited erythromelalgia). These traces show that neurons expressing the mutant channel have a lower current threshold for action potential generation. (c) The average current threshold is notably reduced in cells expressing F1449V compared with cells expressing WT channels (*P <0.05). (d,e) A neuron expressing WT Na_V1.7 responds to a 950 ms stimulation of 150 pA with a lower number of action potentials than does the neuron expressing the F1449V mutant (same cells as in panels a and b). (f) There is a sizeable increase in the frequency of firing of action potentials in response to 100 pA and 150 pA stimuli (950 ms) with expression of F1449V versus expression of WT Na_V1.7 (*P <0.05; **P <0.01). Figure is reproduced, with permission, from ref. 94 © (2005) Oxford University Press.
Figure 5 A model of the putative activation gate of Na_V1.7. The folded structure of the two S6 transmembrane segments presented here were based on the crystal structure of a bacterial sodium channel.³ The carboxy-terminal aromatic residue of each S6 is shown in stick representation for wild-type (WT; a) Na_V1.7 and Na_V1.7 with the F1449V mutation (b). The assembly of aromatic residues at the cytoplasmic C terminus of each of the S6 segments forms the putative activation gate of Na_V1.7. The F1449V mutation in the homologous domain III (DIII) disrupts the hydrophobic ring and destabilizes the pre-open state of the channel.
Figure 5.1 Scheme by which Na_V1.7 mutations, which depolarize resting membrane potential, produce hyperexcitability in dorsal root ganglion (DRG) neurons and hypoexcitability in sympathetic ganglion neurons. Note the enhanced action potential activity below the DRG neuron expressing the Na_V1.7 mutation, in contrast to the electrical silence of the sympathetic ganglion neuron.
Figure 1 L858H renders DRG neurons hyperexcitable. (A and B) Action potentials were evoked from small (≤ 25 µm in diameter) DRG neurons by using depolarizing current injections from the RMP. V_m, membrane potential. (A) Representative traces from a cell expressing WT Na_V1.7 show subthreshold responses to 50- to 130-pA current injections and subsequent all-or-none action potentials evoked by injections of 135 pA (current threshold for this neuron) and 155 pA. (B) In contrast, in a cell expressing L858H, action potentials were evoked by a 60-pA current injection. The voltage for take-off of the all-or-none action potential (approximately –14.5 mV, dashed line) was similar for the neurons in A and B. (C) L858H causes a depolarizing shift in the RMP of DRG neurons. DRG neurons expressing WT Na_V1.7 had an average RMP of –50.1 ± 0.9 mV (n = 20), whereas those expressing L858H mutant channels had a significantly (*, P < 0.001) depolarized RMP of –44.9 ± 1.1 (n = 25). (D) The average current threshold for action potential firing of DRG neurons expressing WT Na_V1.7 channels was 120.6 ± 23.9 pA (n = 20), whereas that of neurons expressing L858H mutant channels was significantly (*, P < 0.01) reduced to 69.2 ± 9.8 pA (n = 25). (E) Action potential overshoot in cells expressing WT Na_V1.7 channels (67.8 ± 3.0 mV, n = 20) was not significantly different from that in cells expressing L858H mutant channels (64.4 ± 2.6 mV, n = 20; P > 0.05). The voltage of action potential take-off was unchanged (WT, –14.5 ± 1.2 mV, n = 20; L858H, –14.5 ± 1.3 mV, n = 25; P > 0.05). n.s., not significant.
Figure 2 L858H renders SCG neurons hypoexcitable. (A and B) Action potentials were evoked by using depolarizing current injections from resting potential. (A) Representative traces from a cell expressing the WT channel show subthreshold responses to 15- to 20-pA current injections and subsequent all-or-none action potentials evoked by injections of ≥25 pA. (B) In contrast, in a cell expressing the L858H channel, action potentials required a ≥70-pA current injection. The voltage for take-off (dashed line) of the all-or-none action potential was unchanged. (C) L858H channels caused a depolarizing shift in the RMP of SCG neurons. SCG neurons expressing WT channels had an average RMP of –46.3 ± 0.8 mV (n = 15), whereas those expressing L858H had a significantly (P < 0.001) depolarized RMP of –41.6 ± 0.8 (n = 17). (D) The average current threshold for action potential firing of SCG neurons expressing WT channels was 22.7 ± 3.6 pA (n = 15), whereas that of neurons expressing L858H channels was significantly (*, P < 0.01) increased to 42.9 ± pA (n = 17). (E) Action potential overshoot in cells expressing WT channels (47.8 ± 3.4 mV, n = 15) was significantly larger (*, P < 0.001) than that in cells expressing L858H (23.8 ± 4.7 mV, n = 20). The voltage of action potential take-off was unchanged (WT, –23.1 ± 1.2 mV, n = 15; L858H, –19.8 ± 1.3 mV, n = 17; *, P > 0.05).
Figure 3 The L858H mutation increases firing frequency in DRG and decreases firing frequency in SCG neurons. (A) Representative DRG neuron expressing WT Na_V1.7 fires a single action potential in response to a 950-ms input of 100 pA from the RMP of this neuron (approximately –50 mV). (Inset) The same neuron fires multiple action potentials in response to a 250-pA stimulus. (B) Representative DRG neuron expressing L858H fires five action potentials in response to a 100-pA current injection from the RMP of this neuron (approximately –42 mV). (C) For the entire population of DRG neurons studied, the firing frequency evoked by 50-pA current stimuli was 0.32 ± 0.13 Hz after transfection with WT channels (n = 20) and 2.06 ± 0.79 Hz after transfection with L858H (n = 24; *, P < 0.05), and the firing frequency evoked by 100-pA stimuli was 0.89 ± 0.28 Hz after transfection with WT and 3.37 ± 1.13 Hz after transfection with L858H (*, P < 0.05). (D) Representative SCG neuron expressing WT Na_V1.7 fires six action potentials in response to a 950-ms input of 40 pA from the RMP (approximately –45 mV). (E) Representative SCG neuron expressing L858H fires only two action potentials in response to a 100-pA current injection from the RMP (approximately –40 mV). (Inset) When the cell was held at –60 mV to overcome the depolarization of the RMP caused by L858H, it produced four action potentials with an identical stimulus. (F) For the entire population of SCG neurons studied, the firing frequency evoked by 30-pA stimuli was 5.33 ± 1.5 Hz after transfection with WT channels (n = 14) and 0.63 ± 0.01 Hz after transfection with L858H channels (n = 15; P < 0.05). The firing frequency evoked by 40-pA stimuli was 7.05 ± 1.86 Hz after transfection with WT and 1.96 ± 1.0 Hz after transfection with L858H channels (*, P < 0.05).
Figure 4 DRG neurons express Na_V1.7 and Na_V1.8; SCG neurons express Na_V1.7 but not Na_V1.8. (A) Restriction analysis of multiplex PCR amplification products from sodium channel domain 1 from adult DRG (lanes 1–9) and SCG (lanes 10–18). M, 100-bp ladder marker (Promega). Lanes 1 and 10 contain amplification products from DRG and SCG, respectively. Lanes 2–9 and 11–18 show results of cutting this DNA with EcoRV, EcoNI, AvaI, AccI, SphI, BamHI, AflII, and EcoRI, which are specific to subunits Na_V1.1, Na_V1.2, Na_V1.3, Na_V1.5/1.9, Na_V1.6, Na_V1.7/1.8, Na_V1.8, and Na_V1.9 (details can be found in table 1, which is published as supporting information on the PNAS web site). Restriction products in lanes 2 and 5–9 show the presence of Na_V1.1, Na_V1.6, Na_V1.7, Na_V1.8, and Na_V1.9 in DRG, in agreement with previous results.¹⁹ Restriction products in lanes 13, 15, and 16 show the presence of Na_V1.3, Na_V1.6, and Na_V1.7 in SCG. (B and C) Immunostaining of Na_V1.7 and Na_V1.8 channels in DRG and SCG neurons in vivo and in cultured neurons. (B) Na_V1.7 (a) and Na_V1.8 (b) proteins are present in adult DRG neurons in vivo; Na_V1.7 (c) and Na_V1.8 (d) proteins are present in cultured DRG neurons from postnatal day 2 (P2) rat pups. (C) Na_V1.7 (a), but not Na_V1.8 (b), protein is present in adult SCG neurons in vivo; Na_V1.7 (c), but not Na_V1.8 (d), protein is present in cultured SCG neurons from P2 rat pups. (Scale bars, 50 µm.)
Figure 5 Coexpression of L858H and Na_V1.8 channels rescues electrogenic properties in SCG neurons. When Na_V1.8 was coexpressed with L858H, current threshold and action potential overshoot were restored, although the depolarization of the RMP induced by L858H persisted. (A) Suprathreshold action potentials recorded from representative SCG neurons transfected with WT (blue), L858H (red), and L858H plus Na_V1.8 (green) channels. (B) Depolarized RMP in cells with L858H channels (–41.6 ± 0.76 mV, n = 17) was maintained with coexpression of Na_V1.8 (–40.5 ± 1.01 mV, n = 17; P > 0.05). n.s., not significant. (C) Current threshold for action potential firing was reduced from 42.9 ± 6.3 pA (n = 17) for L858H to 26.8 ± 4.3 pA (n = 17) for L858H coexpressed with Na_V1.8 (*, P < 0.05). (D) Action potential overshoot in SCG neurons with L858H channel (23.8 ± 4.7 mV, n = 17) was increased when Na_V1.8 was coexpressed with L858H (41.5 ± 4.6 mV, n = 17; *, P < 0.05).
Figure 1 Kinetic model of Na_V1.7 voltage-gated sodium channel based on Hodgkin–Huxley equations. (A) Voltage dependence of conductance/maximal conductance (G/Gmax) at steady-state for inactivation (squares; n = 8) and activation (circles; n = 17) of Na_V1.7 channel. Solid lines are derived from the following equations: h_∞ = α_h/(α_h + ß_h); (m_∞)³ = [α_m/(α_m + ß_m)]³, where m and h are channel activation and inactivation variables and α and ß are forward and backward rate constants (see Materials and Methods). (B) Voltage dependencies of activation (solid circles; n = 8) and deactivation (open circles; n = 14) time constants. Deactivation of the Na_V1.7 current was fitted with a single exponential, whereas channel activation was fitted with a single exponential raised to the 3rd power. (C) Inactivation (solid squares; n = 13) and removal of inactivation (open squares; n = 7) time constant obtained from a single-exponential fit of the respective data. Inset shows data replotted with time constants on a log scale. (D) Time sequences of m³ and h variables along with the resulting open probability (m³h) obtained in the model response to a series of voltage steps ranged from –60 to 40 mV in 5-mV increments from a holding potential of –110 mV. (E) Rising phase of the Na_V1.7 current (bottom) in response to a –30-mV test voltage (top) was fitted with a single exponential function of different powers. The best fit was obtained using 3rd-power exponential as determined based on the residual (middle); the residual of the 3rd-power exponential fit was not substantially different from the background noise, thus 4th-power exponential had not further improved the fit. (F and G) Na_V1.7 current evoked by test pulses ranged from –50 to –25 mV in 5-mV increments (black) and overlaid on the m³h model (blue) at the respective voltages. Vertical dashed line denotes stimulus onset time. (H) Current traces (top) and the current-voltage (I–V) curve (bottom) of Na_V1.7 current (black) and the m³h model (blue) at the respective test voltages.
Figure 2 Measurement of Na_V1.7 contribution to TTX-sensitive (TTX-S) current in small dorsal root ganglia (DRG) neurons. (A) Representative I–V curve family traces of TTX-S sodium currents recorded from wild-type (WT) and Na_V1.7-knockout (KO) DRG neurons, respectively. (B) TTX-S currents in Na_V1.7-KO DRG neurons are significantly smaller than in WT DRG neurons. ***P < 0.001.
Figure 3 Additional Na_V1.7 conductance lowers current threshold for action potential (AP) generation and increases AP firing probability. (A) 10-ms-long test pulses (top) applied at the original threshold without additional conductance (left) and at the threshold after electronic addition of 50% Na_V1.7 conductance (right) elicited APs (bottom). Control stimulus and AP traces are shown in black, and those after addition of 50% Na_V1.7 conductance are shown in blue. (B) Averages of current threshold (top) and current threshold change (bottom) plotted as a function of additional Na_V1.7 conductance (n = 9). The solid line is a linear regression fit (r² = 0.97, top; r² = 0.97, bottom) of the data. Statistical analysis on the bottom was performed between threshold increments obtained at 12.5% and at the respective percentage of conductance increment. *P < 0.05, **P < 0.01, and ***P < 0.001. (C) APs evoked by a 1-s-long, 10-Hz train of current pulses (10-ms pulse width) applied at the original threshold level (100% native Na_V1.7 conductance). Electronic addition of Na_V1.7 conductance (expressed as the incremental increase over the endogenous Na_V1.7 conductance) is noted on the y-axis. Scale bar, 200 ms. (D) Averages of the number of APs evoked by the protocol described in C plotted as a function of dynamically introduced Na_V1.7 conductance (n = 8).
Figure 4 Removal of Na_V1.7 conductance raises current threshold for AP generation and reduces AP firing probability. (A) APs were evoked by 10-ms-long current pulses applied at 1.5× threshold amplitude in control (Ctrl; black) and after electronic subtraction of the incremental values of Na_V1.7 conductance. Dynamic-clamp subtraction of Na_V1.7 conductance (expressed as the incremental decrease over the endogenous Na_V1.7 conductance) is noted on the y-axis. Scale bar: 200 ms. (B, top) Averages (n = 13) of current threshold change in response to the subtraction of respective proportion of endogenous Na_V1.7 conductance. The solid line is a linear regression fit (r² = 0.99) of the data. Statistical analysis on the top was performed between threshold increments obtained at 12.5% and at the respective percentage of conductance increment. (B, bottom) Averages (n = 15) of the number of APs evoked by the protocol described in A and plotted as a function of dynamically subtracted Na_V1.7 conductance; the solid line is a linear regression fit (r² = 0.98) of the data. **P < 0.01 and ***P < 0.001. (C) AP (top) evoked by a 10-ms-long current stimulus of 1.5× threshold intensity in control (black) and after dynamic-clamp subtraction of 12.5% of Na_V1.7 conductance (blue) and the respective Na_V1.7 model current (bottom). (D) APs (top) evoked by the protocol described above in control (black) and after dynamic-clamp subtraction (middle) of 12.5% of endogenous Na_V1.7 conductance. The I–V phase plot of the model Na_V1.7 conductance dynamically subtracted during neuronal repetitive firing is shown on the bottom. Note that the positive-going (outward) dynamic-clamp current shown in C and D is flipped over 0 line to facilitate comparison of Na_V1.7 current across the manuscript.
Figure 5 Kinetic model of L858H Na_V1.7 channel predicts a substantial enhancement of the persistent sodium current (I_Na) during repetitive firing of DRG neuron. (A) Normalized I–V relationships obtained from WT (blue) and L858H (LH; red) Na_V1.7 channel models. Respective traces of the I_Na = g_maxm³h(V_m – E_Na) model, where V_m is membrane voltage potential and E_Na = 65 mV is sodium reversal potential, used to obtain the I–V plot are presented on top. (B) Comparison of steady-state inactivation and steady-state activation (left), steady-state channel open probability (Po; middle), and activation time constant (τ; right) of the kinetic model of WT (blue) and L858H (red) Na_V1.7 channel. (C, top) The trace shows repetitive firing of a spontaneously active small DRG neuron. The neuron spontaneously fired APs under i=o current-clamp conditions, i.e., with no additional injected current. A small –50-pA constant current was injected to stop AP firing. The trace presented in C, top, was recorded in response to the removal of the stabilizing –50-pA current. (C, bottom) Modeled WT (blue) and L858H (red) Na_V1.7 currents obtained in response to the voltage command shaped in the form of the AP shown in C, top. Current-clamp recordings shown in C, top, were obtained from small DRG neuron in primary culture transfected by electroporation with WT Na_V1.7 (Dib-Hajj et al. 2009). D: I–V phase plots of WT (blue) and L858H (red) Na_V1.7 currents presented in C, bottom. Both models were calculated in a 28-pF equipotential sphere of 1 µF/cm² capacitance; conductance density was set to 0.029 S/cm².
Figure 6 L858H mutant lowers current threshold and enhances AP firing probability of DRG neuron. (A) AP (bottom, left) evoked by 10-ms-long stimulus (on top) of threshold intensity in control (black) and after implementation of 25% (green) and 50% (red) WT-to-L858H substitution ratio (SR); traces of the respective AP rate of change are shown in bottom, right. (B) Trajectory plots of AP rate of change (left) and the respective trajectory of stimulus (right) recorded at incremental levels of WT-to-L858H substitution; data are obtained from APs shown in A. (C) Current threshold (top) and current threshold change (bottom) obtained at different levels of WT-to-L858H conductance substitution in dynamically clamped DRG neuron. Solid line represents linear regression fit of the data (n = 5; top, r² = 0.96; bottom, r² = 0.97). Statistical analysis on the bottom was performed between threshold increments obtained at 12.5% and at the respective percentage of conductance increment. *P < 0.05. (D) APs evoked by a 10-Hz train of 10-ms-long current pulses of threshold intensity in control (black) and after incremental levels (the value is depicted on the left to the y-axis) of WT-to-L858H conductance substitution. Scale bar, 200 ms. (E) Averages (n = 5) of the number of APs evoked by the protocol presented in D at different levels of WT-to-L858H conductance substitution. **P < 0.01.
Figure 7 L858H mutant drives depolarization of resting membrane potential (RMP) of DRG neuron. (A) Dynamic-clamp recordings of RMP (bottom) and WT current (top) of DRG neuron in control and after addition of 100% WT Na_V1.7 conductance. (B) Averages (n = 6) of the RMP (top) and the RMP changes (bottom) as a function of addition or subtraction of the incremental values of WT Na_V1.7 conductance. (C and D) Data description is similar to A and B, but the RMP data were obtained as a response of WT-to-L858H substitution (WT conductance was dynamically subtracted while the equivalent amount of L858H conductance was added) at incremental levels ranging from 12.5 to 100% (n = 6). Statistical analysis on the bottom was performed between RMP changes obtained at 12.5% SR and at the respective percentage of conductance SRs. *P < 0.05 and **P < 0.01.
Figure 8 L858H mutant augments DRG neuron excitability by enhancing sodium influx at subthreshold (subthresh) membrane voltages. (A) AP evoked by a 10-ms-long current pulse of threshold intensity in control (black) and after dynamically introduced +12.5% WT conductance (blue). APs are shown on the top (stimulation protocol is shown on top of APs), and the respective Na_V1.7 current is presented in the bottom. (B) AP repetitive firing (top) evoked by a 10-Hz train of 10-ms-long current pulses at threshold intensity in control (black) and after 12.5% WT addition (blue); dynamic-clamp recording of the 12.5% WT current addition is shown on the bottom. (C and D) Same protocols as shown in A and B, but the comparison is made between data obtained in control (black) and after dynamic-clamp substitution of 12.5% WT to 12.5% L858H conductance. (E) I–V phase plots of dynamic-clamp recordings of repetitive AP firing in DRG neuron presented in B and D at +12.5% WT (blue) and at 12.5% WT to 12.5% L858H substitution (red). (F) Modeled sodium currents were recorded in dynamic-clamp mode and subsequently integrated over 3 different time intervals: (1) from stimulus onset to AP threshold (threshold is defined when 2nd differential of AP changes its sign); this interval extends from arrow 1 to 2 in A and C; (2) from threshold to undershoot; this interval extends between arrows 2 and 3 in A and C; and (3) from undershoot to the next stimulus onset. Sodium charge (pA*ms) was normalized to the peak value of the native Na_V1.7 sodium current measured in each DRG neuron in voltage-clamp (see Materials and Methods). The data represent the model channel activity at +12.5% WT addition (n = 8, blue) and 12.5% WT-to-L858H substitution (n = 5, red). **P < 0.01.
Figure 9 A single-allele SCN9A mutation: L858H functional evaluation in small DRG neuron. (A) AP evoked by a current pulse of threshold intensity in control (black) and after dynamic-clamp 50% exchange of WT-to-L858H conductance (red). APs are shown on the top (stimulation protocol is shown on top of APs), and the respective Na_V1.7 current differential is presented in the bottom. (B) AP repetitive firing (top) evoked by a 10-Hz train of current pulses (same as in A) at threshold intensity in control (black) and after 50% WT-to-L858H SR (blue); dynamic-clamp recording of the 50% (L858H – WT) current is shown on the bottom. (C) Dynamic-clamp (L858H – WT) model currents at 50% SR of endogenous Na_V1.7 conductance were integrated over 3 different time intervals: (1) from stimulus onset to AP threshold (arrow 1 to 2 in A); (2) from threshold to undershoot (arrows 2 and 3 in A); and (3) from undershoot to the next stimulus onset. Sodium charge (pA*ms) was normalized to the peak value of the native Na_V1.7 sodium current (n = 6). Kruskal-Wallis ANOVA nonparametric test for 3 populations was used to determine whether the samples come from different populations (P < 0.01).
Figure 1 Human painful neuroma. A montage of low-magnification images of a neurofilament-labeled section from a human painful neuroma. Neurofilament-positive axons within the nerve trunk (right side of montage) are parallel in orientation, whereas within the club-shaped nerve-end neuroma, the axons are tangled and disorganized.
Figure 2 Sodium channel Na_V1.3 accumulates in human painful neuromas. Control human tissue exhibits low levels of Na_V1.3 immunolabeling. Painful neuromas display substantially increased Na_V1.3 immunoreactivity (red) compared with control tissue. Colocalization (magenta) of neurofilament (blue) and Na_V1.3 (red) demonstrates that Na_V1.3 is present within axons. At increased magnification (bottom two panels), axons (blue) within neuromas display Na_V1.3 immunolabeling. Na_V1.3 immunolabeling is exhibited by an apparently blind-ending axon (bottom right panel).
Figure 3 Na_V1.7 and Na_V1.8 accumulate in human painful neuromas. Control human tissue exhibits low levels of both Na_V1.7 and Na_V1.8 immunoreactivity. Human painful neuromas display increased Na_V1.7 and Na_V1.8 immunolabeling (red) compared with control tissue. At increased magnification, both Na_V1.7 and Na_V1.8 immunolabeling (red) is displayed in apparently blind-ending axons (blue) within neuromas (bottom right panels for Na_V1.7 and Na_V1.8); colocalization is indicated by magenta color. (insets) Na_V1.8 (red) immunolabeling is displayed at nodes of Ranvier (bounded by Casprpositive (green) paranodes) in both control nerve and neuromas.
Figure 4 Na_V1.1, Na_V1.2 and Na_V1.9 are not accumulated in neuromas. Control and neuroma tissue sections were reacted with isoform-specific antibodies to Na_V1.1, Na_V1.2 and Na_V1.9. Low levels of Na_V1.1 immunolabeling are exhibited in both control nerves and neuromas, whereas only background levels of Na_V1.2 labeling are present in control tissue and neuromas. Low levels of Na_V1.9 immunolabeling are present in control nerves and neuromas. NF = neurofilament.
Figure 5 Na_V1.6 is expressed at nodes of Ranvier in control nerves and neuromas. Sections of control nerves and neuromas were triple immunolabeled for Na_V1.6, neurofilament (NF), and Caspr. Left column shows Na_V1.6 (red) signal only, whereas the right column shows merged image of Na_V1.6 (red), NF (blue), and Caspr (green) images. Nodes (arrows) in both control nerves and neuromas exhibit Na_V1.6 immunolabeling. Nonmyelinated axons (arrowheads) in control nerves and neuromas display a low level of Na_V1.6 immunoreactivity. There is no apparent accumulation of Na_V1.6 in neuromas compared with control nerves.
Figure 6 Mitogen-activated protein (MAP) kinases accumulate in human painful neuromas. Control human tissue displays low levels of activated (phosphorylated) p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2). In contrast, painful neuromas exhibit substantially increased immunolabeling for p38 and ERK1/2 compared with control tissue. (Insets) At increased magnification, activated p38 and ERK1/2 are localized within neurofilament-positive (blue) axons. In favorable section, activated p38 is accumulated at an apparent axon end-bulb.
Figure 9.1 Small fiber neuropathy can be diagnosed using skin biopsy to demonstrate degeneration of the endings of nerve fibers within the skin. Panel A shows a skin biopsy from a normal individual in whom multiple nerve fibers within the skin (green arrows) can be seen. Panel B shows a skin biopsy from a patient with small fiber neuropathy, in whom there is a depletion of nerve fibers. Panel C shows swellings of nerve fibers (green arrows) in the skin, from another patient with small fiber neuropathy. These are predegenerative changes. Scale bars: 50 μm. From Hoeijmakers, Faber et al. (2012).
Figure 9.2 Diagram showing how, in injured axons (1) energy reserves (ATP) are depleted, leading to failure of the ATPase and collapse of ionic gradients; (2) Na⁺ ions enter through sodium channels; (3) the resultant increase in Na⁺ within the axon causes the Na⁺-Ca²⁺ exchanger to operate in reverse mode, carrying damaging quantities of Ca²⁺ into the axon. MY, myelin. Modified from Stys, Ransom, and Waxman (1992).
Figure 9.3 (A) A micrograph showing small-diameter nerve fibers within the skin, in which red staining indicates the presence of Na_V1.7. Scale bar: 20 μm (Black et al. 2012). (B) A micrograph showing small nerve fibers in the skin, in which red staining indicates the presence of the sodium-calcium exchanger. Scale bar: 20 μm. From Persson et al. (2010).
Figure 9.4 Patch-clamp recording from a small nerve fiber with a diameter of approximately 1 μm extending from a dorsal root ganglion neuron in tissue culture. The micrograph shows (upper right) the tip of the electrode where it contacts the nerve fiber. The top trace shows a nerve impulse (action potential) from this nerve fiber. The bottom trace shows the sodium currents, produced by Na_V1.7 (blue trace) and then Na_V1.8 channels (red), which underlie the action potential. The black trace indicates the summed Na_V1.7 and Na_V1.8 currents. From Vasylyev and Waxman (2012).
Figure 1 *Causes identified for SFN: sarcoidosis, n = 150; medication, n = 9; hemochromatosis, n = 5; diabetes mellitus, n = 4; thyroid dysfunction, n = 4; alcohol abuse, n = 4; gammopathy related, n = 3; hypercholesterolemia, n = 2; vitamin B6 intoxication, n = 1; Lyme disease, n = 1, Wegener granulomatosis, n = 1; antiphospholipid syndrome, n = 1. The Maastricht University Medical Hospital is a referral center for sarcoidosis in the Netherlands. IENFD, intraepidermal nerve fiber density; QST, quantitative sensory testing.
Figure 2 Schematic sodium channel showing the locations of the Na_V1.7 mutations found in patients with idiopathic small nerve fiber neuropathy. Mutation R185H was found in 2 patients.
Figure 3 Electrophysiological analysis of I720K mutation. (A) Representative current traces recorded from HEK 293 cells expressing wild type (WT) (top) or I720K (bottom), evoked by voltage steps (100 milliseconds) from −80 to 40mV in 5mV increments, from a holding potential of −120mV. (B) Activation and steady state fast inactivation for WT (black squares) and I720K (red circles). Fast inactivation was examined using a series of 500-millisecond prepulses from −140 to −10mV followed by test pulses to −10mV. Left inset: midpoint values for fast inactivation (V_{1/2, fast-inact}) of WT (black) and I720K (red). Right inset: midpoint values for activation (V_{1/2, act}) of WT (black) and I720K (red). (C) Steady state slow inactivation of WT (black squares) and I720K (red circles). Slow inactivation was assessed using a 20-millisecond pulse to −10mV after a 30-second prepulse to potentials from −140 to 10mV followed by a 100-millisecond pulse to −120mV to remove fast inactivation. Inset: midpoint values of slow inactivation (V_{1/2, slow-inact}) (WT: black; I720K: red); *p < 0.05. (D) Resting membrane potential (RMP) of dorsal root ganglion (DRG) neurons expressing WT (255.8 ± 1.7, n = 26) or I720K (248.7 ± 1.9, n = 29); *p < 0.05. (E) Current threshold of DRG neurons expressing WT (237 ± 28, n = 26) or I720K (134 ± 30, n = 29) to 500-millisecond stimuli; p < 0.05. (F) Comparison of mean firing frequency in DRG neurons expressing WT and I720K across a range of current injections from 100 to 600pA; *p < 0.05. (G) Bar graph showing the proportion of spontaneous firing cells for DRG neurons expressing I720K (red) and WT channels (black); numbers to the right of the bar graph show mean values for WT (lower value in parentheses) and I720K (upper value). The recording on the right shows spontaneous firing (10 seconds) of representative DRG neuron expressing I720K; the numbers above the trace show average ± standard deviation frequency of spontaneous action potentials. V_1/2 represents voltage midpoint, I/I represents normalized current, and G/G represents normalized conductance for fast-activation, slow-inactivation, and activation. APs, action potentials.
Figure 4 Electrophysiological analysis of D623N mutation. (A) Representative current traces recorded from dorsal root ganglion (DRG) neurons expressing wild type (WT) (top) or D623N (bottom), evoked by voltage steps (100 milliseconds) from −80 to 40mV in 5mV increments, from a holding potential of −100mV. (B) Activation and steady state fast inactivation for WT (black squares) and D623N (red circles). Fast inactivation was examined using a series of 500-millisecond prepulses from −140 to −10mV followed by test pulses to −10mV. Left inset: midpoint values for fast inactivation (V_{1/2, fast-inact}) of WT (black) and D623N (red). Right inset: midpoint values for activation (V_{1/2, act}) of WT (black) and D623N (red). (C) Steady state slow inactivation of WT (black squares) and D623N (red circles). Slow inactivation was assessed using a 20-millisecond pulse to −10mV after a 30-second prepulse to potentials from −140 to 10mV followed by a 100-millisecond pulse to −120mV to remove fast inactivation. Inset: midpoint values of slow inactivation (V_{1/2, slow-inact}) (WT: black; D623N: red); *p < 0.05. (D) Resting membrane potential (RMP) of DRG neurons expressing WT (255.0 ± 1.5, n = 29) or D623N (245.5 ± 1.5, n = 27); **p < 0.01. (E) Current threshold of DRG neurons expressing WT (256 ± 28, n = 29) or D623N (125 ± 19, n = 27) to 200-millisecond stimuli; **p < 0.01. (F) Comparison of mean firing frequency in DRG neurons expressing WT and D623N across a range of current injections from 25 to 500pA; *p < 0.05. (G) Bar graph showing the proportion of spontaneous firing cells for DRG neurons expressing D623N (red) and WT channels (black); numbers to the right of the bar graph show mean values for WT (lower value in parentheses) and D623N (upper value); *p < 0.05. The recording on the right shows spontaneous firing (10 seconds) of representative DRG neuron expressing D623N; the numbers above the trace show the average ± standard deviation frequency of spontaneous action potentials. V_1/2 represents voltage midpoint, I/I represents normalized current, and G/G represents normalized conductance for fast-activation, slow-inactivation, and activation. APs, action potentials.
Figure 5 Electrophysiological analysis of M932L/V991L mutation. (A) Representative current traces recorded from dorsal root ganglion (DRG) neurons expressing wild type (WT) (top) or M932L/V991L (bottom) (unless otherwise noted, protocols are the same as in figures 3 and 4). (B) Activation and steady state fast inactivation for WT (black squares) and M932L/V991L (ML/VL; red circles). Inset shows midpoint values for fast inactivation (V_{1/2, fast-inact}) and activation (V_{1/2, act}) of WT (black) and M932L/ V991L (red), respectively. (C) Steady state slow inactivation of WT (black squares) and M932L/V991L (red circles). Inset: midpoint values of slow-inactivation (V_{1/2, slow-inact}) (WT: black; M932L/V991L: red). (D) Resurgent currents recorded from DRG neurons expressing WT (left) or M932L/V991L (right). Resurgent currents were assessed with a 2-step protocol that initially depolarized the membrane to +30mV for 20 milliseconds before testing for resurgent sodium currents by hyperpolarizing the membrane potential in 25mV increments from 0 to −80mV for 100 milliseconds, then returning to the holding potential of −100mV. Current amplitude (normalized to peak current evoked by a +30mV depolarization) (left) and proportion of cells producing resurgent current (right) are shown below traces; *p < 0.05. (E) Resting membrane potential (RMP) of DRG neurons expressing WT (256.9 ± 1.9mV, n = 20) or M932L/V991L (249.8 ± 1.6mV, n = 23); **p < 0.01. (F) Current threshold of DRG neurons expressing WT (250 ± 23pA, n = 20) or M932L/V991L (145 ± 22pA, n = 23) to 200-millisecond stimuli; **p < 0.01. (G) Comparison of mean firing frequencies of DRG neurons expressing WT and M932L/V991L across the range of current injections from 25 to 500pA; *p < 0.05. (H) Bar graph showing the proportion of spontaneous firing cells for DRG neurons expressing M932L/V991L (red); numbers to the right of the bar graph show mean values for WT (lower value in parentheses) and M932L/V991L (upper value); p = 0.072. The recording on the right shows spontaneous firing (10 seconds) of representative DRG neuron expressing M932L/V991L; the numbers above the trace show average ± standard deviation frequency of spontaneous action potentials. V_1/2 represents voltage midpoint, I/I represents normalized current, and G/G represents normalized conductance for fast-activation, slow-inactivation, and activation. APs, action potentials.
Figure 1 Neurite length of neurons expressing Na_V1.7 wild-type (WT) and I228M, M932L/V991L (ML/VL), and I720K channels. (A) Large-field montage image consisting of a 10 × 10 field-of-view montage image of a dorsal root ganglion culture 3 days after transfection with Na_V1.7 WT + green fluorescent protein (GFP) constructs, with GFP signal as white. Dotted lines distinguish individual field-of-view captures. Scale bar: 1,000µM. (B) Increased magnification of individual neurons transfected with Na_V1.7 WT, I228M, ML/VL, and I720K constructs demonstrates reduced neurite length of I228M-transfected neuron compared to WT. Scale bar: 250µM. (C) Quantifications of the total neurite length/neuron calculated from large-field images and averaged for each condition. Pairwise comparisons between neurites from neurons expressing WT channels and channel variants I228M, ML/VL, and I720K are presented. Data are normalized to WT values and presented as mean ± standard error of the mean. *p < 0.05.
Figure 2 Cell viability of neurons 3 days after transfection with Na_V1.7 wild-type (WT) or I228M channels. Top left: Neurons 3 days after transfection with I228M + green fluorescent protein (GFP) constructs. Top right: Same field after incubation with EthD-1, a marker for dead or dying cells. Lower left: Merged image shows that GFP-positive cells do not colabel with EthD-1. Lower right: Quantification of viable cells at 3 days after transfection (cells positive for GFP and negative for EthD-1 are considered viable). Data are presented as mean ± standard error of the mean, where n = number of large-field images, and number of cells analyzed is indicated in parentheses. Scale bar: 100µM.
Figure 3 Effect of carbamazepine (CBZ) and KB-R7943 on I228M-induced reduced neurite length. (A) Left panel: Indvidual neurons transfected with I228M, untreated or treated with 10µM CBZ. CBZ-treated neurons display increased neurite length compared to untreated neurons. Right panel: Quantification of the total neurite length/neuron calculated for neurons expressing I228M (top) or wild-type (WT; bottom), untreated or treated with CBZ. CBZ significantly increases neurite length in I228M-transfected neurons but does not alter neurite length of WT-transfected neurons. (B) Left panel: Individual neurons transfected with I228M, untreated (top) or treated (bottom) with 0.5µM KB-R7943. KB-R7943–treated neurons exhibit increased neurite length compared to untreated neurons. Right panel: Quantification of the total neurite length/neuron calculated for neurons expressing I228M (top) or WT (bottom) channels, untreated or treated with KB-R7943. KB-R7943 significantly increases neurite length in I228M-transfected neurons but does not affect neurite length of WT-transfected neurons. Data are presented as mean ± standard error of the mean. Scale bar: 200µM. *p < 0.05.
Figure 10.1 The N1768D mutation in the Na_V1.6 sodium channel, from a 15-year-old girl with epilepsy and a neurodevelopmental disorder, impairs channel inactivation, a process that makes channels unoperable after they open. The y-axis of this graph shows the percentage of channels that are not inactivated (and thus are available for operation) as a function of membrane potential. At membrane potentials more depolarized than –80 mV, the fraction of available channels is larger for N1768D mutant channels (red) than for normal, wild-type (WT) Na_V1.6 channels (black). At a membrane potential of –60 mV, close to the resting potential of neurons in the brain, only one-half of the wild-type channels are available for operation; in contrast nearly all of the mutant channels are available. At membrane potentials more depolarized than –30 mV, almost none of the wild-type channels are available and inactivation is complete, while 10% of the mutant channels are available (arrow). Thus, at the membrane potentials of most nerve cells in the brain, more of the mutant Na_V1.6 channels are available for operation, a change that would be expected to make neurons more excitable. Modified from Veeramah et al. (2012).
Figure 10.2 Representative transmembrane currents produced by normal wild-type (WT, top) and N1768D mutant Na_V1.6 channels (bottom). Cells were held at –120 mV, and step depolarizations (–80 to +60 mV in 5-mV increments) were applied every 5 sec. Insets on right show persistent currents (presented as a percentage of maximal transient peak currents) at the end of a 100-msec step depolarization to –80 mV (black) and +20 mV (red). Note the different scales for the y-axes for wild-type and mutant channels on the right. As seen in the lower right panel, the persistent current, a driver of a neuronal excitability, is more than five times larger for the mutant channels. From Veeramah et al. (2012).
Figure 10.3 Currents generated by wild-type (WT) Na_V1.6 channels (top trace, black) and N1768D mutant channels (red trace) in response to a slow, ramp-like stimulus that gradually depolarizes the cell from –120 mV to +40 mV (shown at the bottom of the figure). The stimulus simulates a synaptic input. The ramp response of the mutant channels is more than ten-fold larger, indicating that the mutant channels respond more vigorously to even small, subtle inputs. From Veeramah et al. (2012).
Figure 10.4 These “whole cell current-clamp” recordings show the effect of N1768D mutant channels on neurons from the rat hippocampus, a part of the brain where seizures can originate. Ai shows abnormal spontaneous firing in a hippocampal neuron transfected with N1768D mutant channels. Aii shows examples of abnormal plateau-like depolarizations, similar to the paroxysmal depolarizing shifts characteristics of epileptic neurons, in hippocampal neurons transfected with the mutant N1768D channels. Panel B shows the percentage of neurons that fired spontaneously (spon.) following transfection with mutant N1768D channels (dark bar) or with normal, wild-type (WT) Na_V1.6 channels (open bar). There is much more spontaneous firing in neurons containing the mutant channel. Panel C shows the number of nerve impulses (action potentials, AP) evoked by a 1-second depolarizing step stimulus at a variety of stimulation intensities, in hippocampal neurons transfected with N1768D mutant channels (red) or normal, wild-type Na_V1.6 channels (black). At any given stimulus intensity, neurons containing the mutant channel fire more vigorously. From Veeramah et al. (2012).
Figure 1 iPSCs from IEM subjects and non-IEM donors differentiate into sensory neurons with comparable Na_V1.7 activity. (A) Sanger sequencing of IEM subject–derived iPSCs. The black arrow highlights the heterozygous point mutation in the pherogram. (B) Bright-field images of representative examples of IEM subject–derived and non-IEM donor–derived iPSCs with typical pluripotent-like morphology. Scale bars, 1000 mm. Panels below show immunostaining for nuclear Hoechst stain (blue) and expression of the Oct4 pluripotency marker (green). Scale bars, 100 mm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Bright-field images of representative examples of IEM subject–derived and non-IEM donor–derived iPSCs after differentiation into sensory neurons (iPSC-SNs). Scale bars, 1000 mm. Panels below show immunostaining for expression of the sensory neuron marker Brn3a (blue), Islet1 (red), and peripherin (green). Scale bars, 200 mm. (D) Example sodium current traces measured in the iPSC-SNs derived from the non-IEM donor (D4) and IEM subject EM5 (carrying the F1449V mutation) showing subtracted currents sensitive to the Na_V1.7 blocker. iPSC-SNs were held at −110 mV and stepped to 0 mV to evoke voltage-gated currents, which were partially blocked by 100 nM PF-05153462. (E) Summary of Na_V1.7 current density in the non-IEM donor–derived and IEM subject–derived iPSC-SNs. No significant difference was observed among all the clones (n = 13 to 40).
Figure 2 Excitability of iPSC-SNs from IEM and non-IEM subjects. (A) Representative traces of spontaneous firing in sensory neurons derived from iPSCs from IEM subject EM3 (V400M mutation) and non-IEM control subject D3. (B) Quantification of the number of spontaneous firing iPSC-SNs versus nonspontaneous firing iPSC-SNs from non-IEM and IEM subjects (n = 19 to 98; P < 0.05, linear logistic model). (C) Representative current-clamp traces showing subthreshold responses and subsequent action potentials evoked until reaching current thresholds (rheobase) of 544 pA for non-IEM subject D3 iPSC-SNs and 120 pA for IEM subject EM5 (F1449V mutation) iPSC-SNs. (D) Quantification of action potential rheobase comparing healthy control donor iPSC-SNs and IEM subject iPSC-SNs (n = 16 to 86; P < 0.05, nonparametric ANOVA). (E) Representative traces showing train of action potentials evoked in non-IEM control subject D1 and IEM subject EM1 (S241T mutation) iPSC-SNs after inducing depolarization by 100-pA current injection. (F) Quantification of action potential frequency induced by current injection (n = 10 to 46).
Figure 3 Na_V1.7 channel blockers reduce spontaneous firing and increase action potential rheobase in iPSC-SNs. (A) Representative traces of spontaneous action potentials in IEM subject EM3 (V400M mutation) iPSC-SNs blocked by increasing concentrations of the Na_V1.7 blocker PF-05153462. (B) Concentration-dependent effect of PF-05153462 on spontaneous action potential (AP) firing with a half-maximal inhibitory concentration (IC₅₀) of 2 nM for iPSC-SNs from IEM subjects EM2 (I848T mutation) and EM3 (V400M mutation). (C) Representative traces of spontaneous firing blocked by treatment of iPSC-SNs from IEM subject EM2 with 60 nM PF-05089771. (D) Representative current-clamp traces in iPSC-SNs from non-IEM control subject D2 and IEM subject EM2 (I848T mutation) showing an increase in rheobase after application of PF-05089771 in a concentration-dependent manner. (E) Quantification of the effect of PF-05089771 on rheobase for iPSC-SNs from non-IEM control subjects and IEM subjects (n = 6 to 10; P < 0.05, ANOVA). (F) Quantification of the effect of PF-05153462 on rheobase for iPSC-SNs from non-IEM control subjects and IEM subjects (n = 6 to 10; P < 0.05, ANOVA; comparison at each concentration greater than 10 nM).
Figure 4 Na_V1.7 channel blocker reverses the elevated heat sensitivity of iPSC-derived sensory neurons from IEM subjects. (A) Representative traces of evoked action potentials showing a small increase in rheobase when iPSC-SNs from non-IEM control subject D3 were incubated with extracellular recording solution at an elevated temperature of 40°C (control temperature was 35°C). The rheobase for iPSC-SNs for IEM subject EM5 (F1449V mutation) was decreased relative to the D3 control. Far right panels show an example time course for rheobase changes of iPSC-SNs from non-IEM subject D3 and IEM subject EM5 (F1449V mutation) upon heating of the incubation solution. (B) Quantification of the effect of heating on rheobase for non-IEM and IEM iPSC-SNs. The heating effect was calculated as the change in the rheobase at 40°C versus 35°C (n = 13 to 34; P < 0.01, comparing non-IEM and IEM iPSC-SNs using ANOVA). (C) Representative traces of rheobase showing the effect of heating on rheobase before and after the application of the Na_V1.7 channel blocker PF-05153462. The heat sensitivity of rheobase was reversed by PF-05153462 on iPSC-SNs from IEM subject EM5, but no effect was seen on iPSC-SN from IEM subject EM1. (D) Quantification of the effect of PF-05153462 on heat sensitivity of iPSC-SNs from IEM subjects EM1 (S241T mutation), EM2 (I848T mutation), EM3 (V400M mutation), and EM5 (F1449V mutation) (n = 6 to 10; P < 0.05 for EM1, EM2, and EM5 and P < 0.01 for EM3; paired t test). Only iPSC-SNs demonstrating a positive response to PF-05153462 (where the rheobase showed sensitivity greater than 50 pA at 35°C) were included, and the number of iPSC-SNs excluded was 3 of 12 iPSC-SNs for IEM subject EM1, 2 of 10 iPSC-SNs for IEM subject EM2, 1 of 8 iPSC-SNs for IEM subject EM3, and 1 of 11 iPSC-SNs for IEM subject EM5.
Figure 5 Na_V1.7 contributes to the elevated heat sensitivity of iPSC-SNs from IEM subjects. Correlation of the heat sensitivity changes to rheobase changes at 35°C induced by PF-05153462 (Pearson’s r = 0.22, 0.88, 0.82, and for IEM subjects EM1, EM2, EM3, and EM5, respectively). The regression coefficients were significantly different from zero for IEM subjects EM2, EM3, and EM5. In contrast to figure 4D, iPSC-SNs demonstrating a positive response to PF-05153462 (rheobase changes at 35°C greater than 50 pA, indicating clear Na_V1.7 expression) and iPSC-SNs demonstrating a negative response to PF-05153462 (rheobase changes at 35°C less than 50 pA, indicating low or no Na_V1.7 expression) were included.
Figure 6 Clinical study design overview and maximum pain scores after dosing. (A) Each treatment session consisted of two study periods separated by at least a 72-hour washout period. Subjects received either a single oral dose of the Na_V1.7 channel blocker PF-05089771 or placebo in a crossover manner in each study period. Pain scores were recorded every 15 min up to 10 hours after dosing. Core body temperature was measured regularly throughout the study period. The cooling paradigm (C1 to C4) was used before evoking pain (EP1 to EP4) at specific time points during the study period. Pharmacokinetic (PK) samples were collected before dosing and at 0.5, 2, 6, and 24 hours after dosing. (B) Maximum pain scores recorded by subjects using the PI-NRS after dosing with either the Na_V1.7 channel blocker PF-05089771 or placebo in TS1 and TS2. Individual subject results for maximum pain scores included subjects who used nonpharmacological cooling therapy to alleviate pain. IEM subject EM1 (S241T mutation) did not show any notable difference in pain scores after PF-05089771 treatment compared to placebo between TS1 and TS2. IEM subject EM2 (I848T mutation) had a reduction in pain scores in TS2 after PF-05089771 treatment compared to placebo at the 4- to 5-hour postdose time point. IEM subject EM4 (V400M mutation) had a reduction in pain score at the 4- to 5- and 8- to 10-hour postdose time point in TS1, but no difference in pain scores between drug and placebo in TS2. IEM subjects EM3 (V400M mutation) and EM5 (F1449V mutation) had a reduction in pain scores after a single dose of PF-05089771 at the 4- to 5-hour time point and the 8- to 10-hour postdose time point in both TS1 and TS2. (C) Change from baseline in maximum pain scores (PI-NRS) after PF-05089771 treatment versus placebo in individual IEM subjects. (D) Differences in maximum pain scores after dosing with PF-05089771 including and excluding IEM subjects who used cooling as “rescue” therapy.
Figure 1 Structural modelling of transmembrane domains of human Na_V1.7 channel. (a) Schematic of the human Na_V1.7 channel topology showing the mutations S241T, V400M and F1449V. (b) Intra-membrane view of structural model of Na_V1.7 channel transmembrane domains. Domain I, light blue; Domain II, salmon; Domain III, cyan; Domain IV, lime. (c) Cytosolic view of the structural model of Na_V1.7 channel transmembrane domains. Boxed area containing S241, V400 and F1449 residues is enlarged in panel e. (d) Close-up intra-membrane view of the area containing S241, V400 and F1449 residues. (e) Close-up cytosolic view of the boxed area of panel c. S241, V400 and F1449 are shown as stick and coloured grey, red and yellow, respectively.
Figure 2 Mutant cycle analysis of voltage dependence of activation of Na_V1.7 mutations. (a) Voltage dependence of activation curves of Na_V1.7 WT, V400M, S241T, and V400M/S214T (VM/ST) double mutant channels. Curves were Boltzmann fits of the data. (b) Voltage dependence of activation curves of Na_V1.7 WT, V400M, F1449V and V400M/F1449V (VM/FV) double mutant channels. (c–h) Representative traces of current families recorded from HEK293 cells expressing WT (c), V400M (d), S241T (e), F1449V (f), VM/ST double mutant (g) and VM/FV double mutant (h) channels.
Figure 3 Voltage dependence of activation and steady-state fast inactivation of S241T and F1449V mutant channels. (a–d) Representative traces of current families recorded from HEK293 cells expressing S241T mutant channel treated with DMSO (a), or with CBZ (b); F1449V mutant channel treated with DMSO (c), or with CBZ (d). (e) The averaged voltage dependence of activation of S241T mutant channel treated with DMSO or CBZ (30 µM) was plotted and fitted with Boltzmann equation. A depolarizing shift of activation of 7.1 mV was observed when S241T mutant channel was treated with CBZ (P < 0.01, Student’s t-test). (f) The averaged voltage dependence of activation of F1449V mutant channel treated with DMSO or CBZ was plotted and fitted with Boltzmann equation. No notable shift in activation curve of F1449V mutant channel was observed. (g–h) The voltage dependence of steady-state fast inactivation in response to 500 ms depolarizing potential for S241T (g) or F1449V (h) mutant channel treated with DMSO or CBZ was plotted and fitted with Boltzmann equation. No notable shift was observed.
Figure 4 Current-clamp analysis of DRG neurons expressing WT or S241T mutant channel. (a) Representative DRG neuron expressing Na_V1.7 WT channel showed sub-threshold response to 225 pA current injection and subsequent action potential evoked by injection of 230 pA, which was the current threshold for this neuron. (b) Representative DRG neuron expressing Na_V1.7-S241T mutant channel showed sub-threshold response to 55 pA current injection and subsequent action potential evoked by injection of 60 pA. (c) Comparison of current threshold for DRG neurons expressing WT and S241T mutant channels. Expression of S241T channel reduced current threshold significantly (**P < 0.01, Student’s t-test). Current threshold for WT: (227.6 ± 36.7 pA, n = 19); for S241T: (83.5 ± 18.2 pA, n = 20). (d–f) Responses of a representative DRG neuron expressing WT channel to 1-s-long depolarizing current steps at 100 (d), 300 (e) and 400 (f) pA current injection. (g–i) Responses of a representative DRG neuron expressing S241T mutant channel to 1-s-long depolarizing current steps at 100 (g), 300 (h) and 400 (i) pA current injection. The difference in responses is apparent across this range. (j) The averaged number of action potentials between DRG neurons expressing WT and S241T mutant channel was compared. The response of DRG neurons expressing WT channel to current injection was significantly different compared with DRG neurons expressing S241T mutant channel across a range (125–500 pA) of step current injections (*P < 0.05, Mann–Whitney test). (k) Averaged resting membrane potentials for DRG neurons expressing WT or S241T mutant channel were not statistically different. Results are presented as mean ± s.e.m.
Figure 5 Current thresholds of CBZ- or DMSO-treated DRG neurons expressing S241T or F1449V mutant channels. (a,b) Sub- and supra-threshold responses of representative DRG neurons expressing S241T mutant channel treated with DMSO (a) or 30 µM CBZ (b) are shown. (c) Comparison of current threshold for DRG neurons expressing S241T mutant channel treated with DMSO or 30 µM CBZ. CBZ treatment increased the current threshold significantly (**P < 0.01, Student’s t-test). Current threshold for DMSO-treated DRG neurons: 90.4 ± 13.2 pA (n = 27); for CBZ-treated DRG neurons: 162.7 ± 24.4 pA (n = 28). (d,e) Sub- and supra-threshold responses of DRG neurons expressing F1449V mutant channel treated with DMSO (d) or 30 µM CBZ (e) are shown. (f) Comparison of current threshold for DRG neurons expressing F1449V mutant channel with the treatment of DMSO (153.5 ± 17.9 pA, n = 29) or 30 µM CBZ (165.5 ± 19.7 pA, n = 28). No significant difference was found (P > 0.05, Student’s t-test). Results are presented as mean ± s.e.m.
Figure 6 Firing frequencies and membrane potentials of CBZ- or DMSO-treated DRG neurons expressing S241T. (a–c) Responses of a representative DRG neuron expressing S241T mutant channel treated with DMSO to 1 s long depolarization current steps at 100 (a), 200 (b) and 400 (c) pA current injection. (d–f) Similar recordings from a representative DRG neuron expressing S241T mutant channel treated with 30 µM CBZ at 100 (d), 200 (e), and 400 (f) pA current injection. (g) Averaged response for DRG neurons expressing S241T mutant channel treated with DMSO (n = 27) or CBZ (n = 28) are summarized. CBZ statistically reduced firing frequency starting from 100 pA current injection (*P < 0.05, Mann–Whitney test). (h) Averaged RMP between DRG neurons expressing S241T mutant channel treated with DMSO or CBZ were not statistically different (DMSO: −55.3 ± 1.4 mV, n = 27; CBZ: −54.9 ± 1.3 mV, n = 28, P > 0.05, Student’s t-test). Results are presented as mean ± s.e.m.
Figure 7 Firing frequencies and membrane potentials of CBZ- or DMSO-treated DRG neurons expressing F1449V. (a–c) Responses of a representative DRG neurons expressing F1449V mutant channel treated with DMSO to 1-s-long depolarization current steps at 200 (a), 350 (b) and 425 (c) pA current injection. (d–f) Similar recordings from a representative DRG neuron expressing F1449V mutant channel treated with CBZ at 200 (d), 350 (e) and 425 (f) pA current injection. (g) Averaged firing frequencies for DRG neurons expressing F1449V mutant channel treated with DMSO (n = 29) or CBZ (n = 28) were compared and no statistical difference was found across the entire range (P > 0.05, Mann–Whitney test). (h) Averaged RMPs between DRG neurons expressing F1449V mutant channel treated with DMSO or CBZ were not statistically different (DMSO: −54.8 ± 1.5 mV, n = 29; CBZ: −54.1 ± 1.0 mV, n = 28, P > 0.05, Student’s t-test). Results are presented as mean ± s.e.m.
Figure 8 Alignment of Na_V1.7 structural model with NavAb structure. (a) Intra-membrane view of structural model of Na_V1.7 channel transmembrane domains aligned with NavAb structure (3RVY). Na_V1.7 Domain I, light blue; Domain II, salmon; Domain III, cyan; Domain IV, lime. NavAb, white. (b) Cytosolic view of the structural model of Na_V1.7 channel transmembrane domains aligned with NavAb structure. (c) Close-up intramembrane side view of the area containing S241 (grey), V400 (red) and F1449 (yellow) residues. (d) Close-up cytosolic view of the area containing S241, V400 and F1449 residues.
Figure 1 Inherited erythromelalgia pain rating. An example of rating of pain fluctuations after an episode is elicited with the thermal boot. The rating shown here was recorded after the thermal stimulus was switched off. gLMS indicates generalized Labeled Magnitude Scale.
Figure 2 Pain characteristics in patients 1 and 2. Pain characteristics and effects of carbamazepine treatment vs placebo for patients 1 and 2. (A) Time in pain as reported in patients’ diaries during the 3 phases of treatment ramp-up, maintenance, and taper. Histograms represent means. (B) Same as in panel A for the reported duration of inherited erythromelalgia episodes. (C) Number of awakenings due to pain during 3 phases of ramp-up, maintenance, and taper.
Figure 3 Brain activity modulation with carbamazepine (CBZ). Treatment effects of CBZ vs baseline. (A) Brain activity obtained when contrasting baseline to carbamazepine (baseline > CBZ) (paired t test; n = 2; fixed effects; P < .05, corrected for multiple comparisons). The bar plot shows mean (SEM) brain activity in z scores during pain rating (blue) and visual tracking (white) within the left (L) nucleus accumbens (NAc) (blue arrowhead) plotted for baseline (BL, left), chronic CBZ treatment (CBZ, middle), and chronic placebo (PL, right) treatment, respectively. (B) Brain activity when contrasting CBZ > baseline; the bar plot depicts mean activity within primary somatosensory area (SI) (red arrowhead). MI indicates primary motor cortex; PCC, posterior cingulate cortex; PC, parietal cortex; R, right; rACC, rostral anterior cingulate cortex; and SI, primary somatosensory cortex.
Figure 4 Brain activity modulation by placebo. (A) Treatment effects of placebo vs baseline. Areas shown in red to yellow represent the contrast (baseline > placebo) and areas shown in blue to green represent the contrast (placebo > baseline). Unlike carbamazepine, placebo decreases activity in somatosensory parietal areas and increases activity in the posterior cingulate cortex and medial prefrontal cortex, among others. (B) Contrast results between placebo scans (placebo > carbamazepine, red to yellow) and carbamazepine scans (carbamazepine > placebo, blue to green). Differences in activations are similar to those shown in figure 3 for carbamazepine and baseline.
Figure 5 Brain activity associated with decreased pain. (A) Regression of brain activity during pain rating scans across all visits against pain intensity reported during scanning. (B) Regression of brain activity against time in pain as reported in patients’ diaries after masking with results shown in panel A. Areas in red to yellow represent positive correlations, whereas areas in blue to green represent negative correlations.
Figure 6 Carbamazepine attenuation of warmth-evoked firing in dorsal root ganglion neurons expressing Na_V1.7 S241T mutant channels. (A–C) Heat maps of a representative multielectrode array recording of dorsal root ganglion neurons expressing Na_V1.7 S241T before carbamazepine treatment (upper panels). The firing frequency of each active electrode is color coded with white/red representing high firing frequency and blue/black representing low firing frequency. Each circle corresponds to an active electrode within an 8 × 8 electrode array. There is only 1 active electrode in the heat map at 33°C (A). The number of active electrodes and firing frequency increase at 37°C (B) and 40°C (C). (D–F) Heat maps of the same multielectrode array recording well after (30-µM) carbamazepine treatment (upper panels). The number of active electrodes and firing frequency of neurons are both markedly reduced at all 3 temperatures: 33°C (D), 37°C (E), and 40°C (F). White arrowheads indicate silent neurons after carbamazepine treatment. In the lower panels in A–F, recordings from a representative neuron in the heat map indicated by yellow arrowheads are shown. Note increased firing as temperature increased in the absence of carbamazepine (A–C) and attenuation of firing by carbamazepine (D–F).
Figure 7 Firing frequency. Mean firing frequency of neurons (n = 98) expressing Na_V1.7 S241T before and after carbamazepine treatment at all 3 temperatures.

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