THE ROLE of Nav1.9 and Nav1.1 in SOMATOSENSORY PERCEPTION

THE ROLE OF NaV1.9 AND NaV1.1 IN SOMATOSENSORY PERCEPTION

Juan J. Salvatierra

“A dissertation submitted to the Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy”

Baltimore, Maryland September 2018

Abstract

Voltage-gated sodium (NaV) channels are critical for the electrical signaling of excitable cells. Due to their importance in cellular signaling, mutations in these crucial proteins can lead to a cadre of different diseases and syndromes, such as severe painful neuropathies, seizures, cardiac arrhythmias, along with many other grave conditions. Here, we show that NaV1.1 and NaV1.9 are key players in transmitting visceral pain and itch, respectively. NaV1.1 is functional upregulated in chronic visceral hypersensitivity (CVH) mouse models, and inhibiting NaV1.1 with a NaV1.1-selective inhibitor leads to an attenuation of the visceral pain that develops in these IBS models.

We also demonstrate that NaV1.9 is important for both histamine- and Mrgpr-dependent itch, and loss of NaV1.9 leads to a dramatic reduction in mice for both forms of itch.

Furthermore, patients with a gain-of-function mutation in NaV1.9 experience severe pruritus caused by an enhanced excitability of MrgprA3+ neurons, as evidenced by our experiments. Together, we provide compelling evidence for the functional importance in the pain and itch field for two NaV channel subtypes.

Thesis Advisor: Frank Bosmans

Thesis Reader: Dr. Xinxhong Dong

ii Acknowledgements

I’d like to begin by thanking my advisor Frank Bosmans. He truly has been an ideal mentor. He has taught me how to become an independent scientist, think critically, approach and tackle difficult scientific questions, and how to best present my data. But importantly, he also helped me realized and offered valuable advice on the difficulties in striving to become a successful scientist, whether that was the struggles of applying for grant money, getting your research published, or how to network efficiently. He has strived to advance my career and he has always been graciously supportive and encouraging of my ideas, and for that I will forever be thankful.

I also want to acknowledge several members of my lab, past and present, and members of the Hopkins community who also contributed immensely to my professional and personal life here at Hopkins. People in the Bosmans lab, including John Gilchrist,

JP Llongueras, Marcelo Diaz-Bustamante, and Peilin Shen. We all had very meaningful, thoughtful discussions both about science and personal matters, which I will never forget. It was a pleasure work next to all of them, and I am proud to consider them all my friends. I want to thank our collaborators and people who helped with experiments:

Jimmy Meixiong, Jay Pasricha, Allan Basbaum, Stuart Brierley, Elaine Tierney, and

Barbara Slusher. I would also like to thank the people in charge of the BCMB program

Carolyn Machamer, Arhonda Gogos, and Christina Bailey for all the help they provided during my graduate education. I want to especially thank my thesis committee: Micheal

Caterina, Xinzhong Dong, and Dax Fu for all the help and thoughtful suggestions.

I would also not be here today if not for the guidance and support I received as a burgeoning scientist. Dr. Kathy Wilson was immensely helpful is building my confidence

iii early on in my career. Her commitment and passion for the PREP program at Hopkins ensured my future success here as a graduate student. I also would like to thank Dr.

Seth Blackshaw for taking me on in his lab as a PREP scholar and passing onto me his contagious enthusiasm for science. And I would especially like to acknowledge Dr.

Daniel Lee, who was truly my first hands-on scientific mentor. He took me on during my time in Dr. Blackshaw’s lab, and provided me with my first-hand experience in bench science. He instilled in me a keen sense of scientific rigor and perseverance, and he’s also become one of my greatest role models.

Of course, I could not have done any of this if not for my parents Silvia Villa and

Jose Salvatierra. They have endeavored to provide the most they could for my success in all aspects of my life and have always been encouraging with whatever I decided to pursue in my life. They struggled to make it in this country as immigrants, and their sacrifices ensured by future successes in life. I will never be able to express how deeply grateful I am for them, but I hope I have and will continue to make them proud. My sisters Lorena Salvatierra, Janet Monsour, Leah Gift, and my grandmother Maria

Fajardo have always been there for me, as well. It was an incredible experience to be raised by such wonderful women my entire life. Most of all, I want to thank my girlfriend and best friend: Eliah Shamir. She has made getting through the final stages of my PhD that much easier. She has always been supportive, encouraging, and critical in both my scientific pursuits and in my personal development. She has made me a better person, and I could not fathom continuing in my scientific endeavors without her.

iv Table of Contents

Abstract…………………………………………………………………………………………...ii

Acknowledgements……………………………………………………………………………..iii

List of Figures………………………………………………………………………………….viii

List of Tables……………………………………………………………………………………xi

Chapter 1: Introduction…………………………………………………………………………1

Voltage-gated sodium (NaV) channels…………………………….………………………….2

NaV channels and pain ……………………………………………….………………………..3

NaV channels and itch…………………………………………………….…………………….8

NaV channels and the enteric nervous system……………………………………………..11

Chapter 2: The role of NaV1.1 channel in gastrointestinal physiology….…………….….14

Summary…………………….……………………………………………………………….…15

Introduction……………………………………………………………………………………..16

Results………………………………………………………………………………………….19

Pharmacological screen of compound B. …………………………………………..19

Pharmacokinetic profile of compound B…………………………………………....19

Toxicity of compound B……………………………………………………………….21

Compound B reduces peripheral nerve-injury-induced mechanical

hypersensitivity…………………………………………………………………………21

Sodium current recordings in colon-innervating DRG neurons…………………..22

Colonic nociceptor recordings…………………………………….…….………..…..23

Visceral motor reflex (VMR) in response to colorectal distension (CRD)…..……23

v Rufinamide Derivatives show differential effects on hNaV1.1 gating…….………25

Discussion …………………………………………………………………………..…………26

Materials and Methods………………………………………………….…………………….28

Acknowledgments………………………………………………..……………………………40

Figures………………………………………………...………………………………………..41

Tables…………………………………………………………………………..……………….63

Chapter 3: The functional contribution of NaV1.9 to pruritic stimuli………………………73

Summary…………………….………………………………………………………………….74

Introduction……………………………………………………………………………………..76

Results………………………………………………………………………………………….78

+/WT A newly-identified NaV1.9 p.L811P patient………………………………...…...78

Generation and characterization of a sfGFP-tagged NaV1.9 mouse line………..79

NaV1.9 is predominantly expressed in non-myelinated small diameter

DRGs…………………………………………………………………………………..81

NaV1.9 expression in MrgprA3/MrgprC11 neurons………………………………...83

Loss of NaV1.9 leads to a reduction in itch………………………………………….83

2+ NaV1.9 is important for histamine- and CQ-evoked Ca responses………….…84

Pruritogens influence NaV1.9 currents………………………….……………….…..85

+/WT The NaV1.9 p.L799P mutation leads to gain of itch in mice…………………..88

Discussion…………………………………………………………………………….……..…90

Materials and Methods…………………………………………………………………….….93

vi Acknowledgments …………………………………………………………………...……...104

Figures……………………………………………………….……………….…..………..….105

Chapter 4: The role of NaV1.9 in the ENS……………………………..…………..………124

Summary………………………………………………………………….…………………..125

Introduction………………………………………………………….….………….…..……..126

Results…………………………………………………………….….……………………….127

Expression of NaV1.9 in the enteric nervous system……..….…………………..127

-/- Gut motility in NaV1.9 mice…………………………………...……………………127

-/- Metabolic changes in NaV1.9 mice………………….…………..…………….….128

Discussion……………………………………………………………………………….……129

Acknowledgements ………………………………………………………….………..…….130

Figures………………………………..…………………………………………..…………..132

Chapter 5: Conclusions………………..…………..…………………………………..……137

Summary of findings……………….…………………..……………………………..……..138

Future directions………………………………….………………………………………..…143

REFERENCES……………..……………………………………………………………..….146

CURRICULUM VITAE………….……………..………………………………………….….165

vii List of Figures

Figure 2-1: Effect of 100μM Compound B on NaV1.5, NaV1.7, and NaV1.8……………..41

Figure 2-2: Metabolic stability of Compound B in plasma and liver microsomes……….42

Figure 2-3: Pharmacokinetic (PK) profile of Compound B……………………………...…43

Figure 2-4. Pharmacological blockade of NaV1.1 is antinociceptive…………….……….44

Figure 2-5: Compound B assessment in the rotarod test…………………………..……..46

Figure 2-6. Compound B reduces sodium currents in colon-innervating DRG neurons...... 47

Figure 2-7: Compound B does not affect sodium currents in a subpopulation of colon- innervating DRG neurons………………………………………………………………..……48

Figure 2-8. Effect of Compound B on colonic nociceptive afferents………………..……49

Figure 2-9: Compound B does not affect a subpopulation of colonic nociceptive afferents from healthy control and CVH mice………………………………………..……..51

Figure 2-10: Effect of Compound B and Hm1a on colonic nociceptive afferents..……..52

Figure 2-11. Effect of intracolonic administration of Compound B on VMR in an IBS mouse model of TNBS-induced CVH………………………………………………..………53

Figure 2-12: Colonic compliance……………………………………………...... 55

Figure 2-13. Effect (i.p.) of Compound B in an IBS mouse model of acetic acid–induced

CVH……………………………………………………………………………………..………56

Figure 2-14. Effect (s.c.) of Compound B in an acetic acid–induced IBS mouse model………………………………………………………………………………….….……..58

Figure 2-15. Rufinamide derivatives that significantly alter steady-state activation and inactivation V1/2 values on human NaV1.1……………………………………………..……60

viii Figure 2-16. Rufinamide derivatives that don’t show effects on human NaV1.1……..…61

Figure 2-17. Compound F in an acetic-acid mouse model of IBS……………….….……62

Figure 3-1. Generation and characterization of NaV1.9 mouse lines…………...………105

Figure 3-2. Voltage-clamp recordings in DRGs show a similar TTX-R current in WT and sfGFP-NaV1.9 mice……………………………………………………………………..…...107

Figure 3-3. sfGFP-NaV1.9 expression patterns……………………………………...……108

Figure 3-4: NaV1.9 in vagal ganglia and hairy skin………………………………..……..109

+ + Figure 3-5: NaV1.9 expression in MrgprA3 and MrgprC11 neurons and behavioral models…………………………………………………………………………………………110

Figure 3-6: Loss of NaV1.9 leads to a reduction in histamine and chloroquine but not

BAM8-22 responsive neurons………………………………………………………………112

Figure 3-7: Action potentials are influenced by NaV1.9 and CQ……………….……..…113

Figure 3-8: Application of chloroquine, BAM8-22, and histamine lead to a potentiation of

NaV1.9 current and a shift in activation voltage of TTX-R current………………………116

Figure 3-9. Activation of MrgprA3 and MrgprC11 speed up NaV1.9 activation……..…118

Figure 3-10. MrgprA3 and MrgprC11 activation in ND7/23 cells does not lead to changes in the NaV1.8 activation……………………………………………………...……119

Figure 3-11: sfGFP-NaV1.9L799P/WT mice show higher basal scratching and more chloroquine responsive neurons……………………………………………………………121

L799P/WT Figure 3-12. qRT-PCR analysis of SCN11A RNA in sfGFP-NaV1.9 mice……..123

Figure 4-18. Expression of NaV1.9 in the enteric nervous system……………………...133

Figure 4-19. Gut transit time and stool content in NaV1.9-/- mice compared to littermate controls………………………………………………………………………………………..134

ix -/- Figure 4-20. No effects on metabolism are observed in NaV1.9 mice……………..…136

x List of Tables

Table 2-1. Additional targets tested against Compound B……………………………..…63

Table 2-2. PK data……………………………………………………………………..……...66

Table 2-3. Table providing values for fits of the data presented in Figure 3-15…..…….67

Table 2-4. Table providing values for fits of the data presented in Figure 3-15…..…….68

Table 2-5. Table providing values for fits of the data presented in Figure 3-15…..…….69

Table 2-6. Table providing values for fits of the data presented in Figure 3-16…..…….70

Table 2-7. Table providing values for fits of the data presented in Figure 3-16…..…….71

Table 2-8. Table providing values for fits of the data presented in Figure 3-16…..…….72

xi THE ROLE OF NaV1.9 AND NaV1.1 IN SOMATOSENSORY PERCEPTION

Chapter 1: Introduction

1 Voltage-gated sodium (NaV) channels

Voltage-gated sodium (NaV) channels are crucial components of electrical signaling in nervous and muscle tissues [1]. Therefore, it should come as no surprise that dysregulation of these indispensable channels can lead to a cadre of diseases including epilepsy, arrhythmias, and pain syndromes [2]. They are responsible for allowing rapid and selective flow of Na+ across the membrane in response to changes in the membrane potential and thus generating electrical signals in cells. These channels are made up of a single polypeptide composed of four similar domains. Each domain consists of six membrane spanning hydrophobic alpha-helical segments, with the first four transmembrane segments (S1-S4) forming the voltage-sensing domain (VSD) and the latter two segments (S5-S6) forming the ion-conducting pore domain. The opening and closing, or gating, of the channel is accomplished via the VSDs that detect this difference in voltage and transfers the energy to the pore to modulate gating. It is the

S1-S4 segments from each domain that impart exquisite voltage sensitivity to the channel. The VSD itself is highly conserved across different ion channels and in different species [1, 3].

There are nine known channel isoforms (Nav1.1-1.9; SCN1A-11A), each with a specific tissue distributions and unique electrophysiological properties. Nav1.4 is known to be expressed throughout sarcolemma and T-tubule membranes of the skeletal muscle and is important for skeletal muscle action. Nav1.5 is primarily expressed in cardiac muscle where it is known to be critical for cardiac action potentials [4-6]. Nav1.1,

Nav1.2, and Nav1.6 are primarily expressed in the central nervous system, while

Nav1.7-Nav1.9 are predominantly expressed in the peripheral nervous system. NaV1.3 is

2 primarily expressed in the peripheral nervous system early in development, but upregulation of Nav1.3 RNA beyond development has been observed during chronic pain states [7, 8].

Sodium channels and pain

Many NaV channel subtypes are expressed in the peripheral nervous system and can serve an important role in coding for different somatosensations such as pain. In particular, NaV1.7-NaV1.9 have been widely targeted for drug discovery due to their strategic expression in the dorsal root ganglia (DRG) and trigemal ganglia (TG) where many of the primary nociceptor (pain sensing) and pruritoceptor (itch sensing) neurons are located [6].

Human NaV1.7 was the first NaV channel reported with gain-of-function and loss- of-function mutations that lead to extreme pain disorders or insensitivity to pain, respectively [8-11]. Gain-of-pain mutations lead to inherited erythromelalgia, which is characterized by debilitating burning pain and redness of the distal extremities, and paroxysmal extreme pain, which is typically characterized by mandibular, ocular, and rectal pain and flushing. However, loss-of-function mutation patients typically report congenital insensitivity to pain. Interestingly, NaV1.7 loss-of-function patients also report anosmia, or the inability to smell, due to the expression of NaV1.7 in olfactory sensory neurons and its importance in transmitting olfactory signals [12]. Knockout mouse models of NaV1.7 recapitulated some of the phenotypes seen in human patients with loss of function mutations [13]. With the loss of NaV1.7, mice showed slightly altered heat- and inflammation-induced pain thresholds. However, mechanical and cold

3 thresholds remained unchanged. In addition, visceral pain, which is associated with people suffering from irritable bowel syndrome (IBS), was found to be independent of

NaV1.7. Although many of the visceral afferents innervating the gut expressed NaV1.7

RNA, the loss of NaV1.7 in sensory neurons did not lead to significant reductions in visceral, nociceptive behaviors that develop in response to intracolonic applications of either capsaicin or mustard oil, stimuli known to induce sensitization following tissue damage [14]. The selective importance of NaV1.7 for heat- and inflammation induced pain is further supported by FGF13 KO mouse studies. Selectively knocking out FGF13 in sensory neurons leads to a dramatic increase in heat- and inflammation-induced pain thresholds. This phenotype has been attributed to the importance of FGF13 in regulating membrane localization of NaV1.7 [15].

Although early studies describing the role in NaV1.7 in different pain modalities did not uncover major alterations in neuropathic pain thresholds, recent papers have found NaV1.7 to indeed be important for neuropathic pain. Knockdown of microRNA-

30b, known to regulate NaV1.7 channel expression, resulted in pain relief in neuropathic pain modeled in rats via the spared nerve injury model [16]. Li et al also demonstrated that NaV1.7 was upregulated in chemotherapy-induced peripheral neuropathy (CIPN) and that intrathecal injection of ProTx II, which targets NaV1.7, significantly attenuated behavioral sings of CIPN in human patients [17]. Additionally, mRNA for NaV1.7 is known to be upregulated in diabetes and knockdown of NaV1.7 has been shown to reduce diabetic neuropathy [18]. As expected, selectively inhibiting NaV1.7 increases the firing threshold in both rodent and human DRGs and also reduces neurotransmitter

4 release from peripheral and central nerve terminals, which provides a mechanistic understanding of the importance of NaV1.7 in transmitting pain signals [19-21].

NaV1.8 is another NaV channel expressed in sensory neurons and thought to contribute to the transmission of pain signals from the periphery. Gain-of-function mutations in NaV1.8 have also been described in a cohort of patients suffering from painful neuropathies [22]. Mice deficient in NaV1.8 showed deficits in both inflammatory and visceral pain [23]. Research has shown that NaV1.8 also has an important role for maintaining hypersensitivity and allodynia is several rodent chronic pain models. NaV1.8 mRNA is upregulated in a sciatic nerve injury-induced painful neuropathy in rats, and administration of NaV1.8 shRNA could attenuate SNE-induced pain symptoms [24]. It has also been shown that NaV1.8 is upregulated in A afferent fibers subjected to chronic peripheral inflammation and that blocking NaV1.8 reduced the excitability of A- fibers, which lead to reduced mechanical allodynia thresholds induced by a chronic inflammatory pain model [25]. In addition, Wu et al. also found that the phosphorylation of NaV1.8 increases channel function and is involved in the development of mechanical hyperalgesia in mice [26]. Furthermore, a potent and selective NaV1.8 channel blocker,

A-803467, was able to attenuate neuropathic and inflammatory pain behaviors in rats

[27]. NaV1.8 was also found to be important for the development of diabetic pain [28].

Methylglyoxal, which is enriched in the plasma of diabetic patients suffering from diabetic-related pain, was found to enhance the activity of NaV1.8, which lead to increased electrical excitability in pain neurons. The role of NaV1.8 in visceral pain is still

-/- not fully understood. In one model of acute visceral hypersensitivity, NaV1.8 mice show a dampened behavioral response to intracolonic capsaicin and mustard oil and a

5 lack of hyperexcitability upon Nippostrongylus brasiliensis infection-induced transient jejunitis [29, 30]. However, in a chronic model of visceral hypersensitivity using

-/- cyclophosphamide to induce cystitis, NaV1.8 showed normal behavior [29]. Although this discrepancy could be attributed to the different models used, it’s clear that more research is needed to fully understand the role NaV1.8 plays in visceral pain.

Although NaV1.7 and NaV1.8 have received a lot of attention since mutations in these isoforms are thought to lead to either insensitivity to pain or to extreme pain disorders, NaV1.9 has recently been realized to have a much greater importance for the transmission of pain stimuli. NaV1.9 expression is also restricted to in the peripheral nervous system, mainly in the sensory neurons of the DRG and TG [1].

The first KO mouse models of NaV1.9 found that this channel was not essential for the processing of basal mechanical or thermal noxious stimuli [31, 32]. However, accumulating evidence generated from recordings on acutely dissociated DRGs suggests that NaV1.9-mediated currents may be dramatically enhanced by a variety of inflammatory mediators [31, 33, 34]. In addition, pain hypersensitivity elicited by inflammatory mediators such as bradykinin, ATP, prostaglandin E2, capsaicin, and others is either drastically reduced or absent in NaV1.9 KO mice [31, 33]. Although

NaV1.9 is involved in the processing of hypersensitivity induced via acute inflammation, reports on its involvement in chronic inflammation are somewhat confusing. Several groups have reported an increase in mRNA levels of NaV1.9 and a reduction in inflammatory pain assays after models of chronic inflammation, while others have reported no change in expression patterns and no difference in behavioral assays after

-/- chronic inflammation [35, 36]. It has also been reported that NaV1.9 mice had

6 substantially reduced mechanical hypersensitivity after application of inflammatory soup

(bradykinin, ATP, histamine, PGE2, and 5HT), which would suggest an important role for NaV1.9 is the visceral pain associated with IBS [37]. Recently however, Leipold et. al. discovered a gain-of-function mutation in human Nav1.9 that produced insensitivity to pain, although these patients suffered from varying degrees of gastrointestinal discomfort and itch [38]. Only a short time later, other groups began to identify Nav1.9 gain-of-function mutations in human patients that resulted in extreme pain [39-41].

Additionally, NaV1.9 also appears to be crucial for the processing of cold sensation and cold allodynia, formerly exclusively attributed to NaV1.8. Lolignier et al. showed that

NaV1.9 can act as a subthreshold amplifier in cold-sensitive neurons and that action potentials in these neurons require functional NaV1.9 [42]. Interestingly, they also determined that oxaliplatin-induced cold allodynia is completely absent in NaV1.9 deficient mice. These recent findings have catapulted NaV1.9 into the spotlight for pain, as it becomes increasingly clear that this channel has a much more prominent role to play in pain processing than previously thought.

In addition, other NaV channels previously underappreciated in the pain field have recently been implicated as important players in certain modalities of pain. NaV1.1 mRNA expression is found in predominantly large and medium diameter sensory neurons [43]. Although it was only recently that evidence has been provided for a role of

NaV1.1 in pain transmission. Osteen et al. recently reported NaV1.1 as having an important role in mechanical pain [44]. Hm1a, a toxin from the spider Heteroscodra maculata, was shown to selectively activate NaV1.1 and application of the toxin in mice elicited robust pain behaviors, which were lost in NaV1.1 KO mice. High-threshold

7 mechanosensitive fibers that innervate the guy also showed enhanced toxin sensitivity in a mouse model of IBS.

And as previously mentioned, NaV1.3 is expressed in developing sensory neurons. However, its exact contribution to pain modulation is still controversial. It is known that NaV1.3 is significantly up-regulated following axotomy and inflammation [43,

45, 46]. Additionally, it has been reported that NaV1.3 is up-regulated during spinal cord injury and that there are attenuated pain behaviors associated with chronic constrictive injure (CCI) of the peripheral nerve, a mouse model of spinal cord injury, in NaV1.3 null mice [47, 48]. However, several other researchers have reported normal responses to pain behaviors in acute, inflammatory, and neuropathic pain models after knockdown of

NaV1.3 [7, 49]. Therefore, the exact role of NaV1.3 in pain transmission is still incomplete.

It should come as no surprise that NaV channels have such an important role in the transmission of pain sensation. However, it has been appreciated more now that

NaV channels do in fact have modality specific contributions to pain sensations, and that these channels are not redundant, but that they each have a unique contributing role in the transmission of sensations. Also, although the pain phenotypes in mice don’t seem as striking as in human patients with NaV channel mutations, it’s possible that different compensatory mechanisms exist between species, such as the case with loss of NaV1.7 in humans which leads to upregulation of enkephalin precursor, which may not be upregulated to the same extent in mice [50].

8 NaV channels and itch

Although the role of NaV channels in pain sensations is an important avenue of research, not as much attention has been paid to their role in other forms of somatosensation. Specifically, what functional roles do Nav channels have in terms of pruritus, or itch? Just as there are modality specific nociceptors, it has come to be appreciated that there are many different types of pruriceptors. And since there are certain NaV channels that contribute more to one type of pain versus another, it would be interesting to see if this is the case for itch as well. To date, no NaV channel has been extensively studied with respect to itch sensation. This is primarily due to the lack of knowledge in the itch field that has only recently started to become addressed.

Histamine, which is a compound released by basophils and mast cells in response to allergic or inflammatory insults, was the first compound known to induce an itch response immediately following a local application in human patients [51]. It is known to target a family of G-protein coupled receptors (HRH1-HRH4), with the transmission of itch most likely via HRH1 and HRH4 [52]. Bovine adrenal medulla 8-22

(BAM8-22) and chloroquine (CQ) were two others compounds that were known to induce itch through a histamine-independent pathway, but the receptors they targeted were only recently discovered. As it turns out, these compounds targeted another family of GPCRs called the Mass gene related protein receptors (Mrgpr) [53]. A cluster knockout of several members in this family led to a drastic reduction in induced itch induced by BAM8-22 and CQ, but left histamine-dependent itch in tacked. Liu et al. determined that BAM8-22 and CQ targeted MrgprC11 and MrgprA3, respectively.

Further experiments revealed that specifically activating the MrgprA3+ population led to

9 an increase in itch-type behavior, without evoking pain related behaviors [54, 55]. It is also known that the downstream mediators of histamine and Mrgpr activation include the family of tandem repeat protein (TRP) channels. Specifically, TRPV1 is important for histamine receptor activation while TRPA1 is important for Mrgpr activation [54, 56-58].

However, one of the most vital downstream proteins involved in transmitting these pruritic stimuli would be NaV channels, but very few studies have focused on investigating the contribution these important ion channels in itch.

Global NaV1.7 KO mice showed almost complete absence of scratching bouts in response to histamine injections [59]. In addition, patients with gain-of-function mutations in NaV1.7 and NaV1.8 showed extreme paroxysmal neuropathic itch attacks

[60].

Interestingly, patients with the gain-of-function mutation reported by Leipold et al also complain of extreme itch while at the same time being unable to feel most pain- inducing stimuli. Woods et al. reported that several of the patients with the p.L811P gain-of-function mutations suffered from extreme pruritus, and that they scratched excessively to the point of full-thickness skin loss during infancy [61]. Although the role of NaV1.9 in pain is well establish, its role in itch is unknown. This is primarily due to the difficulty in working with this channel and expressing it in a heterologous system, which is needed for functional analysis. To date, two other unique gain-of-function mutations have been reported in NaV1.9 that have also lead to a loss of pain, but a gain of itch phenotype [40].

However, apart from the histamine response reported in the global NaV1.7 KO mice, and the neuropathic itch reported in several patients with a gain-of-function

10 mutation, no study has explored what the functional role of NaV channels is in terms of the transmission of pruritic stimuli. And given how important NaV channels are to the transmission of most sensory stimuli, it would prudent to begin focusing on how these important ion channels contribute to the sensation of itch.

NaV channels and the enteric nervous system

The enteric nervous system (ENS) is a semi-autonomous part of the nervous system composed of neural circuits intertwined into a mesh-like, self-reinforcing network that are essential for secretion, locomotion, absorption, paracrine and autocrine signaling [62-64]. It is comprised of two layers embedded in the gastrointestinal tract that are termed the myenteric plexus and the submucosal plexus, which are separate by a layer of smooth muscle. The ENS is composed of roughly 500 million neurons, which is significantly less than the brain, but slightly higher than the number of neurons comprising the spinal cord. Although somewhat autonomous, the ENS can still receive input from the central nervous system (CNS) through the parasympathetic (via the vagus nerve) and sympathetic (via the prevertebral ganglia) nervous system.

Interestingly, the ENS synthesizes and utilizes many of the same neurotransmitters found in the CNS with more than 90% of the body’s serotonin and about 50% of the body’s dopamine being used there. The ENS also contains all the necessary components for a polysynaptic reflex arc, such as intrinsic primary afferent neurons

(IPANs), interneurons, and motor neurons [62, 65].

The ENS also contains several members of the NaV channel family, namely

NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 [66, 67]. Specifically, NaV1.3 is expressed

11 in enterochromaffin cells and is a critical player in controlling the excitability and serotonin release from these cells [68, 69]. An estimated 2% of patients with IBS carry mutations in NaV1.5 [70]. And a majority of those mutations are loss-of-function, which is mostly associated with the constipation commonly suffered by IBS patients. Although

NaV1.6 and NaV1.7 are known to be expressed in the ENS, their exact functional relevance is yet to be discerned [71, 72].

A more recent discovery is that NaV1.9 may play an important role in the enteric nervous system (ENS) where the channel shapes action potentials in neurons that communicate with each other and with the principal cells of the ENS-controlled gastrointestinal (GI) tract [73]. Recent evidence has shown robust NaV1.9 expression in both the myenteric and submucosal plexi. However, expression of NaV1.9 differs from that in the DRGs or TGs. NaV1.9 appears to co-localize with peripherin, as it does in DRGs, but also with NF-200, a marker for larger, myelinated and often non-nociceptive neurons

[74]. Although a more thorough characterization of its expression pattern is lacking, it has been hypothesized to be not only functionally important in health and disease, but also in homeostatic mechanisms of the gastrointestinal system. For instance, knocking out NaV1.9 in mice leads to drastic changes in colonic migrating motor complexes

(CMMCs), movements produced entirely by the ENS that drive digestive content through the entire length of the gut. Mice deficient in NaV1.9 produce CMMCs more frequently than controls, but at shorter durations, and with much more force [73].

Furthermore, intrinsic signaling within the ENS typically involves slow EPSPs acting via neurokinin 3 receptors. It has been shown that activation of these receptors leads to a substantial increase in NaV1.9 current in ENS neurons [75]. Other NaV channels, such

12 as NaV1.5, have already been established as contributing to gastrointestinal disorders such as irritable bowel syndrome (IBS) [70]. Given the vastly different environment these neurons are exposed to, such as bacterial metabolites, parasites, antibiotics, and other toxic chemicals, identifying potential drug targets is of great importance.

In addition, there are the complications that arise from visceral hypersensitivity, particularly in the gut. Although this is a hallmark of IBS, this is generally due to nociceptive afferents innervating the gut rather than through neurons of the enteric nervous system, and as such, was discussed in the previous section about NaV channels and pain.

Unfortunately, chronic pain and itch are still major unmet health issues, and many of the current treatments are either inadequate or suffer from terrible side-effects.

Especially worrisome is the current opioid epidemic, with 115 Americans dying every day on average from an opioid overdose. Therefore, identifying new targets to treat these debilitating conditions is of great importance. Knowledge about the functional importance of NaV channels in different modalities of pain and itch will greatly aid in future drug developmental efforts. And hopefully, current and future insights into NaV channels and their importance in pain and itch will help in the development of novel and more potent pain and itch inhibitors.

13 THE ROLES OF NAV1.9 AND NAV1.1 IN SOMATOSENSORY PERCEPTION

Chapter 2: The role of NaV1.1 channel in gastrointestinal physiology

This chapter was adapted from:

Salvatierra, J., et al., NaV1.1 inhibition can reduce visceral hypersensitivity. Journal of

Clinical Investigation Insight, 2018. 3(11).

14 Summary

To understand what role NaV channels serve in both visceral nociception and motor function of the gut, we took advantage of compound B, a selective NaV1.1 channel inhibitor. Inhibition of NaV1.1 led to a reduction in hypersensitivity in three separate mouse models of mechanical hypersensitivity. We find that in two models of visceral hypersensitivity, compound B reduced sodium current densities in colonic- innervating DRG neurons. In addition, the visceral motor reflex (VMR) in response to colorectal distension (CRD) was significantly reduced in both mouse models of irritable bowel syndrome (IBS) after the application of compound B. These findings shed light on a new molecular target that’s important for the development of mechanical hypersensitivity and provides compelling evidence for the development of compounds that target NaV1.1 to ameliorate the pain associated with IBS. In fact, structurally similar compounds that are also selective for NaV1.1 show a similar effect in the acetic-acid IBS mouse model. By tweaking the structure of compound B, we identified new compounds with a greater efficacy in the improvement of mechanical hypersensitivity in a mouse model of IBS.

15 Introduction

Functional bowel disorders (FBDs) such as irritable bowel syndrome (IBS), constipation, diarrhea, and abdominal bloating occur worldwide, and effective treatment is a major unmet clinical need in gastroenterology [76]. Typically, FBDs are associated with alterations in bowel habits that result in a substantially decreased quality of life.

Because of their prevalence, they are also a considerable drain on health care resources [77]. People suffering from FBDs can have an array of symptoms.

Specifically, the most common symptom in ≥40% of the IBS patient population [78] is chronic abdominal pain. A prominent hypothesis for the etiology of this abdominal pain is mechanical hypersensitivity of sensory fibers that innervate the gut [79-83]. Although a range of channels and receptors have been implicated [70, 84-93], our understanding of chronic visceral hypersensitivity (CVH) is still poor. Not surprisingly, therefore, pharmacological management of chronic abdominal pain is nonspecific and ranges from

NSAIDs to opioids, without or with adjuvant analgesics [94]. Anticonvulsants such as gabapentin and pregabalin show promise to treat CVH, but there is insufficient data to support their effectiveness in FBD patients, and all use is considered off-label [95].

Cell membrane–embedded voltage-gated sodium (NaV) channels regulate cellular excitability and initiate action potentials in the peripheral, central, and enteric nervous systems [73, 96-99]. A subset of the 9 NaV channel subtypes is expressed in the enteric nervous system, and mutations can lead to gastrointestinal disorders, including constipation and diarrhea [61, 70]. The discovery that NaV1.1 can regulate the excitability of sensory nerve fibers that mediate mechanical pain [44] led to the

16 hypothesis that this channel subtype may also underlie the development of abdominal pain in FBD states. In our previous studies, consistent with this, a spider toxin (Hm1a) that activates NaV1.1 increased mechanically evoked spiking in high-threshold colonic nociceptors in gut-nerve preparations from healthy control mice. Furthermore, baseline mechanosensory responses of colonic afferents from CVH mice were significantly increased compared with healthy control mice, and application of Hm1a enhanced mechanically evoked spiking beyond this already elevated level. Finally, in contrast to healthy control animals, toxin application evoked a pronounced increase in the electrical excitability of colonic dorsal root ganglion (DRG) neurons from CVH mice, suggesting that NaV1.1 channels are functionally upregulated in CVH states [44]. Taken together, these experiments suggest that inhibiting NaV1.1 function could reduce chronic abdominal pain related to FBDs. This hypothesis remains to be tested, since we previously employed a nonspecific NaV channel inhibitor in isolated mouse colonic afferents, not in behavioral experiments.

Here, we administered a selective NaV1.1 inhibitor— 1-(phenylmethyl)-1H-1,2,3- triazole-4-carboxamide-5-methyl (Compound B) [100] — in a mouse model for peripheral afferent–mediated mechanical pain and 2 CVH paradigms in order to test whether mechanical hypersensitivity can be alleviated. We demonstrate that NaV1.1 inhibition significantly reduces mechanical pain in CVH states, thereby substantiating an important contribution of this NaV channel subtype to FBD-associated chronic abdominal pain.

17 We also sought to modify the structure of Compound B to identify parts of the structure important for the selectivity of NaV1.1 and for future development compounds with a greater efficacy. In line with this, we synthesized 19 compounds and found 9 compounds that altered either the steady-state activation or inactivation of NaV1.1. Out of the 10 compounds that had a significant effect, one – Compound F - showed a similar shift in the steady-state activation of NaV1.1 as Compound B. Compound F was shown to have a more potent effect than Compound B in a CVH mouse model.

18 Results

Pharmacological target screen of compound B

Compound B (100 μM) was previously shown to inhibit NaV1.1 opening by depolarizing its conductance-voltage (G-V) relationship, whereas other channel gating properties were unaffected [100]. Here, we tested compound susceptibility of additional

NaV channel subtypes that have also been found in gut nerves [29, 70, 101]. When applying 100 μM Compound B to NaV1.5, NaV1.7, and NaV1.8, none of the channels showed an altered G-V relationship (Fig. 3-1). At 1 μM, Compound B still inhibited

NaV1.1 activation similar to 100 μM, which is likely to be a saturating concentration. To further investigate potential nonspecific activity of Compound B at 100 μM, we carried out a compound safety screen on 44 commonly tested targets (Table 3-1). With 1 exception, Compound B did not interact with any of the receptors, ion channels, or transporters. Of the enzymes, only phosphodiesterase 4D isoform 2 (PDE4D2) shows a mildly significant inhibitory response to 100 μM Compound B. This protein has 3′,5′- cyclic-AMP phosphodiesterase activity and degrades cAMP, a signal transduction molecule in various cell types. PDE4 inhibitors are known to possess procognitive, neuroprotective, and antiinflammatory effects. Emetic effects caused by PDE4 inhibition have also been reported [102] but were not observed in our experiments.

Pharmacokinetic profile of Compound B

To make optimal use of our animal models, we determined the pharmacokinetic (PK) profile of Compound B. We found that the molecule was metabolically stable in mouse and human plasma over a period of 60 minutes (Fig. 3-2). Additionally, in both mouse

19 and human liver microsomal incubations fortified with NADPH, the compound was stable (>95% remaining), suggesting a resistance to CYP-450–dependent oxidation. A positive control with testosterone was completely metabolized, confirming assay validity

(Fig. 3-2).

We next examined the plasma PK, brain, and CSF distribution of Compound B in male

C57BL/6J mice. Following a single i.v. administration, Compound B (10 mg/kg) showed low plasma clearance (~11 ml/min/kg) with an elimination half-life of ~1.5 hours (Fig. 3-

3 and Table 3-2). The brain/plasma concentration ratio ranged between 0.6 and 1.7, and the CSF/plasma concentration ratio ranged between 0.3 and 0.5. After a single i.p. dose administration of Compound B (10 mg/kg), plasma, CSF, and brain concentrations were detected up to 24 hours with a time at maximum (Tmax) of 0.5 hours. The brain/plasma concentration ratio ranged from 0.8–1.8, and the CSF/plasma concentration ratio fluctuated between 0.3 and 0.8. A single s.c. dose administration allowed detection of Compound B in plasma, CSF, and brain concentrations up to 8 hours with a Tmax of 1 hour. The brain/plasma ratio ranged between 0.8 and 1.2, and the

CSF/plasma ratio was found to be between 0.3 and 0.5. Following a single per os (p.o.) administration of the compound, plasma, CSF, and brain concentrations were detected up to 8 hours with a Tmax of 0.5 hours in plasma and 0.25 hours in the brain and CSF.

The brain/plasma ratio ranged between 0.8 and 1.2, whereas the CSF/plasma ratio oscillated between 0.1 and 0.7. The oral solution bioavailability was 79%.

20 Toxicity

To examine the possibility of adverse side effects interfering with the interpretation of our animal model experiments, we performed a comprehensive toxicity study. In Phase

A, single i.v. doses of 10, 25, 50, or 100 mg/kg Compound B were well tolerated in male and female Sprague Dawley rats. There were no treatment-related effects on survival or clinical signs such as body/organ weight changes, food consumption discrepancies, or visible inflammation. In Phase B, daily i.v. doses of 25, 50, or 100 mg/kg Compound B over the course of 7 days were well tolerated in male and female Sprague Dawley rats.

There were no consistent or treatment-related effects on endpoints, including survival, clinical signs, body weight, clinical chemistry, blood chemistry, hematology, urinalysis parameters, absolute or relative organ weights, or histopathology.

Compound B reduces peripheral nerve injury–induced mechanical hypersensitivity

We previously demonstrated that spider toxin–mediated (Hm1a-mediated) activation of

NaV1.1 in peripheral sensory neurons elicits robust pain behaviors [44]. Interestingly, profound mechanical but not thermal hypersensitivity was produced by Hm1a without neurogenic inflammation, indicating that Hm1a does not target unmyelinated peptidergic nociceptors. Conversely, partially eliminating NaV1.1 from sensory neurons using a genetic approach significantly attenuated the toxin-evoked pain behaviors, indicating that this channel subtype regulates the excitability of primary afferent fibers that mediate mechanical pain. Before embarking on FBD mouse model trials, we sought to corroborate the contribution of NaV1.1 to peripheral afferent-mediated mechanical pain.

21 In these studies, we tested whether inhibiting NaV1.1 with Compound B can ameliorate the hypersensitivity that occurs in a mouse model of neuropathic pain. For this, we used the spared nerve injury (SNI) model in which 2 of 3 branches of the sciatic nerve are transected. SNI mice rapidly exhibited profound and long-lasting mechanical hypersensitivity in response to von Frey hairs (vfh; see Methods). As expected, 3 days after SNI, we recorded a significant reduction (~57%) of the mechanical thresholds leading to mechanical hypersensitivity, ipsilateral to the injury side (Fig. 3-4). At 7 days, this mechanical hypersensitivity was significantly reduced 30 minutes after systemic injection of a single dose of Compound B (i.p., 60 mg/kg). Contralateral mechanical thresholds were unaffected by the compound. One month after SNI, mice received another single dose of Compound B, and again, we recorded a significant reduction in mechanical hypersensitivity ipsilateral to the injury side (i.p., 60 mg/kg; Fig. 3-4).

Importantly, the same dose did not affect baseline thresholds, nor did it impair motor functions in naive mice, despite the compound penetrating the blood-brain barrier (Fig.

3-5; rotarod test). Taken together, these results show that NaV1.1 contributes, at least in part, to the mechanical hypersensitivity that develops after peripheral nerve injury.

Sodium current recordings in colon-innervating DRG neurons

In addition to eliciting behaviors indicative of pain, Hm1a-induced activation of NaV1.1 also evokes neuronal hypersensitivity in a subpopulation of retrograde-labeled colon- innervating DRG neurons [44]. Here, we determined if the inhibitory effect of Compound

B on NaV1.1 reduces peak sodium current density in subpopulations of these neurons

(Figure 3-6 and Fig. 3-7). We found that Compound B inhibited sodium currents from 17

22 of 39 (44%) colonic-innervating DRG neurons tested and caused an overall ~23% decrease in peak sodium currents in affected neurons, likely originating from inhibiting the NaV1.1 current component, whereas other NaV channel subtypes are unaffected.

Colonic nociceptor recordings

We showed that the NaV1.1 activator Hm1a also induces mechanical hypersensitivity of colonic nociceptive afferents, an effect that is enhanced in a mouse model of IBS induced by intracolonic (i.c.) administration of trinitrobenzenesulfonic acid (TNBS) [44].

Therefore, we tested whether NaV1.1 inhibition by Compound B reduces colonic nociceptor mechanical responses in both healthy and CVH states (Fig. 3-8). A substantial population of colonic nociceptors (6 of 9) from healthy mice was inhibited by application of Compound B, reducing responses by ~25% relative to baseline responses in affected afferents (Fig. 3-8 and Fig. 3-9). In CVH mice, 75% of colonic nociceptors were inhibited by Compound B, and their response was reduced by ~35% relative to

CVH baseline responses. In both control and CVH mice, Hm1a (100 nM) was unable to overcome Compound B inhibition of colonic nociceptor mechanical responses (Fig. 3-

10). Based on these findings, we next asked whether NaV1.1 inhibition also reduces behavioral measures of mechanical hypersensitivity in representative FBD animal models.

Visceral motor reflex (VMR) in response to colorectal distension (CRD)

In these studies, we monitored the VMR to CRD in 2 commonly used mouse models of

IBS representing FBDs. The VMR technique involves placement of a flexible inflatable

23 balloon wrapped around a pliable catheter into the descending colon [103]. To quantify the effect of balloon pressures (in mmHg), we measured abdominal electromyogram

(EMG) activity of the abdominal musculature via surgically implanted electrodes. As expected, IBS mice with TNBS-evoked CVH displayed significantly enhanced VMRs compared with healthy control animals. I.c. administration of Compound B (100μM) (i.e., directly applied to the peripheral endings of the colonic nociceptors) to IBS mice reduced VMRs to CRD to healthy control levels (Fig. 3-11). Compared with healthy control mice, colonic compliance was unaltered in CVH mice or in CVH mice that were administered 100 μM Compound B (Fig. 3-12), suggesting that changes in the VMR to

CRD are not due to variations in smooth muscle function. These findings indicate that pharmacologically inhibiting NaV1.1 could be a viable approach to reverse visceral hypersensitivity in vivo.

In a second experiment, we systemically applied Compound B at 2 doses (i.p.) in control and acetic acid–evoked IBS mice and then measured the VMR to CRD. At 20 mg/kg, acute treatment with Compound B in IBS mice returned pain thresholds to those of the saline-injected healthy controls (Fig. 3-13). At a higher dose (75 mg/kg),

Compound B reduced mechanical pain thresholds in IBS mice to below the level of control mice. Based on the PK experiments, Compound B absorption via s.c. injection leads to a lower peak concentration in plasma with a longer Tmax of 1 hour. Therefore, higher concentrations may be needed to normalize mechanical pain thresholds in CVH mice. Indeed, acute treatment with Compound B at a dose of 75 mg/kg (s.c.) significantly reduced the increased pain thresholds in CVH mice to a level noted in saline-injected controls (Fig. 3-14). Together, these in vivo experiments suggest that

24 NaV1.1 inhibition can be an effective approach to reduce mechanical hypersensitivity associated with CVH states.

Rufinamide Derivatives show differential effects on hNaV1.1 gating

Compound B was developed based on the structure of rufinamide. Therefore, we wanted to see how other derivatives of rufinamide effected hNaV1.1. Out of the 19 compounds that were synthesized, we determined that 9 of the 19 compounds altered either SSI or GV of hNaV1.1 (Fig. 3-18 and Tables 3-3, 3-4, and 3-5), while the remaining 10 had no effect on either SSI or GV (Fig. 3-19 and Tables 3-6, 3-7, and 3-8).

Although 9 out of the 19 compounds had a significant effect on hNaV1.1, we found only

1 out of the 9 that had a similar depolarized shift on the GV of hNaV1.1 as compound B.

We tested this compound – which we refer to as Compound F – in the acetic-acid evoked mouse model of IBS and measured VMR to CRD. We found that a lower dose of compound F was needed to reduce the VMR back to baseline when compared to compound B (Fig. 3-20). At 5 mg/kg, acute treatment with Compound F in IBS mice returned pain thresholds to those of the saline-injected healthy controls (Fig. 3-20).

25 Discussion

FBDs represent a major clinical problem in gastroenterology, with a large population of patients experiencing chronic abdominal pain secondary to visceral hypersensitivity to mechanical stimuli [76]. Unfortunately, the mechanisms underlying

CVH are unclear, and pharmacological management of chronic abdominal pain is therefore challenging [104-107]. Accumulating evidence implicates a subset of

NaV channel subtypes in normal gut sensory function and in the development of mechanical hypersensitivity and pain associated with injury [44, 70, 96, 98].

Together with previously reported observations [44], the results presented here suggest that NaV1.1 is functionally upregulated under CVH conditions (Figure 3-11) and that pharmacologically inhibiting this channel subtype can reduce CVH in 2 mechanically distinct mouse IBS models (Fig. 3-11, 3-13, and 3-14) without altering colonic compliance (Fig. 3-12). We show that Compound B, a NaV1.1-targeting inhibitor of channel gating [100], reduces colonic nociceptor mechanical responses and can reduce the significantly enhanced VMRs in 2 mouse models of IBS to levels that are observed in healthy control animals. Importantly, as Compound B has ~79% oral bioavailability, without noticeable signs of toxicity upon acute or chronic dosing, our findings suggest a possible pathway toward the design of novel NaV1.1-inhibiting therapeutics for FBD-related visceral pain (Fig 3-3 and Table 3-2). Although extrapolating preclinical findings in rodents to human chronic conditions such as IBS is challenging, the ability to target the etiology at the level of the peripheral afferent may have many advantages. Therefore, it is worth exploring whether synthesis of compound

26 derivatives that do not penetrate the blood-brain barrier could avoid potential adverse side effects that might occur when even higher but even more effective doses are introduced.

It is also of interest that mutations in NaV1.1 have been linked to an array of epilepsy phenotypes [108]. Via a subtle action on NaV1.1 function, Compound B increases the threshold to action potential initiation in hippocampal neurons, thereby reducing the frequency of seizures in various animal models [100]. Given the role of

NaV1.1 in both epilepsy and CVH, it is therefore reasonable to assume that a subset of epilepsy patients may be prone to FBDs. Indeed, an observational study on 65 people with epilepsy showed a significantly increased prevalence of IBS compared with controls [109]. Conversely, a large-scale study found that IBS patients had greater cumulative incidence of epilepsy compared with the control cohort [110]. As people with epilepsy are not routinely screened for FBDs, appropriate treatment may be delayed; it is also conceivable that gastrointestinal complaints may be erroneously attributed to administration of antiepileptic drugs. Alternatively, the central contribution of NaV1.1 in both disorders may provide a rationale to administer clinically used anticonvulsants in

FBD patients to treat CVH.

27 Materials and Methods

Two-electrode voltage-clamp recording from Xenopus oocytes.

Human NaV1.5 (hNaV1.5), hNaV1.7, and hNaV1.8 (SCN5a, SCN9a, and SCN10a, respectively) and hβ1 (SCN1b) clones were obtained from OriGene Technologies Inc. and expressed in Xenopus laevis oocytes (Xenopus 1). The DNA sequence of all constructs was confirmed by automated DNA sequencing. RNA was synthesized using

T7 polymerase (Invitrogen). Channels were expressed with hβ1 in a 1:5 molar ratio, and currents were studied following incubation for 1–4 days after cRNA injection (incubated at 17°C in (mM) 96 NaCl, 2 KCl, 5 HEPES, 1 MgCl2, 1.8 CaCl2, and 50 μg/ml gentamycin pH 7.6 with NaOH) using 2-electrode voltage-clamp recording techniques

(OC-725C, Warner Instruments). Data were filtered at 4 kHz and digitized at 20 kHz using pClamp10 (Molecular Devices). Microelectrode resistances were 0.5–1 MΩ when filled with 3M KCl. The external recording solution (ND100) contained (mM) 100 NaCl, 5

HEPES, 1 MgCl2, and 1.8 CaCl2 (pH 7.6 with NaOH). All experiments were performed at ~22°C. Leak and background conductances were subtracted by blocking

NaVchannels with 10 μM tetrodotoxin. Chemicals were obtained from MilliporeSigma, unless otherwise stated. Voltage-activation relationships were obtained by measuring peak currents and calculating conductance (G). Compound B was dissolved in DMSO

(~5 mg/ml stock) and diluted to 100 μM with ND100. Oocytes were incubated in solutions containing 100 μM drug (final DMSO concentration of ≤1%) for 1 hour. Data analysis was performed using Microsoft Excel and Origin 8 (OriginLab).

28 Compound safety screen on 44 targets. A saturating Compound B concentration (100

μM, LifeTein) was tested on 44 targets using a binding/competition assay with scintillation counting. Method and target are shown in Supplemental Table 1. The study was carried out by Eurofins Pharma Discovery Services.

Metabolic stability of Compound B in plasma and liver microsomes.

Plasma stability was evaluated using plasma from mice and humans as described previously [111, 112]. Briefly, Compound B (10 μM) was spiked in plasma and incubated in an orbital shaker at 37°C. At predetermined times, aliquots of the mixture, in triplicate, were removed and the reaction quenched by the addition of 3× the volume of ice-cold acetonitrile spiked with the internal standard losartan (500 nM). The samples were vortexed for 30 seconds and centrifuged at 12,000 g for 10 minutes. Compound disappearance was monitored over time using a Liquid chromatography–tandem mass spectrometry (LC/MS/MS) method. Phase I metabolic stability assay was conducted in mouse and human liver microsomes as described previously [113]. The reaction was carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of a

NADPH regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM

MgCl2, 0.4 U/ml glucose-6-phosphate dehydrogenase, 50 μM sodium citrate). Reactions in triplicate were initiated by the addition of liver microsomes to the incubation mixture

(compound final concentration was 10 μM; 0.5 mg/ml microsomes). Negative controls in the absence of NADPH were performed to determine the specific cofactor–free degradation. Testosterone was used as a positive control. Chromatographic analysis was performed using an Accela ultra high–performance system consisting of an analytical pump and an autosampler coupled with a TSQ Vantage mass spectrometer

29 (Thermo Fisher Scientific). Separation of analyte was achieved at ambient temperature using an Agilent Eclipse Plus column (100 × 2.1 mm i.d.) packed with a 1.8 μm C18 stationary phase. The mobile phase used was composed of 0.1% formic acid in acetonitrile and 0.1% formic acid in H2O with gradient elution.

PK profiling of Compound B.

Healthy male C57BL/6J mice (8–12 weeks old) weighing between 20–35 g were obtained from In Vivo Biosciences. Temperature and humidity were maintained at 22°C

± 3°C and 40%–70%, respectively, and illumination was controlled to give a sequence of 12-hour light and 12-hour dark cycle. Animals were administered with compound solution formulation (10 mg/kg) prepared in 40% w/v hydroxypropyl-β-cyclodextrin in normal saline through p.o., i.p., i.v., and s.c. route. A group of 12 mice was used in each study. Blood samples were collected under light isoflurane anesthesia at 0.1, 0.5, 1, 2,

4, and 8 hours in labeled micro centrifuge tube containing K2EDTA as anticoagulant.

Immediately after blood collection, plasma was harvested by centrifugation and stored at –70°C until bioanalysis. Following blood collection, animals were euthanized by

CO2 asphyxiation, and brain and CSF were collected at each time point. Collected brain was dipped in 20 ml fresh phosphate buffer saline (pH 7.4) 3 times and dried on blotted paper. Brain was weighed and homogenized using ice-cold phosphate buffer saline (pH

7.4), and homogenates were stored below –70°C until bioanalysis. Total homogenate volume was 3× the brain weight. All samples were processed for analysis by protein precipitation using acetonitrile and analyzed with a fit-for-purpose LC/MS/MS method

(lower limit of quantification (LLOQ) = 5 ng/ml in plasma, brain, and CSF).

30 Compound toxicity study in rats.

This study consisted of 2 phases: A and B. In Phase A, the maximum tolerable dose

(MTD) of the NaV1.1 compound was determined. In Phase B, the dose range of the compound was investigated together with the toxicokinetic (TK) profile. Phase A consisted of 4 treatment groups of 3 male/female Sprague Dawley rats (Envigo) dosed with the compound orally, once daily for 5 days at 10 ml/kg. Phase A rats were treated with 10 mg/kg, 25 mg/kg, 50 mg/kg, and 100 mg/kg compound. Phase B consisted of 3 compound treatment groups of 5 male and 5 female — and 1 vehicle control group of 3 male and 3 female — Sprague Dawley rats dosed orally once daily for 7 days at 10 ml/kg. Phase B rats were treated with 25 mg/kg, 50mg/kg, and 100 mg/kg compound, and the group of 3 males/females received the vehicle, 15% DMSO, 35% PEG 400, and

50% sterile water and served as the vehicle control. Phase B included a TK cohort with

6 males/females in each treatment group and 3 males/females in the vehicle control group that were bled at 6 time points following dosing on study day 1 and day 7. Body and organ weights were collected, as well as urine for urinalysis. Food consumption was also monitored. Necropsies allowed 43 tissue types to be collected for clinical pathology examination. Tissues examined include adrenal mammary gland with skin, aorta ovaries with oviduct, brain, pancreas, cecum Peyer’s patches, colon, pituitary gland, duodenum, prostate, epididymis, rectum, esophagus, salivary gland

(mandibular), eyes with optic nerve, skeletal muscle (thigh) with sciatic nerve, femur with BM (articular surface of the distal end to include femorotibial joint), seminal vesicles with coagulating glands, heart, spinal cord (cervical, thoracic, and lumbar), ileum, spleen, sternum, jejunum, stomach, kidney, testes, lacrimal gland, thymus, larynx,

31 thyroid with parathyroids, tongue, liver (sections from 2 lobes), trachea, lung with bronchi, urinary bladder, lymph node (mandibular), uterus with cervix, lymph node

(mesenteric), and vagina. Animals were obtained from Envigo and were housed in an environmentally controlled room that maintained temperatures of 20°C–24°C and a relative humidity of 30%–70% with a 12-hour light/12-hour dark cycle. Animals were group housed based on group/sex designation, except for overnight urine collection, when animals were individually housed in metabolic caging for no more than 18 hours.

The animals had ad libitum access to drinking water and to Rodent Diet 2916 (Harlan

TEKLAD). The animals were acclimated for 48 or 25 days (Phase A and Phase B, respectively) prior to dosing.

Behavioral analysis in the SNI model of neuropathic pain.

For these experiments, we used adult, male C57BL/6J mice from The Jackson

Laboratory. Mice were anesthetized with isoflurane (2.0%). After skin and muscle incision at the level of the popliteal fossa, we tightly ligated the sural and superficial peroneal branches of the sciatic nerve with 8-0 silk sutures (Ethicon), leaving the tibial nerve intact. Next, the ligated branches were transected distal to the ligature, and ~2.0 mm of each distal nerve stump was removed. Particular care was taken not to stretch or contact the intact spared branch. The overlying muscle and skin were sutured, and the animals were allowed to recover and then returned to their cages. We assessed mechanical sensitivity in this mouse model of neuropathic pain [114] by placing animals on an elevated wire mesh grid and stimulating the hind paw with vfh. We used an up- down paradigm [115] to define threshold. Animals were tested 3 times, once every other

32 day before surgery to determine baseline threshold and once 3 days after surgery to assess the magnitude of the mechanical hypersensitivity. On day 7, mice received an i.p. injection of Compound B (60 mg/kg), and behavioral testing was performed 15, 30, and 60 minutes after the injection. On day 28 after SNI, mice received a single dose (60 mg/kg) of Compound B, and mechanical thresholds were measured 1 hour after. For behavioral tests, the investigator was blind to treatment. Motor performance of the mice injected with Compound B (60 mg/kg) was evaluated with the rotarod test.

Retrograde labeling to identify colonic neurons in DRG and dissociated DRG cell culture.

Healthy, male C57BL/6J mice (The Jackson Laboratory) of 16 weeks were anesthetized with isoflurane, and — following midline laparotomy — five 2-μl injections of a fluorescent retrograde neuronal tracer (cholera toxin subunit B conjugated to

AlexaFluor-488, Thermo Fisher Scientific) were made subserosally within the wall of the descending colon. Mice were administered analgesic (buprenorphine; 0.4 mg/10 kg s.c.) following completion of the surgery. Four days after tracer injection, mice were culled by

CO2 inhalation, and DRG from thoracolumbar (T10-L1) and lumbosacral spinal levels

(L5-S1) were surgically removed. DRGs were digested with 4 mg/ml collagenase II

(GIBCO, Invitrogen) and 4 mg/ml dispase (GIBCO) for 30 minutes at 37˚C, followed by

4 mg/ml collagenase II for 10 minutes at 37˚C. Neurons were mechanically dissociated into a single-cell suspension via trituration through fire-polished Pasteur pipettes.

Neurons were resuspended in DMEM (GIBCO) containing 10% FCS (Invitrogen), 2 mM

L-glutamine (GIBCO), 100 μM MEM nonessential amino acids (GIBCO), and 100 mg/ml

33 penicillin/streptomycin (Invitrogen). Neurons were spot-plated on 15-mm coverslips coated with poly-D-lysine (800 μg/ml) and laminin (20 μg/ml) and maintained in an incubator at 37˚C in 5% CO2.

Neuronal whole-cell electrophysiological recordings.

Male C57BL/6J mice (The Jackson Laboratory) were used in all experiments. Twenty to

48 hours after plating, whole-cell recordings were made from fluorescent colon- innervating DRG neurons using fire-polished glass electrodes with a resistance of 0.7–2

MΩ (≥75% of series resistance was compensated). All recordings were performed at room temperature (20°C–22°C). Signals were amplified with an Axopatch 200A amplifier, digitized with a Digidata 1322A, recorded using pCLAMP 9 software

(Molecular Devices), sampled at 20kHz, filtered at 5kHz, and analyzed in Clampfit 10.7

(Molecular Devices) and GraphPad Prism 7. Voltage-clamp intracellular solution contained (in mM) 60 CsF; 45 CsCl; 2 MgCl2; 5 EGTA-Na; 10 HEPES-Cs; 30 TEA-Cl; and 2 MgATP adjusted to pH 7.2 with CsOH, 280 mOsm. Extracellular solution contained (in mM) 70 NaCl; 50 NMDG; 40 TEA-Cl; 4 CsCl; 2 MgCl2; 2 CaCl2; 10

HEPES; and 5 Glucose adjusted to pH 7.4, approximately 300 mOsm. Current-voltage

(INa-V) relationships were determined by application of a prepulse to –100 mV (100 ms), followed by a series of step pulses from –65 mV to +60 mV (5 mV increments (100 ms)), before returning to hold at –70 mV (repetition interval of 3 sec, P/8 leak subtraction). Neurons inhibited by Compound B (LifeTein) were determined as a >15% reduction from baseline response. Extracellular (bath) solution containing Compound B

34 at 100 μM (2-minute incubation) was applied with a gravity-driven multibarrel perfusion system positioned within ~1mm of the neuron.

Colonic nociceptor afferent recordings.

Male C57BL/6J mice (The Jackson Laboratory) were used in all experiments. In vitro single-unit extracellular recordings of action potential discharge were made of splanchnic colonic afferents from healthy control or IBS mice using standard protocols

[44, 86, 116, 117]. Baseline mechanosensitivity was determined in response to a 3- second application of a 2 g vfh probe to the afferent receptive field. This process was repeated 3–4 times, separated each time by 10 seconds. Mechanosensitivity was then retested after application of Compound B (100 μM). Afferents were considered inhibited by Compound B if we recorded a >10% reduction from baseline response. In some instances, data are presented as percentage change from baseline. This value was calculated as the percentage change in mechanosensitivity of individual afferents between the baseline responses compared with the respective mechanical responses following compound addition. This difference is then averaged across all afferents to obtain a final mean ± SEM of percentage change in response from baseline. The spider toxin Hm1a was purified as previously described [44].

Animal models of FBDs.

Male C57BL/6J mice (The Jackson Laboratory) were used in all experiments. In a first assay, colitis was induced by administration of TNBS as described previously [117,

118]. Briefly, 13-week-old anesthetized mice were administered an i.c. enema of 0.1 ml

35 TNBS (3.8 mg per mouse in 30% EtOH) via a polyethylene catheter. Histological examination of mucosal architecture, cellular infiltrate, crypt abscesses, and goblet cell depletion confirmed that TNBS induced significant damage by day 3 after treatment, largely recovered by day 7, and fully recovered at 28 days. High-threshold nociceptor recordings at the 28-day time point revealed significant mechanical hypersensitivity and lower mechanical activation thresholds. Based on these properties, we consider these mice an IBS model of TNBS-induced CVH.

An abdominal EMG allows assessment of visceral sensitivity in vivo in fully awake animals. Under isoflurane anesthesia, the bare endings of 2 Teflon-coated stainless- steel wires (Advent Research Materials Ltd.) were sutured into the right abdominal muscle, tunnelled s.c., and then exteriorized at the base of the neck for future access.

At the end of the surgery, mice received prophylactic antibiotic (Baytril; 5 mg/kg s.c.) and analgesic (buprenorphine; 0.09 mg/kg s.c.), were housed individually, and were allowed to recover for at least 3 days before assessment of the VMR. On the day of

VMR assessment, mice were briefly anesthetized using isoflurane and received a 100 μl enema of vehicle (sterile saline) or Com pound B (100 μM). A lubricated balloon (2.5 cm length) was gently introduced through the anus and inserted into the colorectum, up to

0.25 cm past the anal verge. The balloon catheter was secured to the base of the tail and connected to a barostat (Isobar 3, G&J Electronics) for graded and pressure- controlled balloon distension. Mice were allowed to recover from anesthesia in a restrainer for 15 minutes prior to initiation of the distension sequence. Distensions were applied at 20, 40, 60, and 80 mmHg (20-second duration) at a 4-minute interval. The last distension was performed 30 minutes after i.c. treatment. The EMG electrodes were

36 relayed to a data acquisition system, and the signal was recorded (NL100AK headstage), amplified (NL104), filtered (NL 125/126, Neurolog, Digitimer Ltd., bandpass

50–5000 Hz), and digitized (CED 1401, Cambridge Electronic Design) to a PC for offline analysis using Spike2 (Cambridge Electronic Design). The analogue EMG signal was rectified and integrated. To quantify the magnitude of the VMR at each distension pressure, the AUC during the distension (20 seconds) was corrected for the baseline activity (AUC predistension, 20 seconds). After the final distension, the mice were killed by cervical dislocation. Colonic compliance was assessed by applying graded volumes

(40–200 μl, 20-second duration) to the balloon in the colorectum of fully awake mice, while recording the corresponding colorectal pressure as described previously.

In a second assay, pain sensitivity was measured by VMR in response to CRD. As opposed to TNBS in the first assay, acetic acid was used to evoke mechanical hypersensitivity. A sterilized multistranded, Teflon-insulated, 40-gauge stainless steel wire (Cooner Wire) was implanted in the external oblique muscle and then s.c. tunneled through the back and fixed on the neck skin. One week after surgery, a balloon (made from 3 cm2 polyethylene membrane and attached to soft Tygon (PE60) tubing) was implanted into the colorectum. To test the VMR, the mouse was placed in a restrainer and allowed to adapt for 30–45 minutes. CRD was induced by applying pressure of 15,

30, 45, and 70 mmHg for 10 seconds each. There were at least 4-minute intervals between stimulations. The EMG was recorded from 2 externalized electrodes implanted in the external oblique muscle 20 seconds before, 10 seconds during, and 20 seconds after CRD. The EMG in the 10 seconds before (baseline) and during CRD were analyzed using CED 1401 plus and Spike 2 software. The AUC of the EMG 10 seconds

37 before and during the CRD was measured. CRD response data were normalized to baseline. We used VMR response to CRD to determine the effect of Compound B on visceral hypersensitivity in this mouse model of IBS. This model was generated by colorectal infusion of 20 μl 0.5% acetic acid or saline (control) on postnatal days 9–12.

The mice were weaned at 3 weeks. At 8 weeks of age, we conducted 2 studies. First,

IBS mice (n = 6) were treated with Compound B (75 mg/kg, 10 ml/kg) or vehicle (20% m/V 2-hydroxypropyl-β-cyclodextrin in saline) s.c. A group of mice (neonatal saline treated) was used together with the vehicle as control. In the second study, IBS mice were treated with vehicle, 20 or 75 mg/kg of Compound B i.p. alongside a control group that was saline treated. Thirty minutes after Compound B treatment, EMG responses to

CRD were recorded. Analysis methods are specified in the appropriate sections. For these behavioral tests, the investigator was blind to treatment.

Statistics.

PK parameters were calculated using the noncompartmental analysis tool of Phoenix

WinNonlin (Version 6.3). For rat toxicity tests, animals were randomized but assigned a unique identification number. For Phase A, data were analyzed by parametric 1-way

ANOVA if normally distributed and variances were homogeneous. Post hoc analysis was conducted by making all possible comparisons among the treatment groups with the Holm-Sidak test. If the assumptions of parametric analysis were not met, data were analyzed by Kruskal-Wallis 1-way ANOVA on ranks. Dunn’s test was used to assess any post hoc differences by comparisons among all groups if the Kruskal-Wallis 1-way

ANOVA was significant. For Phase B, data were analyzed by 1-way ANOVA followed by

Dunnett’s test if data were normally distributed and variances were homogeneous. Post

38 hoc comparisons with Dunnett’s test were only employed if treatment effect in the 1-way

ANOVA was significant. If the assumptions of parametric analysis were not met, data were analyzed by Kruskal-Wallis 1-way ANOVA on ranks. Dunn’s test was used to assess any post hoc differences among groups if the Kruskal-Wallis 1-way ANOVA was significant. For all statistical procedures, differences were considered significant if P <

0.05. All statistical analyses were performed with SigmaPlot 13.0 (Build 13.0.0.83),

Systat Software Inc. For electrophysiological recordings, data are presented as mean ±

SEM and analyzed using GraphPad Prism 7. More details about statistical methods used can be found in the text. If used, Student’s t test was 2-tailed. For IBS mouse model experiments, data were statistically analyzed by generalized estimating equations followed by LSD post hoc tests when appropriate using SPSS 23.0. Analysis was typically carried out in GraphPad Prism 7 Software.

Study approval.

PK profiling of Compound B was conducted at Sai Life Sciences Limited in accordance with Study Plan SAIDMPK/PK-16-05-213 and the guidelines of the Institutional Animal

Ethics Committee (IAEC). Rat toxicity study was carried out by Sobran Inc. with an approved IACUC protocol, number SOB-051-2017. Behavioral analysis in the SNI model of neuropathic pain were approved by the UCSF IACUC. The Animal Ethics

Committees of The Flinders University, The University of Adelaide, and the South

Australian Health and Medical Research Institute (SAHMRI) approved experiments involving animals in FBD studies. The Animal Care and Use Committee of Johns

Hopkins University approved the relevant IBS experiments.

39 Acknowledgements

This work was supported by a Ruth Kirschstein NIH predoctoral Fellowship

(F31NS084646 to JG), a Department of Defense (DoD) National Defense Science and

Engineering Graduate (NDSEG) Fellowship (JS), the Maryland Innovation Initiative (MII)

Tedco (FB), the Abell Foundation (FB), a Blaustein Pain Research grant to FB (Johns

Hopkins University), NIH R35 NS097306 to AIB, the Amos Food Body and Mind Center at Johns Hopkins University, a National Health and Medical Research Council of

Australia (NHMRC) RD, Wright Biomedical Research Fellowship APP1126378 to SMB, and NHMRC Australia Project grants 1083480, 1139366, and 1140297 to SMB. We would like to thank Jiachen Chu (Johns Hopkins University) for helpful comments.

Andrew Escayg (Emory University) for help with the PK profiling, and Katie Hamel

(UCSF) for performing some of the behavioral studies.

Figure 2-1: Effect of 100μM Compound B on NaV1.5, NaV1.7, and NaV1.8. (A-C)

Figure shows G/Gmax (G: conductance) and I/Imax (I: current) relationships before (green,

DMSO control) and after (red) addition of 100μM Compound B to NaV1.5 (A), NaV1.7

(B), and NaV1.8 (C). n=5–8, and error bars represent SEM. No effects were observed.

D) Effect of 1μM Compound B (red) on NaV1.1 (black; control) activation voltage.

Figure 2-2: Metabolic stability of Compound B in plasma and liver microsomes.

Compound B was completely stable in both plasma as well as microsomes from mouse and human fortified with NADPH suggesting that the compound is not susceptible to hydrolysis or CYP-450 dependent metabolism. * Testosterone (Testo) was used as a positive control.

Figure 2-3: Pharmacokinetic (PK) profile of Compound B. Figure shows mean concentration (ng/ml) – time (hrs) profiles of plasma, brain, and CSF (cerebrospinal fluid) pharmacokinetics of Compound B in male C57BL/6 mice following a single intravenous (I.V.), intraperitoneal (I.P.), subcutaneous (S.C.), and oral (P.O.) dose administration at 10mg/kg with 6 time points over 8hrs. Deduced values are shown in

Supplementary Table 2.

Figure 2-4. Pharmacological blockade of NaV1.1 is antinociceptive. Systemic administration of Compound B (comp B; 60 mg/kg) has no effect on baseline mechanical thresholds of naive mice (baseline, 0.760 ± 0.03 g, vs. Compound B, 0.836

± 0.105 g, 2-way ANOVA, P = 0.501, n = 7). Three days after spared nerve injury (SNI), mice exhibit mechanical hypersensitivity (~57%) ipsilateral to the injury (baseline, 0.760

± 0.03 g, vs. SNI, 0.330 ± 0.034 g, 2-way ANOVA, P = 0.0001). A systemic injection of a single dose of Compound B (i.p., 60 mg/kg) significantly reduces mechanical hypersensitivity 30 minutes (i.p., 60 mg/kg; 0.515 ± 0.072 g, 2-way ANOVA, P = 0.039, n = 7), but not 15 minutes after injection. Contralateral mechanical thresholds were unaffected by the compound (0.703 ± 0.051 g). One month after SNI, mice still exhibited

44 mechanical hypersensitivity ipsilateral to the injury. Again, a single dose of Compound B

(i.p., 60 mg/kg) significantly reduced mechanical hypersensitivity (60 minutes; SNI baseline, 0.277 ± 0.031 g, vs. Compound B, 0.530 ± 0.061 g, 2-way ANOVA, P = 0.003, n = 7). Data are presented as mean ± SEM with *P ≤ 0.05 and **P ≤ 0.005.

Figure 2-5: Compound B assessment in the rotarod test. Systemic administration of Compound B (I.P., 60mg/kg) has no effect on motor performance on the rotarod test

(baseline: 256sec ± 5 vs Compound B: 261sec ± 4, two-way ANOVA, p=0.990, n=7).

Figure 2-6. Compound B reduces sodium currents in colon-innervating DRG neurons. (A) Group data showing that sodium current density (pA/pF) in a population of colon-innervating DRG neurons was reduced when applying Compound B (100 μM).

****P < 0.0001, n = 14 neurons, paired t test. (B) Individual data from that the group data presented in A. ****P < 0.0001, n= 17 neurons, paired t test.(C) Compound B caused ~23% decrease in peak sodium currents in affected neurons (**P < 0.001, Mann

Whitney U test). (D) Current-voltage (I-V) plots of sodium current density before

(vehicle; black) and after (blue) Compound B application (100 μM) in inhibited colon- innervating DRG neurons. Data represent ± SEM.

Figure 2-7: Compound B does not affect sodium currents in a subpopulation of colon-innervating DRG neurons. (A) Group data showing that sodium current density (pA/pF) in a population of colon innervating DRG neurons was not inhibited

(defined as a ≥15% reduction from baseline responses) by Compound B (p>0.05, n=22 neurons, paired t-test). (B) Individual data from the group data presented in A).

C) I-V (current-voltage) plots of sodium current density before (vehicle; black) and after

(blue) Compound B application (100 M) in uninhibited colon innervating DRG neurons.

Figure 2-8. Effect of Compound B on colonic nociceptive afferents. (A) Top panel:

Application of 100 μM Compound B inhibited a large subpopulation of colonic nociceptors from healthy control mice (n = 6, ****P < 0.0001, paired t test). Middle panel: In inhibited afferents, Compound B reduced responses to ~75% of healthy control baseline levels. *P < 0.05, unpaired t test. Lower panel: Representative examples of ex vivo healthy control colonic nociceptor recordings showing nociceptors in the absence and presence of Compound B. *P < 0.05, paired t test. (B) Top panel: In an IBS mouse model of TNBS-induced chronic visceral hypersensitivity (CVH),

49 Compound B (100 μM) inhibited a large subpopulation of CVH colonic nociceptors (n =

6, ****P < 0.0001, paired t test). Middle panel: In inhibited afferents, Compound B reduced responses to ~67% of CVH baseline levels. Lower panel: Representative examples of ex vivo CVH colonic nociceptor recordings showing nociceptors in the absence and presence of Compound B. vfh, von Frey hair. The vfh with upward arrow indicates start of the application, and a downward arrow signifies removal.

Figure 2-9: Compound B does not affect a subpopulation of colonic nociceptive afferents from healthy control and CVH mice. (A) Healthy control mice: application of Compound B (100μM) did not inhibit a population of nociceptors (n=3, p>0.05, paired t-test). (B) TNBS colitis-induced CVH mouse model of IBS: application of Compound B

(100μM) did not inhibit a population of CVH nociceptors (n=3, p>0.05, paired t-test).

Figure 2-10: Effect of Compound B and Hm1a on colonic nociceptive afferents.

Left: Healthy control mice with application of 100 M Compound B inhibiting colonic nociceptors from control mice (n=9 afferents). Addition of 100nM Hm1a does not overcome Compound B inhibition. Right: In a TNBS colitis-induced CVH mouse model of IBS, application of Compound B inhibited colonic nociceptors from CVH mice (n=9 afferents). Co-application of 100nM Hm1a does not overcome Compound B inhibition.* p<0.05; ** p<0.01, repeated measures one-way ANOVA.

53 Figure 2-11. Effect of intracolonic administration of Compound B on VMR in an

IBS mouse model of TNBS-induced CVH. (A) Representative EMG recordings at increasing colorectal distension pressures (mmHg) in healthy control mice with intracolonically (i.c.) administered vehicle (black), or IBS mice i.c. administered with vehicle (orange) or Compound B 100 μM (blue), 30 minutes before recordings. (B)

Upper panel: Group data showing that IBS mice with CVH display increased VMRs

(visceromotor reflexes) to CRD (colorectal distension) compared with healthy control mice, particularly at a distension pressure of 60 mmHg (***P < 0.001) and 80 mmHg

(*P < 0.05). Lower panel: I.c. Compound B administration significantly reduced the VMR to CRD in IBS mice, normalizing responses to healthy control levels; 60 mmHg (***P <

0.001) and 80 mmHg (*P < 0.05). Significance of differences were analyzed by the

Generalized Estimating Equation (GEE), followed by the Least Significant Difference

(LSD) post hoc test. HC, healthy control; CVH, chronic visceral hypersensitivity.

Figure 2-12: Colonic compliance. Compared with healthy control mice, colonic compliance is unaltered in a TNBS colitis-induced CVH mouse model of IBS or IBS mice dosed with 100μM Compound B, suggesting that changes in the VMR to CRD are not due to changes in smooth muscle function. HC = Healthy Control.

Figure 2-13. Effect (i.p.) of Compound B in an IBS mouse model of acetic acid– induced CVH. (A) Shown is the effect of Compound B (i.p. injection) at 20 and 75 mg/kg as measured by VMR response to CRD. Acute treatment at a dose of 20 mg/kg normalized the increased pain sensitivity in IBS mice, whereas 75 mg/kg compound reduced pain sensitivity in IBS mice to a level that is lower than control mice. (B) Two- way ANOVA showed main effect of treatment F(3,76) = 31.93, P < 0.001; main effect of pressure F(3,76) = 44.09, P < 0.001; interaction of treatment × pressure F(9,76) = 2.4, P =

56 0.017. Data (n = 7) are presented as mean ± SEM. *P < 0.05, significantly different from saline-vehicle at the same pressure; #P < 0.05, significantly different at the same pressure from IBS vehicle; and ^P < 0.05, significantly different from saline-vehicle and

IBS-vehicle at the same pressure (Student Newman-Keuls post hoc test). F represents

F statistic obtained after 2-way ANOVA to test whether the means between 2 populations are significantly different.

Figure 2-14. Effect (s.c.) of Compound B in an acetic acid–induced IBS mouse model. (A and B) Effect on hyperalgesia of s.c. Compound B treatment in an IBS mouse model measured by VMR response to CRD. Data are presented as mean ±

SEM. *P < 0.05, significantly different from saline-vehicle at same pressure; @P < 0.05, significantly different from IBS-vehicle (Student Newman-Keuls post hoc test). Two-way

ANOVA analysis showed the main effect of treatment F(2,56) = 14.02, P < 0.001; main effect of pressure F(3,56) = 17.61, P < 0.001; interaction of treatment x pressure F(6,56) =

58 1.13, P = 0.35 (n = 7); these P values were obtained from the 2-way ANOVA test for the data shown in the figure.

Figure 2-15. Rufinamide derivatives that significantly alter steady-state activation and inactivation V1/2 values on human NaV1.1. Significant depolarized shifts in steady-state inactivation are seen with five compounds (A-C, G-H). Significant depolarized shifts in steady-state activation are seen with four compounds (D-F, I). V1/2

Values are reported in Tables 3-3, 3-4, and 3-5.

Figure 2-16. Rufinamide derivatives that do not show effects on human NaV1.1. (A-

J) Ten rufinamide derivatives did not show a statistically significant effect on either steady-state activation or inactivation of human NaV1.1. V1/2 values are reported in

Tables 3-6, 3-7, and 3-8.

Figure 2-17. Compound F in an acetic-acid mouse model of IBS. Effect on hyperalgesia of s.c. Compound F treatment in an IBS mouse model measured by VMR response to CRD. Data are presented as mean ± SEM. *P < 0.05, significantly different from saline-vehicle at same pressure; #P < 0.05, significantly different from IBS-vehicle

(Student Newman-Keuls post hoc test). Two-way ANOVA analysis showed the main effect of treatment F(2,56) = 36.07, P < 0.001; main effect of pressure F(3,56) = 59.54, P <

0.001; interaction of treatment x pressure F(6,56) = 2.64, P = 0.01 (n = 5); these P values were obtained from the 2-way ANOVA test for the data shown in the figure.

62 Assay Source Ligand Conc. Kd Non- Incubation Detection Result specific method Receptors 3 A2A (h) human [ H]CGS 6nM 27nM NECA 120min RT Scintillation Negative (agonist recombinant 21680 (10 µM) counting radioligand) (HEK-293) α1A (h) human [3H]prazosin 0.1nM 0.1nM epinephrine 60min RT Scintillation Negative (antagonist recombinant (0.1mM) counting radioligand) (CHO) 3 - α2A (h) human [ H]RX 1nM 0.8nM epinephrine 60min RT Scintillation Negative (antagonist recombinant 821002 (100µM) counting radioligand) (CHO) 3 β1 (h) human [ H](-)CGP 0.3nM 0.39nM alprenolol 60min RT Scintillation Negative (agonist recombinant 12177 (50µM) counting radioligand) (HEK-293) 3 β2 (h) human [ H](-)CGP 0.3nM 0.15nM alprenolol 120min RT Scintillation Negative (antagonist recombinant 12177 (50µM) counting radioligand) (CHO) 3 CB1 (h) human [ H]CP 0.5nM 3.5nM WIN 55212- 120min 37°C Scintillation Negative (agonist recombinant 55940 2 counting radioligand) (CHO) (10µM) 3 CB2 (h) human [ H]WIN 0.8nM 1.5nM WIN 55212- 120min 37°C Scintillation Negative (agonist recombinant 55212-2 2 counting radioligand) (CHO) (5 µM) 125 CCK1 (CCKA) human [ I]CCK-8s 0.08nM 0.24nM CCK-8s 60min RT Scintillation Negative (h) recombinant (1µM) counting (agonist (CHO) radioligand) 3 D1 (h) human [ H]SCH 0.3nM 0.2nM SCH 23390 60min RT Scintillation Negative (antagonist recombinant 23390 (1µM) counting radioligand) (CHO) 3 D2S (h) human [ H]7-OH- 1nM 0.68nM butaclamol 60min RT Scintillation Negative (agonist recombinant DPAT (10µM) counting radioligand) (HEK-293) 125 ETA (h) human [ I]endothe 0.03nM 0.03nM endothelin-1 120min 37°C Scintillation Negative (agonist recombinant lin-1 (100nM) counting radioligand) (CHO) 3 H1 (h) human [ H]pyrilami 1nM 1.7nM pyrilamine 60min RT Scintillation Negative (antagonist recombinant ne (1µM) counting radioligand) (HEK-293) 125 H2 (h) human [ I]APT 0.075nM 2.9nM tiotidine 120min RT Scintillation Negative (antagonist recombinant (100µM) counting radioligand) (CHO) 3 M1 (h) human [ H]pirenzep 2nM 13nM atropine 60min RT Scintillation Negative (antagonist recombinant ine (1µM) counting radioligand) (CHO) 3 M2 (h) human [ H]AF-DX 2nM 4.6nM atropine 60min RT Scintillation Negative (antagonist recombinant 384 (1µM) counting radioligand) (CHO) 3 M3 (h) human [ H]4-DAMP 0.2nM 0.5nM atropine 60min RT Scintillation Negative (antagonist recombinant (1µM) counting radioligand) (CHO) N neuronal SH-SY5Y cells [3H]cytisine 0.6nM 0.3nM nicotine 120min 4°C Scintillation Negative α4β2 (h) (human (10µM) counting (agonist recombinant) radioligand) δ (DOP) (h) human [3H]DADLE 0.5nM 0.73nM naltrexone 120min RT Scintillation Negative (agonist recombinant (10µM) counting radioligand) (CHO) κ (KOP) rat [3H]U 69593 1nM 2nM naloxone 60min RT Scintillation Negative (agonist recombinant (10µM) counting radioligand) (CHO) μ (MOP) (h) human [3H]DAMGO 0.5nM 0.35nM naloxone 120min RT Scintillation Negative (agonist recombinant (10µM) counting radioligand) (HEK-293 cells) 3 5-HT1A (h) human [ H]8-OH- 0.5nM 0.5nM 8-OH-DPAT 60min RT Scintillation Negative (agonist recombinant DPAT (10µM) counting radioligand) (HEK-293) 125 5-HT1B rat cerebral [ I]CYP 0.1nM 0.16nM serotonin 120min 37°C Scintillation Negative (antagonist cortex (+ 30µM (10µM) counting radioligand) isoproteren ol) 125 5-HT2A (h) human [ I](±)DOI 0.1nM 0.3nM (±)DOI 60min RT Scintillation Negative

63 (agonist recombinant (1µM) counting radioligand) (HEK-293) 125 5-HT2B (h) human [ I](±)DOI 0.2nM 0.2nM (±)DOI 60min RT Scintillation Negative (agonist recombinant (1µM) counting radioligand) (CHO) GR (h) IM-9 cells [3H]dexamet 1.5nM 1.5nM Triamcino- 6hr 4°C Scintillation Negative (agonist (cytosol) hasone lone counting radioligand) (10µM) AR (h) LNCaP cells [3H]methyltri 1nM 0.8nM testosterone 24hr 4°C Scintillation Negative (agonist (cytosol) enolone (1µM) counting radioligand) 3 V1a(h) human [ H]AVP 0.3nM 0.5nM AVP 60min RT Scintillation Negative (agonist recombinant (1µM) counting radioligand) (CHO) Ion channels BZD rat cerebral [3H]flunitraz 0.4nM 2.1nM diazepam 60min 4°C Scintillation Negative (central) cortex epam (3µM) counting (agonist radioligand) NMDA rat cerebral [3H]CGP 5nM 23nM L-glutamate 60min 4°C Scintillation Negative (antagonist cortex 39653 (100µM) counting radioligand) 3 5-HT3 (h) human [ H]BRL 0.5nM 1.15nM MDL 72222 120min RT Scintillation Negative (antagonist recombinant 43694 (10µM) counting radioligand) (CHO) Ca2+ channel rat cerebral [3H]nitrendip 0.1nM 0.18nM nitrendipine 90min RT Scintillation Negative (L, dihydro- cortex ine (1µM) counting pyridine) (antagonist radioligand) Potassium human [3H] 3nM 6.6nM Terfenadine 60min RT Scintillation Negative channel recombinant Dofetilide (25µM) counting hERG (HEK-293) (human)- [3H] Dofetilide 125 KV channel rat cerebral [ I]α- 0.01nM 0.04nM α- 60min RT Scintillation Negative (antagonist cortex dendrotoxin dendrotoxin counting radioligand) (50nM) Transporters Nor human [3H]nisoxe 1nM 2.9nM desipramine 120min 4°C Scintillation Negative epinephrine recombinant tine (1µM) counting transporter (CHO) (h) (antagonist radioligand) dopamine human [3H]BTCP 4nM 4.5nM BTCP 120min 4°C Scintillation Negative transporter recombinant (10µM) counting (h) (CHO) (antagonist radioligand) 5-HT human [3H]imipra 2nM 1.7nM imipramine 60min RT Scintillation Negative transporter recombinant mine (10µM) counting (h) (CHO) (antagonist radioligand) Enzymes MAO-A rat cerebral [3H]Ro 41- 10nM 14nM clorgyline 60 min 37°C Scintillation Negative (antagonist cortex 1049 (1µM) counting radioligand) Kinases and other enzymes Assay Source Substrate/stimulus/ Incu- Measured Detection Result tracer bation component method Lck kinase human ATP + Ulight-Poly GAT[EAY(1:1:1)]n 10 min RT phospho-Ulight- LANCE Negative (h) recombinant (25nM) Poly (Sf9 cells) GAT[EAY(1:1:1)]n COX1 (h) human Arachidonic acid (3µM) + ADHP 3min RT Resorufin Fluorimetry Negative recombinant (25µM) (oxydized ADHP) COX2 (h) human Arachidonic acid (2µM) + ADHP 5min RT Resorufin Fluorimetry Negative recombinant (25µM) (oxydized ADHP) (Sf9 cells) PDE3A (h) human [3H]cAMP + cAMP (0.5µM) 20min RT [3H]5'AMP Scintillation Negative recombinant counting (Sf9 cells) PDE4D2 (h) human [3H]cAMP + cAMP (0.5µM) 20min RT [3H]5'AMP Scintillation Positive

64 recombinant counting (Sf9 cells) acetylcholin human Acetylthiocholine (400µM) 30min RT 5 thio 2 Photometry Negative esterase (h) recombinant nitrobenzoic acid (HEK-293) Table 2-1. Additional targets tested against Compound B.

Route Matrix Tm C0/C AUClast AUCinf T1/2 CL Vss % ax max (ng*hr/m (ng*hr/m (hrs) (ml/min/kg) (l/kg Fa (hrs (ng/ml l) l) ) ) ) I.V. Plasm - 14442 15319 15479 1.4 10.8 0.8 - a - 11116 13861 14151 1.6 11.8 1.1 - Brainb - 4795 5016 5111 1.8 32.1 2.5 - CSF I.P. Plasm 0.5 5523 10694 11134 - - - - a 0.5 6579 13505 13878 - - - - Brainb 0.5 2776 5179 5277 - - - - CSF P.O. Plasm 0.5 5681 12097 12437 - - - 79 a 0.25 6371 11525 11828 - - - - Brai 0.25 3397 4426 4453 - - - - nb CSF S.C. Plasm 1.00 4570 14237 14839 - - - - a 1.00 4041 14010 14010 - - - - Brai 1.00 2248 6190 6190 - - - - nb CSF Table 2-2. PK data. a AUClast was used for bioavailability calculation. b Brain concentration and exposure expressed as ng/g and ng/g*hr, density of brain tissue was considered to be 1, which is equivalent to plasma density.

66 DMSO AS-34 AS-43 AS-35(2) AS-39

Activation (V1/2) -20.75 ± -21.90 ± -19.55 ± -20.56 ± -18.41 ± h1.1 0.97 1.82 2.04 2.18 1.21

Inactivation (V1/2) 32.92 ± -28.91 ± -29.30 ± -30.12 ± -30.18 ±

0.47* 0.39* 0.40* 0.33* 0.29*

Table 2-3. Table providing values for fits of the data presented in Figure 3-15. V1/2

provides the midpoint voltage of the calculated curve (in mV). *p<0.05. student’s

unpaired t-test was used for analysis. Data are presented as mean  SEM.

67 DMSO DK0.6

Activation (V1/2) -29.19 ± 1.18 -26.16 ± 2.26 h1.1 -40.83 ± 1.63 -35.07 ± 1.08*

Inactivation (V1/2)

Table 2-4. Table providing values for fits of the data presented in Figure 3-15. V1/2 provides midpoint voltage of the calculated curve (in mV). *p<0.05. student’s unpaired t- test was used for analysis. Data are presented as mean  SEM.

DMSO 10.B 10.D 13.B 13.C

Activation (V1/2) -29.19 ± -24.65 ± -24.62 ± -22.93 ± -21.15 ± h1.1 1.18 1.75* 1.75* 2.16* 1.41*

Inactivation (V1/2) -40.83 ± -40.81 ± -37.13 ± -39.19 ± -36.16 ±

1.63 1.05 0.92 1.17 1.70

Table 2-5. Table providing values for fits of the data presented in Figure 3-15. V1/2 provides midpoint voltage of the calculated curve (in mV). *p<0.05. student’s unpaired t- test was used for analysis. Data are presented as mean  SEM.

69 DMSO AS-47 AS-36 AS-38

Activation (V1/2) -20.75 ± -21.74 ± -18.23 ± -25.96 ± h1.1 0.97 3.16 1.83 5.85

Inactivation (V1/2) 32.92 ± -32.07 ± -29.97 ± -33.61 ±

0.47 0.98 0.67 2.06

Table 2-6. Table providing values for fits of the data presented in Figure 3-16. V1/2 provides midpoint voltage of the calculated curve (in mV). *p<0.05. student’s unpaired t- test was used for analysis. Data are presented as mean  SEM.

70 DMSO AS05B AS12A AS20 AS18 AS28 AS15

Activation (V1/2) -20.44 -22.88 ± -26.17 ± -16.28 ± -18.63 ± -17.01 ± -17.32 ± h1.1 ± 2.25 2.83 2.11 2.17 1.10 3.07 1.70

Inactivation (V1/2) -33.19 -34.14 ± -32.70 ± -37.59 ± -30.87 ± -33.08 ± -35.89 ±

± 1.49 1.16 0.60 1.43 0.54 0.80 1.74

Table 2-7. Table providing values for fits of the data presented in Figure 3-16. V1/2

provides midpoint voltage of the calculated curve (in mV). *p<0.05. student’s unpaired t-

test was used for analysis. Data are presented as mean  SEM.

71 DMSO DK.04

Activation (V1/2) -29.19 ± -30.59 ± h1.1 1.18 2.25

Inactivation (V1/2) -40.83 ± -38.47 ±

1.63 1.08

Table 2-8. Table providing values for fits of the data presented in Figure 3-16. V1/2 provides midpoint voltage of the calculated curve (in mV). *p<0.05. student’s unpaired t- test was used for analysis. Data are presented as mean  SEM.

72 THE ROLE OF NaV1.9 AND NaV1.1 IN SOMATOSENSORY PERCEPTION

Chapter 3: The role of NaV1.9 in the transmission of pruritic stimuli

This chapter was adapted from:

Salvatierra, J., et al., A disease mutation reveals a role for NaV1.9 in acute itch. Journal

of Clinical Investigation, 2018. (accepted).

73 Summary

To investigate the functional relevance of NaV1.9 in acute itch, we leveraged our newly generated sfGFP-tagged NaV1.9 mouse line to determine the percentage of

+ putative itch neurons that co-expressed NaV1.9. We discovered that most MrpgrA3 and

+ MrpgrC11 neurons co-expressed NaV1.9. Although other putative itch neurons, such as

DRG neurons that expressed substance P, had very few neurons that co-expressed

-/- NaV1.9. In line with this evidence, we used a newly generated NaV1.9 mouse line and discovered that the loss of NaV1.9 led to a complete absence of histamine- and BAM8-

22 (MrgprC11 agonist)-induced itch, and to about a 50% reduction in chloroquine

(MrgprA3 agonist)-induced itch. In addition, there were a lower percentage of histamine

-/- and chloroquine responsive neurons in NaV1.9 DRGs compared to littermate controls, which underlies the importance of this channel in the initiation and propagation of itch stimuli. Moreover, we found that activation of either of these receptors lead to a drastic hyperpolarizing shift in the activation of NaV1.9, but not NaV1.8, and that the action potentials (AP) evoked via activation of MrpgrA3 revealed that several AP parameters

-/- that were altered in WT DRGs, were unaffected in DRGs from NaV1.9 mice. This helps to explain the contribution of this channel to the excitability of these neurons in response to a pruritic stimulus.

Furthermore, we determined that mice with an orthologous gain-of-function mutation observed in human patients also exhibited severe pruritus. This mutation sits on the distal end of the sixth transmembrane segment of domain II and leads to a dramatic slowing of channel deactivation and a hyperpolarizing shift in steady-state

74 inactivation, which leads to excess sodium flux and, in turn, leads to inactivation of other ion channels. DRGs expressing this hyperactive channel undergo progressive conduction block. However, we determined that a subset of MrgprA3+ neurons behave opposite that phenotype, and instead become hyperexcitable, which could help explain the spontaneous itch effecting both human and mice containing this mutation. The results here underscore the importance of NaV1.9 not only in pain signaling, but also in itch. This could have important future implications for drug development targeting ways of reducing both acute and chronic itch.

75 INTRODUCTION

The somatosensory nervous system detects sensory modalities such as pain, itch, and temperature sensitivity and transfers them from the periphery to the spinal cord and eventually to the sensory cortex in the brain [119]. Of these signals, itch (pruritis) has evolved to alert us to potentially dangerous external stimuli [120, 121]. Although acute itch may guard against environmental threats, pathological itch is a distressing physical sensation and can dramatically affect quality of life. Yet, compared to other sensory modalities, itch is much less understood. Histamine was the first compound discovered to elicit itch by activating a subset of peripheral sensory neurons (pruriceptors) [122]

[123, 124]. Later, additional receptors were identified as key players in mediating itch sensation. The discovery of Mas-related G protein-coupled receptors as critical contributors to itch helped identify potential itch-selective neurons [125]. These four families of histamine-independent Mrgprs (MrgprA-D) can be activated by pruritic compounds such as chloroquine (CQ) and BAM8-22 and are expressed in trigeminal

(TG) and dorsal root ganglia (DRG) [53, 55, 126]. Although these receptors and others have been established as initial detectors of pruritic stimuli [54, 127-131], the downstream mechanisms involved in mediating itch are still poorly understood.

Although NaV channels (NaV1.1-NaV1.9) are critical for the propagation of action potentials [96] in sensory neurons, little is known about how they contribute to the transmission of pruritic stimuli. Specifically, the relationship between NaV1.7, NaV1.8, and NaV1.9 and pain has been studied extensively, with knockout mouse models for all three channels showing effects in various pain assays [11, 22, 32, 38, 42, 132-136].

However, the contribution of these and other NaV channel subtypes to itch has yet to be

76 explored.

Here, we report a new clinical case of unbearable itch and distorted pain sensation stemming from the heterozygous de novo p.L811P mutation in NaV1.9 channels, encoded by the SCN11a gene. This mutation was previously published in relation to several cases of complete insensitivity to pain in which abnormal NaV1.9 function was thought to result in nociceptor depolarization and subsequent conduction block [38, 40]. Although there is a consensus that NaV1.9 contributes to pain [134, 137,

+/WT 138], the role of this NaV channel subtype and the p.L811P mutation in itch is unknown [61]. Compared to other NaV channel subtypes, NaV1.9 expression patterns, functional properties, and pharmacological sensitivities are less defined. To investigate the role of NaV1.9 in itch, we assessed its functional role in new mouse models in which channel expression was spatiotemporally manipulated. We also tagged NaV1.9 with a fluorescent reporter to facilitate reliable identification and biophysical characterization of

NaV1.9-expressing cells. We found that the channel is present in a subset of non- myelinated, non-peptidergic small diameter dorsal root ganglia (DRG). In wild-type

-/- DRGs but not in NaV1.9 mice, pruritogens altered action potential parameters and NaV

-/- channel gating properties. Compared to control animals, NaV1.9 mice displayed reduced scratching behavior upon application of histamine, CQ, and BAM8-22.

Combined with the observation that disease-related p.L799P+/WT mice exhibited amplified scratching behavior in rest conditions, our data provide compelling evidence for NaV1.9 participation in itch.

77 RESULTS

A newly-identified NaV1.9 p.L811P+/WT patient

Whole-exome sequencing uncovered the de novo p.L811P (c.T2432C) mutation in

NaV1.9 in a female patient reporting severe pruritus without a family history. In addition to itch, the patient reported a partial loss-of-pain sensation with remaining back, neck, and side pain. Past medical history included fractures in her lower extremities with little trauma, diurnal and nocturnal enuresis, constipation, intermittent diarrhea, developmental delay, heterotrophic ossification with bilateral hip disease, scoliosis, hyperhidrosis, asthma, eczema, gastroesophageal reflux, hypoglycemia, vitamin D deficiency, headaches, and picking the skin on her fingers.

Regarding itch experienced, the patient reported that itch was worse at night, even in the absence of topical skin pathology such as eczema, and that itching, tingling, sweating, and movement of lower extremities commonly prevented her from falling asleep. The patient had excoriations and marks on her legs from scratching and her fingers bled from picking pieces of skin. She used compression bandages and pressed on her lower extremities to lessen itching sensations and wore mitts to bed to prevent scratching herself while asleep. The patient reported no itch relief from diazepam or oxycodone and only a minor benefit from diphenhydramine and acetaminophen.

Physical examination revealed that the patient had a lack of position sense in the toes, had distal movement sense in both ankles, and detected von Frey 0.07 g filament on the dorsum of her feet. Further examination with a pin showed that she had decreased sensation bilaterally that was dull initially but turned painful after repeated touch. The

78 patient was also diagnosed with restless leg syndrome (RLS) and anxiety disorder not otherwise specified. She was treated with cyproheptadine after reporting partial improvement of itch with diphenhydramine. Gabapentin was added to her treatment due to: 1) the reported decrease in discomfort in individuals with small fiber neuropathy

[139], and 2) the reduction in lower extremity movements in patients with RLS [140].

Subsequently, she no longer damaged her lower extremity skin by rubbing or scratching, and her evening discomfort lessened drastically, allowing lesions to heal.

After healing, severe wounds from scratching left marks that resembled bruising (Fig. 2-

1A, black arrows). The observation that no other mutations were found in these patients, and that all reported patients with the NaV1.9 p.L811P mutation complained of severe pruritus [61], suggests an important contribution of NaV1.9 to itch.

Generation and characterization of a sfGFP-tagged NaV1.9 mouse line

To determine the expression pattern of NaV1.9, we used a mouse line in which the channel was fused to a fluorescent reporter. To limit impact on NaV1.9 function and expression we: 1) used superfolder Green Fluorescent Protein (sfGFP), a generation of

GFP with enhanced folding kinetics [141]; 2) targeted the N-terminus of the channel for sfGFP fusion [96, 142]; and 3) codon optimized [143] sfGFP. Using the F.A.S.T. technique [144], we inserted sfGFP immediately after the endogenous start codon (Fig.

1B). The original knock-in cassette contained a construct (loxP-FRT- Neo-STOP-FRT-

-/- tetO-loxP) which led to a global NaV1.9 knockout phenotype (NaV1.9 F.A.S.T. mice).

This strain had the additional benefit of being capable of 1) tTA-mediated overexpression; and 2) tetracycline-controlled transcriptional silencer (tTS)-mediated

79 conditional knock-down (Fig. 2-1B).

By crossing sfGFP-NaV1.9 F.A.S.T. mice (C57BL/6J) with a global Cre-mouse

(B6.C-Tg(CMV- cre)1Cgn; Jackson laboratories, USA), we obtained a line in which endogenous NaV1.9 was N- terminally fused to sfGFP. As seen in Fig. 2-1C, a subset of dissociated DRG displayed robust fluorescence signal that overlapped with a GFP antibody whereas wild-type (WT) tissue only showed weak background fluorescence.

Biochemical analysis of DRGs showed the presence of a GFP-positive band at the appropriate size for sfGFP-tagged NaV1.9, which was not observed in either WT or

F.A.S.T. cassette-containing mouse DRGs (Fig. 2-1D). Correspondingly, qRT-PCR did not detect NaV1.9 RNA in DRGs of F.A.S.T. mice. Blotting for GFP and then stripping and re- probing the Western blot with a NaV1.9 antibody [37] revealed a positive band in both DRG and trigeminal ganglia (TG). Aside from these tissues, NaV1.9 expression was not observed in the brain or other major organs (Fig. 2-1E).

After confirming expression of fluorescence, we next sought to determine if the gating properties of sfGFP-tagged NaV1.9 were altered when compared to non-tagged

NaV1.9. Patch- clamp recordings from fluorescent sensory neurons in the presence of tetrodotoxin (TTX) and Cs in the patch pipette revealed a functional sfGFP-NaV1.9 channel with WT gating behavior and kinetics as reported in the literature [34, 38, 42,

74, 135, 145-147] (Fig. 2-2).

To further validate proper channel function, we constructed mouse WT NaV1.9 and sfGFP- NaV1.9 stable rodent DRG-derived ND7/23 cell lines by combining the Flp

Recombination Target (FRT)-based Flp-In system (ThermoFisher®, USA) with intron- mediated enhancement of gene expression, a technique that leads to increased

80 accumulation of mRNA and protein relative to unaltered cDNA [148]. An additional benefit of this approach was the reduction of unwanted rearrangement events when propagating NaV channel cDNA in bacteria. The mouse SCN11a gene is encoded by 24 exons with known exon/intron boundaries [149]. Although we did not obtain sufficient expression with mouse cDNA lacking an endogenous intron or with the addition of C- terminal GFP [150], we consistently saw NaV1.9 current with cDNA containing intron 2 that increased after differentiating the ND7/23 cells in media containing NGF (100ng/ml) and 1% FBS (Fig. 2-1F and 2-1G). RT-PCR and sequencing confirmed the correct excision of intron 2 to form mature channels [149]. Patch-clamp recordings from these cells showed sfGFP-NaV1.9 and WT channel currents with virtually identical gating properties and kinetics (Fig. 2-1H-J).

NaV1.9 is predominantly expressed in non-myelinated small diameter DRGs

We exploited the sfGFP tag to determine NaV1.9 expression in mouse DRG subtypes.

Using IHC with a GFP antibody, we found that all sfGFP positive cells overlapped with peripherin, a marker for peripheral sensory neurons (Fig. 2-3A). We also observed strong overlap between sfGFP and IB4 (Fig. 2-3B, 86%) and c-Ret (Fig. 2-3C, 89%), two markers for small diameter, unmyelinated non- peptidergic fibers. We detected a small portion of sfGFP+ cells that overlapped with calcitonin- gene related peptide

+ (CGRP), indicating a subpopulation of NaV1.9 neurons that are peptidergic (Fig. 2-3D,

12%). We rarely spotted commonality of NaV1.9 with NF200, a marker for large diameter neurons (Fig. 2-3E, 1.5%). NaV1.9 has been implicated in thermal hyperalgesia under inflammatory conditions [31, 32, 136], and we indeed found

81 substantial overlap with TRPV1 (Fig. 2-3F, 35%), a marker for thermosensitive

+ nociceptor subgroups [151]. However, these TRPV1 cells had low NaV1.9 expression

(Fig. 2-3F arrowheads). Markedly, mouse DRGs show little to no overlap with TRPV2

(Fig. 2-3G, 5%). Finally, we did not observe co-expression of NaV1.9 and tyrosine hydroxylase (TH) [152] found in cells that have been implicated in allodynia [153] (Fig.

2-3H, 0.7%). In line with previous reports, we detected NaV1.9 in small- to medium- sized neurons, which make up most nociceptors and pruriceptors (Fig. 2-3I). In addition, we crossed our homozygous sfGFP-NaV1.9 model with NaV1.8-Cre-tdTomato mice that express the Cre recombinase under control of the NaV1.8 promoter to induce tdTomato fluorescent protein in NaV1.8-expressing neurons [154]. As a result, we found extensive expression overlap between NaV1.8 and NaV1.9 (Fig. 2-4A). We also explored NaV1.9 expression in vagal ganglia responsible for transmitting stimuli from organs such as the heart, larynx, lungs, and alimentary tract, to the central nervous system [119]. Here, we

+ found that all Nav1.9 neurons expressed the pan-neuronal marker PGP9.5 (Fig. 2-4B) and detected extensive expression of NaV1.9 in the jugular ganglia (Supplemental Fig.

2-4C); however, we did not find cells with high levels of NaV1.9 expression in the nodose ganglia (Fig. 2-4D). Finally, we determined NaV1.9 expression at central and peripheral ends of the DRG. The fluorescent signal overlaps extensively with IB4 at the inner portion of layer II of the dorsal horn where connecting interneurons may be involved in influencing pruritic circuits [155, 156] (Fig. 2-3K). We also observed extensive fluorescence along the axon of the saphenous nerve (Fig. 2-3L). Although we saw strong staining for PGP9.5, we could not detect significant sfGFP signal at the nerve terminals of either the glabrous skin or hairy skin of the mouse hind paw in whole-

82 mount skin preparations (Fig. 2-3M, Fig. 2-4E).

NaV1.9 expression in MrgprA3/MrgprC11 neurons

GPCRs MrgprA3 and MrgprC11 are expressed in a subpopulation of nociceptors linked to non- histamine-related itch [126]. To examine if NaV1.9 is found in MrgprA3- and

MrgprC11-expressing neurons, we used the MrgprA3-GFPCretdTomato/+ mice [55] in which

MrgprA3+ neurons, which include both MrgprA3 and MrgprC11 receptors, are labeled with tdTomato fluorescent protein. During patch-clamp recordings, we were able to

+ identify NaV1.9 currents in all tdTomato DRG neurons (Fig. 2-5A-B). Next, we sought to determine co-expression of NaV1.9 with MrgprC11 using a validated antibody [157]. In

+ this case, we found that 76% of MrgprC11 neurons also expressed NaV1.9 (Fig. 2-5C).

Channel expression was rarely observed in Substance P-positive cells (10%), which are another subset of neurons which have been implicated in coding for itch sensation [158]

(Fig. 2-5D). Although we found NaV1.9 in a majority of pruriceptors thought to code for itch via members of the Mrgpr family, we did not see this NaV channel subtype in all putative itch neurons.

Loss of NaV1.9 leads to a reduction in itch

To examine the extent of NaV1.9 participation in itch, we carried out behavioral tests with our homozygous F.A.S.T. mice which contain a Neo-STOP cassette to generate a

-/- global NaV1.9 knockout line (NaV1.9 ) and we compared these results to WT littermate controls. We assessed scratching behavior in the F.A.S.T. mice by subcutaneously injecting pruritic compounds into the nape. The number of bouts of hind paw scratching

83 directed toward the injection site was tallied and binned every 5 minutes. Notably, our

-/- NaV1.9 F.A.S.T. mice exhibited a strong reduction in acute scratching behavior upon histamine application (Fig. 2-5E). Moreover, injection of CQ (MrgprA3 activator) and

BAM8-22 (MrgprC11 activator) into these mice also led to a robust decrease in bouts of scratching compared to WT littermates, thereby supporting a key role for NaV1.9 in both histamine-dependent and histamine-independent itch (Fig. 2-5F and 2-5G). It should be noted that although there is no strong response throughout the 30 minutes of recording

-/- to either histamine or BAM8-22 in the NaV1.9 mice (Fig. 2-5E and 2-5G), there appears to be residual scratching that is delayed in response to CQ in the Nav1.9-/- mice when compared to the littermate WT controls (Fig. 2-5F). This residual scratching behavior could occur due to off-target effects of CQ. Indeed, Liu et al. [53] reported that

CQ can also activate mast cells, which may cause an itch behavioral response that is independent of direct MrgprA3 activation. Overall, our results support the importance of

NaV1.9 in both histamine-dependent and histamine-independent itch.

2+ NaV1.9 is important for histamine- and CQ-evoked Ca responses

Histamine, CQ, and BAM8-22 are known to signal via GPCRs, with subsequent activation of TRP channels and increases in internal Ca2+ concentrations. Therefore, we

2+ wanted to determine whether Ca responses were altered after loss of NaV1.9. We compared Ca2+ signals after the application of pruritogens in isolated DRGs from

-/- 2+ NaV1.9 and WT littermate controls using Fura- 2 Ca imaging. After the application of

-/- histamine (100μM), we observed that the peak responses in NaV1.9 mice DRGs were

-/- like WT controls (WT: 42.8 ± 3.0; NaV1.9 : 39.8 ± 5.0; p=0.59) (Fig. 2-6A-B). However,

84 we found a significant reduction in the total number of neurons that responded to

-/- -/- histamine in NaV1.9 mice when compared to WT (WT: 8.0% ± 0.8; NaV1.9 : 3.2% ±

0.6; p=0.0014; Fig. 2-6A-B). Similar effects were seen with CQ (1mM), with a

-/- comparable peak magnitude after the application of CQ in NaV1.9 and WT DRGs (WT:

-/- 48.2 ± 3.1; NaV1.9 : 44.8 ± 3.2; p=0.44); however, there were significantly less

-/- responsive neurons (WT: 10.1% ± 1.6; NaV1.9 : 6.0% ± 0.6; p=0.04, Fig. 2-6C-D).

Although we did see a similar trend after BAM8-22 application, there were no significant

-/- differences, with cells from WT and NaV1.9 mice showing a comparable magnitude in

-/- response to BAM8-22 (WT: 55.2 ± 15.8; NaV1.9 : 26.9 ± 3.7; p=0.09) and an akin

-/- percentage of neurons responding after compound application (WT: 1.1 ± 0.4; NaV1.9 :

0.8 ± 0.2; p=0.5; Fig. 2-6E-F). These observations highlight the importance of NaV1.9 in

Ca2+ signaling in both histamine and CQ responsive neurons.

Pruritogens influence NaV1.9 currents

Next, we evaluated the overall electrophysiological properties of DRGs from WT and

-/- 2+ NaV1.9 animals identified by Ca imaging (Fluo-4) following a brief CQ application and

-/- washout. NaV1.9 DRGs exhibited a significant depolarized shift in the resting membrane potential (RMP) of 8.4 ± 3.0mV (p=0.09) (Fig. 2-7A). Action potential amplitude was not affected, yet the threshold to elicit an action potential trends towards

-/- more positive values from -13.1 ± 1.8mV to -7.4 ± 3.5mV in NaV1.9 neurons (p=0.67)

(Fig. 2-7B). Action potential kinetics are also affected as determined by a higher time to reach the maximum peak of 0.5 ± 0.2ms (T to peak; p=0.016), and a lower time to reach the minimum value in the repolarization phase of 3.6 ± 1.2ms (T to min; p=0.004).

85 These results are largely in line with a previous report from Leipold et al., who measured action potential kinetics in a large heterogeneous DRG population [38]. In the presence of CQ (100μM), WT DRGs showed a lower amplitude of 10.4 ± 2.7mV

(p=0.002) and a higher action potential threshold of 4.4 ± 2.4mV (p=0.033) whereas the

-/- T to peak and T to min were not affected (Fig. 2-7C). Notably, NaV1.9 DRGs were not affected by the addition of CQ in all parameters tested (Fig. 2-7D), suggesting a key role for NaV1.9 in CQ-mediated signaling. Pro-inflammatory mediators such as PGE2, bradykinin, histamine, and ATP can potentiate NaV1.9 currents [34], which is consistent with its role in inflammatory induced thermal and mechanical hyperalgesia. Therefore, we determined whether application of either histamine or MrgprA3/MrgprC11 agonist

(CQ/BAM8-22) would also lead to increases in DRG TTX-Resistant (TTX-R) current density or changes in activation voltage. Since MrgprC11+ neurons are a subset of the

MrgprA3+ population, we employed MrgprA3-GFPCretdTomato/+ mice to isolate DRG neurons expressing the receptors for CQ and BAM8-22. We performed patch-clamp experiments in the presence of TTX (300nM), which blocks all NaV channel subtypes except for NaV1.8 and NaV1.9. Additionally, it is established that F- in the pipette permits the partial separation of both Na+ currents with NaV1.9 showing augmented currents and more hyperpolarized activation whereas NaV1.8 is unaffected [159]. Following CQ application, we observed a larger current density at voltage ranges where only NaV1.9 is active (Fig. 2-8A and 2-8B). Furthermore, we also noted a 15mV hyperpolarizing shift in channel activation voltage of the TTX-R Na+ current (Fig. 2-8C). Although we saw

+ significantly larger NaV1.9 current density (Fig. 2-8D, we observed reduced Na current

+ densities at more depolarized voltages where Na current is mainly driven by NaV1.8

86 (Fig. 2-8E and 2-8F). With the application of BAM8-22 (10μM), we noted a similar potentiation in NaV1.9 current density at more hyperpolarized potentials (Fig. 6G-H, J), but no significant differences in NaV1.8 current density (Fig. 2-8H and 2-8K-L). The application of BAM8-22 also led to an 11.5mV hyperpolarized shift in activation voltage of the TTX-R current (Fig. 2-8I). Finally, CQ and BAM8-22 accelerated NaV1.9 activation but not that of NaV1.8 (Fig. 2-9). Extensive overlap between CQ and histamine responsive neurons has been reported (9).So, we explored if histamine application

(100μM) affected TTX-R currents in MrgprA3+ (tdTomato+) neurons. Although we occasionally saw larger NaV1.9 current densities, we did not find significant potentiation of NaV1.9 or NaV1.8. However, histamine application did result in a 6.5mV hyperpolarized shift of activation voltage in the TTX-R current (Fig. 2-8M-R). The observation that not all cells showed this effect in response to histamine can be explained by the population of histamine responsive neurons only partially overlapping with CQ- and BAM8-22-responsive cells. Therefore, it is possible that the non- responsive cells lacked the appropriate receptors for histamine. To further assess if CQ or BAM8-22 influenced NaV1.8 currents, we transiently transfected mouse NaV1.8 along with MrgprA3 or MrgprC11 in ND7/23 cells. In cells containing NaV1.8 and MrgprA3, we did not see altered channel activation kinetics after the application of CQ; however, we did observe a similar reduction in current densities at a range of voltages (Fig. 2-10A,

C). In cells transfected with NaV1.8 and MrgprC11, we noted comparable current densities and channel activation properties before and after application of BAM8-22

(Fig. 2-10B, D).

87 +/WT The NaV1.9 p.L799P mutation leads to gain of itch in mice

We next used a CRISPR/Cas9 strategy to introduce an orthologous p.L811P

+/WT (c.2432T>C) gain-of- function mutation into mouse NaV1.9 (p.L799P ; c.2396T>C) against our tetO cassette background (mouse and human NaV1.9 proteins are 73% identical). After five back crossings (first with the B6.C-Tg(CMV-cre)1Cgn global Cre mouse from Jackson laboratories, USA), we performed itch assays on the p.L799P+/WT mouse line and compared the data to those obtained with WT NaV1.9 mice (Fig. 2-11A).

As is the case with human p.L811P+/WT patients, the p.L799P+/WT mice exhibited robust spontaneous scratching when compared to their WT littermate controls (Fig. 2-11B). In virtual agreement with previous observations [38], qRT-PCR analysis of DRGs from

+/WT sfGFP- NaV1.9L799P mice revealed a 68% reduction in overall SCN11A RNA levels when compared to WT controls (Fig. 2-12).

Leipold et al. [38] reported a significantly depolarized RMP in DRGs from NaV1.9

+/WT p.L799P mice, which in turn could lead to nerve conduction block as other NaV channel subtypes expressed in the DRG would inactivate. However, it is possible that some histamine and/or MrgprA3+ DRG neurons are hyper-excitable at depolarized

RMPs and continue to signal itch stimuli. To test this hypothesis, we first measured Ca2+

+/WT responses in DRGs from NaV1.9 p.L799P mice and WT littermate controls in response to histamine, CQ, and BAM8-22. We observed no difference in the number of cells or in the magnitude of the Ca2+ response after the application of either histamine or

BAM8-22 (Fig. 2-11C-F). Although we did not observe a difference in the peak response after the application of CQ, we noticed that the percentage of responsive neurons was

+/WT higher in cells from NaV1.9 p.L799P mice when compared to WT (Fig. 2-6C-D, Fig.

88 2-11G-H). The depolarized RMP caused by the mutation would reduce the driving force

2+ for Ca it is not unreasonable to assume that the depolarized RMP in NaV1.9 p.L799P+/WT mice pushes the weakly responding CQ neurons to respond more strongly.

tdTomato/WT L799P/WT Next, we crossed MrgprA3-EGFPCre mice with NaV1.9 mice to investigate the excitability of MrgprA3+ neurons. This resulting mouse line allowed us to perform current- clamp experiments on MrgprA3+ DRGs expressing the mutant channel.

Similar to what was reported before [38], we found a significantly depolarized RMP in

+ L799P/WT MrgprA3 neurons from NaV1.9 mice compared to littermate controls (WT = -40.0

± 1.3mV and mutant = -23.9 ± 1.8mV; p = 0.0007, n = 21; Fig7I). Most tested MrgprA3+ neurons containing the mutant channel were unable to generate action potentials, even in response to large current injections (17 out of 21 cells, Fig. 2-11K). However, a small

DRG subset (4 out of 21 cells) could fire at a depolarized RMP, even in response to current injections as low as 10pA, and repetitive action potentials at 50pA, which was substantially less than the current needed to evoke action potentials for WT MrgprA3+ neurons (>50pA; Fig. 2-11J-L).

89 DISCUSSION

While the symptom of itch is prevalent and has a substantial disease burden, it remains challenging to address. As such, new therapeutics for pathological itch are needed. While NaV1.9 has a proven role in pain [61], the role of this NaV channel subtype in itch is less understood. Here, we report a clinical case of a patient with a heterozygous NaV1.9 p.L811P mutation that suffers from unbearable pruritis (Fig. 1A).

To better understand NaV1.9 involvement in itch, we developed an N-terminally sfGFP- tagged NaV1.9 mouse line. Using this mouse line, we determined that this channel subtype was expressed primarily in a subset of non-myelinated, non-peptidergic small diameter DRGs that typically contain MrgprA3 or MrgprC11 (Fig. 2-1B, 2-2, 2-3, 2-4, 2-

5).

In our studies, itch evoked by administering histamine, CQ, or BAM8-22 in

-/- NaV1.9 animals, revealed a strong reduction in acute scratching behavior compared to WT controls (Fig. 2-6). Moreover, several action potential parameters in WT DRGs are altered by CQ but not in the absence of NaV1.9 (Fig. 2-7). It is noteworthy that

-/- NaV1.9 mice still scratched moderately in response to administration of CQ, a possible off-target effect of this compound. Indeed, Liu et al. measured CQ-induced scratching bouts in SASH mice, which lack mast cells, and still found a significant reduction in the total bouts of scratching compared to controls [53].

While it is challenging to anthropomorphize the obtained results, NaV1.9 p.L799P+/WT mice displayed an increased frequency of spontaneous scratching as is the case with human patients (Fig. 2-11). Previous work has shown that this mutation can lead to a slowing of deactivation as well as a hyperpolarizing shift in channel

90 availability, which in turn would lead to excess Na+ influx that can subsequently inactivate other NaV channel subtypes to cause conduction block [38]. Indeed, we

+ L799P/WT found that most MrgprA3 neurons from NaV1.9 mice were unable to fire action potentials even in response to large current injections. However, a subset of MrgprA3+ neurons with depolarized RMPs could still fire action potentials in response to current injections as small as 10pA, much less than for most WT MrgprA3+ neurons (Fig 2-11I-

L). Therefore, it is possible that this small subset of hyper-excitable MrgprA3+ neurons

L799P/WT (∼20%) may contribute to the increase in spontaneous itch seen in the NaV1.9 mice and in human patients. Although other NaV channel subtypes such as NaV1.7 [60] and NaV1.8 [22] may also be involved in transmitting pruritic stimuli, it is striking that loss of NaV1.9 leads to such drastic reductions in acute itch (Fig. 2-5). All three pruritic compounds potentiate NaV1.9 currents in mouse DRGs without affecting NaV1.8, except for CQ which influences both NaV1.8 and NaV1.9 currents (Fig. 2-8. 2-9, 2-10).

However, BAM8-22 activity predominantly affects NaV1.9 activation. Furthermore, CQ and BAM8-22 speed up the kinetics of activation of NaV1.9, making it more likely to contribute to the upstroke of the action potential. Finally, activation of MrgprA3+ neurons with either of these compounds leads to significant hyperpolarized activation of TTX-R currents. Thus, it is conceivable that NaV1.9 inhibitors administered to p.L811P patients can decrease itch while restoring, at least in part, pain sensations

[38, 40]. To identify such compounds, a dependable NaV1.9 cell line is of great value in drug screening experiments. Therefore, we developed such a tool using intron- mediated enhancement of gene expression, an approach that may also be useful to enhance heterologous expression of other ion channels (Fig. 2-1F-G). The

91 peripherally-restricted expression of NaV1.9 should be beneficial in limiting adverse side-effects of therapeutics. Altogether, the insights reported here can help us better understand itch and show that NaV1.9 constitutes an attractive pharmacological target to relieve pathological itch.

92 MATERIALS AND METHODS

Case report and subject details

Written authorization for the release of health information in the case report was obtained from the patient's parent (her legal guardian) in accordance with the guidelines of the Johns Hopkins Medicine Institutional Review Board. The patient verbally assented, the parent supplied the photograph used in this publication, and the parent and patient approved the written content of the paragraph describing her clinical features and the layout of the picture showing the scars. All personal information that could lead to identification has been removed.

Mouse Lines

F.A.S.T. NaV1.9 mice were generated by iTL technologies (USA). NaV1.8-Cre mice were provided by Dr. Michael Caterina (Johns Hopkins University School of Medicine).

Ai9(RCL-tdTomato) and SCN11atm1Dgen/J were acquired from Jackson Laboratories

(USA). All experiments were performed using protocols approved by the Animal Care and Use Committee of Johns Hopkins University School of Medicine. 2-4 months old male and female mice were backcrossed to C57BL/6J in our mouse colony for at least five generations. We housed 4-5 mice in each cage in the vivarium with a 12hr light/dark cycle and an ad libitum food and water supply.

Behavioral itch assays

All mouse behavior tests were performed and analyzed with the experimenter blinded to genotype. Male and female mice (8-12 weeks old, 20 to 30g each) were used for

93 experimentation. All itch behavior was performed between 8a.m. and 12p.m. On the day before the experiment, animals were placed in the test chamber for 30 minutes before undergoing a series of three mock injections with 5-minute breaks in between.

On the day of the experiment, animals were allowed to acclimatize to the test chamber for 10 minutes before injection. Pruritic compounds were subcutaneously injected into the nape and scratching behavior was observed for 30 minutes. A bout of scratching was defined as a continuous scratching, not wiping, movement by either hind paw directed at the area of the injection site. Scratching behavior was quantified by counting the number of scratching bouts over the 30-min observation period.

Concentrations used for all compounds were 1mM dissolved in physiological saline.

Cultures of dissociated DRG neurons

Adult mice (8-12 weeks old) were anesthetized with CO2 followed by cervical dislocation. The spinal column was removed and trimmed of excess muscle. The vertebral column was then bisected, and one side was placed into a Petri dish with cold bicarbonate-free (bf)DMEM (Thermo-Scientific, USA), while the DRGs were collected from the other side. In a separate Petri dish, DRGs were collected into cold bfDMEM. Once all ganglia were collected, forceps and fine scissors were used to trim excess nerve roots from the ganglia. Trimmed ganglia were placed into a 15ml falcon tube with 4ml of enzyme mix for 30 minutes at 37°C. The enzyme mix consisted of

0.78 mg/ml of protease (Worthington, USA) and 1.25 mg/ml Collagenase I

(Worthington, USA) in 4ml of bfDMEM. After incubation, the ganglia were centrifuged at 50 RPM for 5 minutes. The enzyme solution was removed, and ganglia were

94 washed with complete DMEM (10% FBS, 1X penn/strep) and then centrifuged at 600

RPM. After the centrifugation step, ganglia were triturated with 1ml pipette tips 20-30 times or until no large chunks of tissue remained. The dissociated cells were put through a 100μm strainer (Fisher Scientific, USA). The cells were then centrifuged for

5 minutes at 600 RPM. After this step, the overlying solution was removed and replaced with 200-400μl or in a volume of cell culture media (complete DMEM,

10%FBS, 1X penn/strep) necessary to drop the desired density of cells onto poly-D- lysine coated coverslips in 24-well plates (Thermo-Scientific, USA). About 60-80μl of cell solution was applied to the center of the coverslip. Cells were allowed to settle for

30 minutes and then the well was flooded with 500μl of additional cell culture media.

Immunofluorescence

Adult mice (8-12 weeks old) were anesthetized with CO2 and perfused with 15ml

0.1M PBS (pH 7.4, 4°C) followed by 25ml of fixative (4% paraformaldehyde (vol/vol)

and 14% sat. picric acid (vol/vol) in PBS, 4°C). Spinal cord and DRGs were dissected

from perfused mice. DRG was post- fixed in fixative at 4°C for 30 minutes, and spinal

cords were fixed for 2hrs. Skin was dissected from non-perfused mice and fixed in 2%

paraformaldehyde (vol/vol) for 2-4hrs at 4°C. After fixation, tissues were washed with

0.1M PBS 3 times for 5 minutes each. All tissues were cryoprotected in 20% sucrose

(weight/volume) for more than 24hrs and were sectioned with a cryostat at 20μm for

DRG and nerves and 30μm for spinal cord and skin. DRGs and nerve sections were

dried at 37°C for 30 minutes. An antigen retrieval step was also conducted with

sodium citrate (10mM, pH 6, and 0.05% Tween-20) for 10 minutes at 85°C, followed

95 by three washes 5 minutes each with 0.1M PBS. Tissue was pre-incubated with blocking solution (10% fetal bovine serum (volume/volume), 0.1% Triton X-100

(volume/volume) in PBS, pH 7.4) for 1-2hrs, and then incubated overnight at 4°C with primary antibodies. Secondary antibody incubation was performed for 2hrs in blocking solution at 22-23°C. Three washes with PBS (0.1% Triton X-100) for 5 minutes each were performed in between incubations. For spinal cord and skin, cryostat sections were cut to 30μm and placed in 0.1M PB for whole-mount immunostaining. Sections were then incubated in blocking solution for 1hr at room temperature. Next, sections were incubated in primary antibody overnight at 4°C with gentle shaking for at least

24hrs. Primary incubation was followed by three washes for 5 minutes each, followed by incubation with secondary antibody for 2hrs at room temperature. Finally, sections were washed three more times for 5 minutes each, followed by 2 washes with H2O and mounted onto coverslips. The following primary antibodies were used: chicken anti-GFP ( 1:2000, Aves labs, GFP-1020, USA) rabbit anti-sfGFP (1:2000, gift from

Ramanujan Hegde; University of Cambridge, UK); rabbit anti- GFP (1:2000, Abcam, ab290); rabbit anti-NF200 (1:500, Millipore, AB1989, USA); rabbit anti- Substance P

(1:1000, Millipore, AB1566, USA); rabbit anti-tyrosine hydroxylase (1:500, Pel-

Freeze, P40101-150, USA); goat anti-c-RET (1:500, Novusbio, AF482, USA); rabbit anti-PGP9.5 (1:500, Thermo-Scientific, PA5-29012, USA); rabbit anti-CGRP (1:500,

Peninsula labs, T-4239, USA); anti-peripherin (1:200, Abcam USA, ab4666). To detect IB4 binding, sections were incubated with biotinylated Griffonia simplicifolia isolectin GS-IB4 (1:500, Vector Labs, B-1205, USA). For secondary antibodies, we used donkey anti-rabbit (A31572, Alexa 555 conjugated), Goat anti-chicken (A21103,

96 Alexa 488 conjugated), rabbit anti-goat (Alexa 555 conjugated, A214310) and for biotinylated IB4 we used streptavidin (DyLight 549, SA-5549, Vector Labs, USA). All secondary antibodies were diluted at 1:500 in blocking solution.

Western blot

Adult mice (8-12 weeks old) were anesthetized with CO2 followed by cervical dislocation. DRG and TG were dissected out and placed in 300μl lysis buffer (in mM:

320 Sucrose, 10 HEPES, 2 EDTA pH 8.0, 1.25 % Triton X-100, 50U/ml benzonase,

Roche protease inhibitor mini tablet; Roche, USA). Tissue was homogenized in a 1ml dounce homogenizer for 5-10 minutes on ice. The samples were then allowed to rotate at 4°C for 1hr. Following this step, tissue solution was centrifuged at 15,000

RPM for 20 minutes at 4°C. The supernatant with protein was removed and placed into a new tube and stored at -20°C until further use. All other tissue was placed into lysing beads (Matrix D, MP, USA) in 2ml tubes and kept on ice. The tubes were then placed into a benchtop homogenizer (FastPrep®-24 Classic Instrument homogenizer,

MP, USA) and homogenized using a custom-made program (speed: 4M/s for 20sec).

This step was repeated if the tissue was not completely homogenized. The samples were then centrifuged at 15,000 RPM for 20 minutes. Supernatant with protein was removed and placed into a new tube and stored at -20°C until further use. Protein concentration was determined using a BCA assay (Pierce, USA). All protein samples were appropriately diluted in 1X LDS (Thermo-Scientific, USA) plus reducing agent

(Thermo-Scientific, USA). 10μg of protein was run on 3-8% Tris-Acetate gels (Thermo

Fisher Scientific, USA) with Tris-Acetate running buffer and analyzed by Western

97 analysis. Nitrocellulose membranes were incubated in blocking buffer (5% BSA, 0.1%

Tween-20, in 1X TBS) and then probed overnight at 4°C with appropriate primary

antibodies and 2hrs at room temperature with 1:10000 goat anti-mouse HRP-

conjugated antibody as secondary antibody (Thermo-Fisher Scientific, USA).

Membranes were incubated for 5 minutes with an enhanced chemiluminescent

substrate before imaging (Thermo-Scientific, USA). Primary antibodies used: rabbit

anti sfGFP (1:2000, gift from Ramanujan Hegde; Cambridge, UK); rabbit anti-NaV1.9

(1:5000, Alomone labs, ASC-017, Israel); rabbit anti HSP90 (1:1000, Cell Signaling,

C45G5, USA)

Generation of ND7/23 stable cell lines

ND7/23 FRT cell lines (Sigma-Aldrich, USA) were generated using the Flp-InTM system (Thermo- Scientific, USA) as per protocol instructions. For the generation of sfGFP-NaV1.9 and NaV1.9 ND7/23 stable cell lines with intron 2 (synthesized by

Genscript, USA), ND7/23 FRT cells were grown to 20% confluency and co-transfected with the pOG44 vector (Thermo-Scientific, USA) and either sfGFP-NaV1.9 or NaV1.9 inserted into the pcDNA5/FRT vector (Thermo-Scientific, USA) at a ratio of 9:1, using the LipofectamineTM 3000 protocol (Thermo-Scientific, USA) as per protocol instructions. 24hrs after transfection, cells were washed and fresh cell culture media

(complete DMEM) was added. 48hrs after transfection, the cells were split onto a fresh

6-well plate so that they were no more than 25% confluent. After allowing the cells to settle for 2-3hrs, fresh culture media with the appropriate amount of hygromycin

(150ug/ml) (Thermo-Scientific, USA) was added. Non-transfected ND7/23 FRT cells

98 were used as a negative control. The cell culture media was replaced every 2-3 days and the transfected cells could grow until no cells remained in the control plates. After this, cells were split and allowed to grow for an additional passage, before using for future experiments or freezing. Transient transfections of mouse NaV1.8

(NM_001205321; Origene Technologies) with either MrgprA3 or MrgprC11 were performed with LipofectamineTM 3000 (Thermo-Scientific, USA) as per protocol instructions.

Ca2+ imaging

DRG neurons from male and female mice (8-12 weeks old) from NaV1.9-/-,

L799P/+ NaV1.9 , and littermate controls were dissociated using an established DRG

dissociation protocol (see above) and used for Ca2+ imaging studies within 36hrs after

dissociation. All recordings and appropriate compound dilutions were done in

modified Ringer’s solution (in mM: 140 NaCl, 5 KCl, 10 HEPES, 2 MgCl2, 2 CaCl2, 10

glucose, pH 7.4). Neurons were loaded with Fura-2-acetomethoxyl ester (2.5mM,

Thermo-Scientific, USA) plus 0.02% pluronicTM F-127 (Thermo-Scientific, USA) for

1hr in the dark at 22°C. After washing twice, cells were imaged at 340- and 380-nm

excitation to detect intracellular free Ca2+. The protocol for all imaging experiments

included a brief wash, followed by 90s of either histamine (100μM, Sigma-Aldrich,

USA), chloroquine (1mM, Sigma-Aldrich, USA), and BAM8-22 (10μM, Sigma-Aldrich,

USA) application, another brief wash, and finally 30s with high KCl (75mM) to identify

neurons. Cells that did not show a response to high KCl were discarded from our

analysis. Cells were counted as responding that had a peak 340/380 ratio response

99 15% above baseline. All experiments were done in triplicate. For each compound, a

total of 700-1000 neurons from 3 mice of the specified genotype were used for

analysis. Analysis of the images was performed with the experimenter blind to the

genotype.

Electrophysiological recordings of DRGs and ND7/23 cells

Pipettes were fabricated from borosilicate glass (A-M Instruments, USA) and pulled to

1.0-2.5MΩ using a P-1000 puller (Sutter Instruments, USA). A multiclamp 700B

(Molecular Devices, USA) amplifier with pClamp 10 software (Molecular Devices, USA) was used to acquire data. Series resistance for all cells was corrected electronically up to 70%. All recordings were done at room temperature. Current- and voltage-clamp recordings were obtained using whole-cell configurations in isolated DRGs using the dissociation protocol described above. Linear leak and capacitive transients were subtracted electronically using the -P/4 protocol. Data was acquired at 20kHz and filtered at 2kHz. 2% agar bridges filled with bath solution served as the reference electrode. For voltage-clamp recordings, bath solutions contained (in mM): 3 KCl, 10

Glucose, 10 HEPES, 10 TEA Cl, 40 NaCl, 90 Choline Cl, 2.5 CaCl2, 1 MgCl2, 0.05

CdCl2 pH 7.35, 308mOsm. Intracellular solutions contained (in mM): 100 CsCl, 30 CsF,

5 NaCl, 2.4 CaCl2, 1 MgCl2, 5 EDTA pH 8, 10 HEPES, 4 MgATP pH 7.3 303 mOsm.

Osmolarity and pH were adjusted with sucrose and NaOH, respectively. For heterologous expression of mNaV1.8 (clone obtained from Origene, USA) with either

MrgprA3 or GFP-MrgprC11, ND7/23 cells were transfected two days before recording with LipofectamineTM 3000 (Thermo-Fisher, USA) as per protocol instructions. Cells

100 transfected with mNaV1.8 and MrgprA3 were also co-transfected with mCherry to help identify successfully transfected cells. For recordings, cells were allowed to equilibrate for 10 minutes before obtaining seals. Recordings were done 10 minutes after obtaining whole-cell configuration to allow dilution of cell interior with the intracellular solution. Peak currents were measured using either 300-ms or 100-ms pulses between

-100mV or +60 mV every 10s from a holding potential of -120mV. The peak current was also normalized to cell capacitance and plotted against the voltage to obtain current density-voltage relationships. The normalized G-V curves were fit with the

Boltzman function: G = 1/(1 + exp(V – V1/2) /k) to determine V1/2. Time to peak was obtained by measuring the time from beginning of voltage step to peak current.

Activation time constant was obtained via a single-exponential fit (Clampfit 10,

Molecular Devices, USA). Other data analysis software used included Microsoft Excel, and Origin 8 (Originlabs, USA). For current-clamp recordings, bath solutions contained

(in mM): 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 Glucose, 10 HEPES, pH 7.4 adjusted with NaOH, 310 mOsm. Intracellular solutions contained (in mM): 140 K- gluconate,

13.5 NaCl, 1.6 MgCl2, 0.09 EDTA pH 8, 9 HEPES, 4 MgATP, 14 Tris Creatine PO4, 0.3

NaGTP, pH 7.4 adjusted with KOH, 311 mOsm. Resting membrane potential was measured in I=0 (zero current mode) for 30s immediately after establishing whole- cell configuration. Action potential thresholds were measured by a series of 100ms, 10pA depolarizing current injections. At the minimal current injection needed to elicit an action potential, firing threshold was determined by calculating the potential at which the rate of rise crossed 10V/s. Amplitude of the action potential was determined as the difference between the voltage value at the peak and the baseline before stimulation.

101 Time to peak corresponded to the time between the threshold and the maximum peak, and time to minimum to the time between the maximum and the minimum value observed in the hyperpolarization phase. Data were analyzed offline with Clampfit 10 and Axograph X (Molecular Devices, USA).

qRT-PCR analysis

DRGs were extracted from 8-12-week-old mice. Total RNA was extracted from samples using RNeasy Mini Kit according to manufacturer’s instructions (Qiagen,

Germany). TURBO DNA- freeTM kit was used to remove contaminating DNA

(Invitrogen, USA). Samples were then converted to cDNA using SuperScript III First-

Strand Synthesis System for RT-PCR (Invitrogen, USA). Taqman primers for mouse

SCN11A and 18S rRNA were designed and ordered from Invitrogen, USA. qRT-PCR reactions consisted of 1µl of 1/100 cDNA dilution, 5µl Taqman PCR master mix

(Invitrogen, USA), 0.5µM primers, and up to 10µl with deionized water. All samples were run in triplicate using Applied Biosystems QuantStudio 6 Flex Real-Time PCR system. The delta-delta Ct method was used to quantify relative amounts of cDNA for each gene of interest normalized to 18S rRNA.

Quantification and statistical analysis

Data are presented as mean ± SEM. ‘n’ represents the number of samples analyzed.

Statistical analysis was performed with Student t test, Mann-Whitney, or one-/two-way

ANOVA as indicated in figure legends. No data were excluded. Differences were considered statistically significant if p<0.05. Representative data are from experiments

102 that were replicated biologically at least three times with similar results. Detailed statistical analyses are mentioned in Fig. legends. Statistical analysis was done with

GraphPad (Prism 7.04, USA) and Microsoft Excel (USA).

103 Acknowledgements

This work was supported by a Department of Defense (DoD) National Defense Science and Engineering Graduate (NDSEG) Fellowship (J.S.), a Blaustein Pain Research grant, a Johns Hopkins Catalyst Award, and Johns Hopkins Bridge Funding Award to

F.B., the Hugo W Moser Research Institute at Kennedy Krieger Inc. (E.T.), and NIH grants R01DE022750 and R01NS054791 to X.D. X.D. is an Investigator of the Howard

Hughes Medical Institute. We would like to thank Peilin Shen for expert help with managing the mouse colonies, C. Hawkins and the staff of the Transgenic Mouse Core at Johns Hopkins University for assistance with transgenic mouse lines, Liang Han for the MrgprC11 antibody, and Roger Reeves, Michael Caterina, Matthias Ringkamp, and the members of the Bosmans lab for helpful discussions.

104

Figure 3-1. Generation and characterization of NaV1.9 mouse lines. (A) After healing, wounds from scratching left marks that resembled bruising (black arrows) in the p.L811P patient. (B) Schematic diagram of the flexible accelerated Neo-STOP tetracycline inducible (F.A.S.T.) cassette illustrating the generation of global knockout

NaV1.9 mice (red box) and Cre-mediated expression of sfGFP- tagged NaV1.9 mice

(green box). (C) A DRG section from an sfGFP-tagged NaV1.9 (top panel) and WT mouse (bottom panel) showing the overlap between endogenous fluorescent signal

(green), followed by staining with an antibody against GFP (red). (D) Western blot of

-/- DRG tissue from a WT, sfGFP-NaV1.9, and NaV1.9 mouse stained for GFP. A HSP90 antibody was used as a loading control. TG = trigeminal ganglia. (E) Western blot of tissues taken from a sfGFP-NaV1.9 mouse and stained for GFP, stripped, and re-probed

105 for NaV1.9 using a commercial antibody. A HSP90 antibody was used as a loading control. (F, G) Shown are representative current traces from ND7/23 cell lines expressing WT (black) or sfGFP-NaV1.9 (green) channels. (H) Current-voltage (I-

V) and deduced conductance-voltage (G-V) (I), and steady-state inactivation (SSI) (J) relationship of WT (black) and sfGFP-NaV1.9 (green). (G-V: WT-NaV1.9 V1/2 = -25.5 

0.5mV, n=16; GFP-NaV1.9 V1/2 = -24.0  0.2mV, n=11, p=0.52; SSI: WT-NaV1.9 V1/2 = -

29.3  3.5mV, n = 7; GFP-NaV1.9 V1/2 =-26.6  1.7mV, n = 7, p = 0.49) . *p<0.05,

**p<0.01 two-tailed unpaired Student’s t test. Data are represented as mean  SEM.

Scale bars represent 50μm.

106

Figure 3-2. Voltage-clamp recordings in DRGs show a similar TTX-R current in WT and sfGFP-NaV1.9 mice. (A) Current traces evoked at hyperpolarized voltages for WT

(black) and sfGFP-NaV1.9 (green) show similar kinetics (left) and voltage-dependence of activation (right): V1/2 = -61 ± 2mV, slope = 6.5 for WT and V1/2 = -65 ± 2mV, slope = 7.6 for sfGFP-NaV1.9 (n=3-5). Due to NaV1.8 current interference, the voltage range is cut off at -60mV (right). Tail currents are visible due to the channel still being open when hyperpolarizing again to -120mV. (B) Representative example of sfGFP-NaV1.9 currents

(left) in a DRG with undetectable NaV1.8 current up to -20mV (right). Data in (A) is represented as mean ± SEM.

107

Figure 3-3. sfGFP-NaV1.9 expression patterns. (A-H) DRG sections from sfGFP-

NaV1.9 mice stained for the indicated markers. (I) Graph of cell area in DRGs with the total number of neurons by cell size (black bar), using the pan-neuronal marker PGP9.5, as well as the neurons positive for GFP staining (grey bars, n = 200 GFP+ neurons and n = 600 PGP9.5+). (J) Graph showing the fraction of neurons positive for the markers indicated that were also positive for GFP (n = 200 neurons). (K) Sections of the dorsal horn stained with GFP and IB4, (L) the saphenous nerve stained with GFP and PGP9.5, and (M) glabrous skin of the hind paw stained with GFP and PGP9.5. Panels to the right of each image (K-M) show enlarged pictures of the corresponding section in the white box. Scale bars represent 50μm.

108

Figure 3-4: NaV1.9 in vagal ganglia and hairy skin. (A) DRG section from a

NaV1.8CretdTomato/+;sfGFP-NaV1.9 mouse stained against GFP shows large overlap between NaV1.8+ and NaV1.9+ neurons. (B-C) NaV1.9+ neurons overlap with a small subset of TRPV1+ in the jugular ganglia population. (D) NaV1.9 is only seen in the jugular ganglia with no or little expression in the nodose ganglia. (E) Little to no NaV1.9-specific staining is seen in hairy skin of sfGFP-NaV1.9 mice stained against GFP. Scale bars represent 50μm.

109

Figure 3-5: NaV1.9 expression in MrgprA3+ and MrgprC11+ neurons and behavioral models. (A) Schematic diagram showing the breeding strategy for the GFP-

NaV1.9;MgrprA3-EGFP- CretdTomato/+ mice, as well as DRGs from MgrprA3-EGFP-

CretdTomato/+ mice showing tdTomato signal (fluorescence was visualized directly without staining). (B) Trace showing NaV1.9 currents evoked in tdTomato+ neurons at the specified voltages. (C) DRG section showing the overlap between neurons stained for GFP and MrgprC11. (D) DRG section illustrating the overlap between neurons stained for GPF and Substance P. (E) Itch assays with the indicated compound injected

110 -/- into the nape of the neck, recorded for 30 minute intervals, performed in NaV1.9 mice and littermate controls for histamine (E, mice per genotype = 8, p=0.02), chloroquine (F,

-/- mice per genotype = 12, p=0.006), and BAM8-22 (G, WT n=11, NaV1.9 n=8, p=0.002).

Panels located directly below each graph (E-G) are the data binned and graphed for every 5 minutes of the recording. *p<0.05. **p<0.01 two-tailed unpaired Student’s t test.

Data are represented as mean  SEM. Scale bars represent 50um.

111

Figure 3-6: Loss of NaV1.9 leads to a reduction in histamine and chloroquine but not BAM8-22 responsive neurons. Fura-2 ratiometric Ca2+ imaging studies were

-/- performed in NaV1.9 mice and littermate controls. The total percentage of responsive neurons and the magnitude of the Ca2+ response were quantified and representative traces for the Ca2+ response after application of each compound are shown. For

- histamine, the total percentage of responsive neurons was reduced (A, WT and NaV1.9

/- n = 800 cells, p=0.0014), but the magnitude of the response was the same in WT and

-/- -/- NaV1.9 neurons (B, WT and NaV1.9 n = 800 cells, p=0.59). For chloroquine (CQ), the

-/- percentage of responsive cells was also reduced (C, WT and NaV1.9 n = 800 cells,

-/- p=0.038), but the magnitude of the response was similar in WT and NaV1.9 DRGs (D,

-/- WT and NaV1.9 n = 800 cells, p=0.44). For BAM8-22, no differences were observed

-/- for either the percentage of responsive cells (E, WT and NaV1.9 ; n = 800 cells, p=0.51)

-/- or the magnitude of the response (F, WT and NaV1.9 n = 800 cells, p=0.099). *p<0.05,

**p<0.01 two-tailed unpaired Student’s t test for all data represented as mean ± SEM.

112

113 Figure 3-7: Action potentials are influenced by NaV1.9 and CQ. (A) Representative

-/- action potentials for WT (blue), NaV1.9 (green), WT in the presence of CQ (100μM,

-/- red) and NaV1.9 mice in the presence of CQ (100μM, purple). DRGs were identified with Ca2+ imaging (Fluo-4) in response to CQ. Dotted lines represent the resting

-/- membrane potential (RMP) for WT (blue) and NaV1.9 (green); -40.0 ± 1.3mV and -

-/- 31.5 ± 2.7mV respectively (n=13 for WT and n=12 for NaV1.9 , p=0.009). (B)

-/- Quantification of action potential parameters in WT and NaV1.9 DRGs. Amplitude of action potential is not affected (96.6 ± 2.1mV for WT (n=16) and 90.1 ± 5.2mV for

-/- NaV1.9 (n=20), p=0.44) but the threshold to elicit an action potential trends up in

-/- -/- NaV1.9 cells (-13.1 ± 1.8mV for WT (n=20) and -7.4 ± 3.5mV for NaV1.9 (n=20), p=0.67). Absence of NaV1.9 affects action potential kinetics with a slower time to peak

-/- (T to peak, 3.0 ± 0.1ms for WT (n=17) and 3.5 ± 0.1 ms for NaV1.9 (n=20), p=0.016) and a faster time to minimum (T to min, 14.8 ± 0.7ms for WT (n=21) and 11.2 ± 1.0ms

-/- for NaV1.9 (n=18), p=0.004). (C) Chloroquine (CQ) treatment (100μM) affects the action potential amplitude and threshold, but not the kinetics. Amplitude of action potentials is 96.6 ± 2.1mV for WT (n=16) and 86.2 ± 1.8mV with CQ (n=16, p=0.002).

The threshold to elicit an action potential ranges from -13.1 ± 1.8mV for WT (n=20) to

-8.7 ± 1.5mV with CQ (n=16, p=0.033). T to peak, 3.0 ± 0.1ms for WT (n=17) and 3.2

± 0.1ms with CQ (n=17, p=0.063) and T to min, 14.8 ± 0.7ms for WT (n=21) and 16.5

± 1.8ms with CQ (n=18, p=0.732, were not significantly different. (D) These effects do

-/- -/- not occur in NaV1.9 DRGs: action potential amplitude is 90.1 ± 5.2mV for NaV1.9

(n=20) and 98.0 ± 6.1mV with CQ (n=12, p=0.5). The threshold to elicit an action

-/- potential ranges from -7.4 ± 3.5mV for NaV1.9 (n=20) to -9.9 ± 4.1mV with CQ (n=12,

114 -/- p=0.855). T to peak, 3.5 ± 0.1ms for NaV1.9 (n=20) and 3.7 ± 0.1ms with CQ (n=12,

-/- p=0.219) and T to min, 11.2 ± 1.0ms for NaV1.9 (n=18) and 13.5 ± 1.2ms with CQ

(n=16, p=0.157. *p<0.05, **p<0.01 two-tailed unpaired Student’s t test was used for

RMP and Mann- Whitney was used for all other data comparisons which are represented as mean ± SEM.

115

Figure 3-8: Application of chloroquine, BAM8-22, and histamine lead to a potentiation of NaV1.9 current and a shift in activation voltage of TTX-R current.

Voltage-clamp recordings from dissociated DRGs taken from MrgprA3CretdTomato/+ mice.

(A) Example current trace at -70mV shows that chloroquine (CQ) application potentiates

NaV1.9 current. (B) Current-Voltage (I-V) relationship plotted for the TTX-R current before and after chloroquine application. (C) Conductance-Voltage (G-V) plot for TTX-R current shows a 15.7mV hyperpolarized shift in activation voltage (before V1/2= -38.4 ±

4.8mV, after V1/2 = -54.2 ± 3.9mV, n=9, p=0002). (D) Quantification of NaV1.9 current density shows a it to be significantly higher after chloroquine (CQ) application compared to before compound (before I=-5.6pA/pF ± 2.0, after I=-8.1pA/pF ± 2.5, n=9, p=0.004).

(E) Example trace of NaV1.8 current and (F) quantification of the current density shows a significant decrease after administration of CQ (before I=-26.3pA/pF ±7.8, after I=-

4.6pA/pF ± 2.5, n=9, p=0.005). (G) Representative current trace at -70mV shows a larger NaV1.9 current density after BAM8-22 application. (H) I-V plotted for TTX-R

116 current before and after BAM8-22 application. (I) G-V plot for TTX-R current shows an

11.5mV hyperpolarized shift in activation voltage after BAM8-22 application (before V1/2

= -32.0 ± 2.2mV, after V1/2 = -43.5 ± 1.9mV, n=9, p=0.012). (J) Quantification of NaV1.9 current density shows it to be significantly higher after BAM8-22 application compared to before (before I = -3.5pA/pF ± 0.6, after I = - 10.6pA/pF ± 3.3, n=9, p=0.049). (K)

Example trace of NaV1.8 current and (L) quantification of the current density shows no differences before and after the administration of BAM8-22 (before I=-23.7pA/pF ± 4.1, after I=-24.8pA/pF ± 4.9, n=9, p=0.68). (M) A current at -60mV shows a moderate

NaV1.9 potentiation after histamine administration. (N) I-V plot for TTX-R before and after histamine administration. (O) G-V plot for TTX-R current after histamine shows a

6.5mV hyperpolarized shift in activation voltage (before V1/2 = -28.5 ± 4.8mV, after V1/2 =

-35.0 ± 4.2mV, n=10, p=0.029). (P) Quantification of NaV1.9 current density shows no significant change after histamine (before I=-5.0pA/pF ± 1.2, After I=-10.3pA/pF ± 3.7, n=10, p=0.14). (Q) Example trace of NaV1.8 current and (R) quantification of the current density shows no differences before and after histamine application (before I=-

27.8pA/pF ± 3.9, after I=-24.3pA/pF ± 5.0, n=9, p= 0.41). Current traces show NaV1.9 and NaV1.8 current before (black) and after (red) compound application. *p<0.05, ** p<0.01. Two-tailed unpaired and paired Student’s t test was used for all analyses. Data are represented as mean = SEM.

117

Figure 3-9. Activation of MrgprA3 and MrgprC11 speed up NaV1.9 activation.

Electrophysiological recordings from tdTomato+ neurons from MrgprA3tdTomato/+ mice. Activation of either MrgprA3 (A) or MrgprC11 (B) with their respective agonist shortened time to peak. Activation time constant is lower for NaV1.9 after MrgprA3 or

MrgprC11 activation. These effects are not observed with mouse NaV1.8 co-expressed with either MrgprA3 or MrgprC11, except at 0mV (C, D). Black color is before the addition of compound and red after. Asterisks indicate the results from a two-way

ANOVA test followed by Holm-Šídák post hoc analysis. *P˂0.05, **P˂0.01, and

***P˂0.001. Data are represented as mean ± SEM.

118

Figure 3-10. MrgprA3 and MrgprC11 activation in ND7/23 cells does not lead to changes in the NaV1.8 activation. Voltage-clamp recordings from ND7/23 cells transfected with mouse NaV1.8 and MrgprA3 or GFP-MrgprC11. (A) Current trace at

+20 mV shows effects of CQ on NaV1.8. (A, middle and right panel) Normalized current- voltage (I-V) and (C) conductance- voltage (G-V) relationships before and after CQ application show a similar inhibition of MrgprA3 activation on NaV1.8 as in DRGs, but with no shift in activation voltage (Before V1/2 = 7.6 ± 5.3mV; After V1/2 = 4.3 ± 5.6mV; n

= 8, p = 0.39). (B) Current trace at +20 mV shows no effect of MrgprC11 activation on

NaV1.8. (B, middle and right panel) Normalized current-voltage (I-V) and (F) conductance-voltage (G-V) relationships before and after BAM8-22 application show no change in NaV1.8 function in ND7/23 cells (Before V1/2 = 6.9 ± 4.2mV; After V1/2 = 13.0 ±

119 4.5mV; n = 8, p = 0.84). Black color is before the addition of compound and red after.

Two-tailed paired Student’s t-test was used for all. Data are represented as mean ±

SEM.

120

L799P/WT Figure 3-11: sfGFP-NaV1.9 mice show higher basal scratching and more chloroquine responsive neurons. (A) Schematic diagram showing the initial cassette

L799P/WT inserted to generate sfGFP-NaV1.9 mice and subsequent breeding to generate

121 L799P/WT mice for experimentation. (B) sfGFP-NaV1.9 mice showed a higher level of

L799P/+ scratching compared to their littermate controls (WT n=8, sfGFP-NaV1.9 n=9, p=0.043). Fura-2 ratiometric Ca2+ imaging studies were performed in sfGFP-

L799P/+ NaV1.9 mice and littermate controls. No differences were seen after histamine application in either the total percentage of responsive neurons (C, WT and sfGFP-

L799P/WT NaV1.9 n = 700 cells, p=0.15) or in the magnitude of the response (D, WT and

NaV1.9-/- n = 700 cells, p=0.36). For BAM8-22, no differences were observed for either

L799P/WT the percentage of responsive cells (E, WT and sfGFP-NaV1.9 n = 700 cells,

L799P/WT p=0.19) or the magnitude of the response (F, WT and sfGFP-NaV1.9 n = 700 cells, p=0.83). For chloroquine (CQ), the percentage of responsive cells was

L799P/WT significantly higher (G, WT and sfGFP-NaV1.9 n = 700 cells, n=0.042), but no difference was seen in the magnitude of the response (H, WT and sfGFP-

L799P/WT + NaV1.9 n = 700 cells, p=0.59). (I) RMP in MrgprA3 DRGs from WT (-40.0 ±

L799P/WT 1.3mV, n = 13)and NaV1.9 (-23.9 ± 1.8mV, n = 21) mice differ significantly (p =

0.0007). (J) WT MrgprA3+ neurons require ≥50pA current injection to spike whereas

+ L799P/WT (K-L) MrgprA3 ;NaV1.9 DRGs consist of a subset (17/21) that does not fire action potential at large current injections (≥500pA; RMP = -42mV) and a smaller group

(4/21) that fires in response to current injections as low as 10pA, and repetitive action potentials at 50pA (RMP = -26mV). *p<0.05, two-tailed unpaired Student’s t test for

Ca2+ imaging/current-clamp experiments and Mann-Whitney test for behavioral comparisons. Data are represented as mean  SEM.

122

L799P/WT Figure 3-12. qRT-PCR analysis of SCN11A RNA in sfGFP-NaV1.9 mice. (A)

L799P/WT qRT- PCR analysis on DRGs of sfGFP-NaV1.9 mice showed there was about a

68% reduction in SCN11A RNA when compared to littermate controls expressed as change in fold expression compared to WT levels (WT 1.1 ± 0.2, N = 3; sfGFP-

L799P/WT NaV1.9 0.36 ± 0.1, N = 3, p = 0.046).*p<0.05, two-tailed unpaired Student’s t test.

Data are represented as mean ± SEM.

123 THE ROLE OF NaV1.9 AND NaV1.1 IN SOMATOSENSORY PERCEPTION

Chapter 4: The role of NaV1.9 in the ENS

124 Summary

We find that NaV1.9 is expressed throughout the enteric nervous system (ENS) and that the loss of this channel leads to changes in gut motility. However, we observe

-/- no gross differences in the metabolism, in otherwise healthy mice, between NaV1.9 mice and littermate controls. The extensive expression of this channel in the ENS, however, underscores the need for a more thorough investigation of this channel in

ENS function.

125 Introduction

Patients with several reported gain-of-function mutations in NaV1.9 report gastrointestinal complications, ranging from abdominal pain to irregular bowel movements [61]. Although this NaV subtype could be partially responsible for the hypersensitivity experienced by IBS patients due to its expression in afferents innervating the gut [37], similar to what we observed with NaV1.1, this channel has also been shown to be expressed in both the myenteric plexus and submucosal plexus [73].

Therefore, NaV1.9 could also contribute to the on and off bouts of constipation and diarrhea experienced by a cohort of IBS patients. And targeting NaV1.9 could alleviate both the hypersensitivity and the irregular bowel movements of IBS patients.

As mentioned previously, knocking out NaV1.9 in mice leads to drastic changes

-/- in colonic migrating motor complexes (CMMCs). The CMMCs in NaV1.9 mice are more frequently and shorter durations than controls [73]. Additionally, activation of NK3R, which is an important intrinsic signaling mechanism within the ENS, leads to a potentiation of NaV1.9 [75]. However, there is a paucity of studies concerned with the functional relevance of NaV1.9 in the ENS.

Here, we also sought to determine the expression pattern of NaV1.9 in the

-/- myenteric and submucosal plexus, as well as determine if NaV1.9 mice have complications in guy motility, to help establish a role for this channel in gastrointestinal function. Additionally, due to all the gastrointestinal complains in human patients with gain-of-function mutations in this channel, we also wanted to explore any potential

-/- metabolic differences in our NaV1.9 mice.

126 Results

Expression of NaV1.9 in the enteric nervous system

The majority of myenteric neurons can be categorized into two broad groups of either inhibitory or excitatory neurons based on the expression of nitric oxide (NO) and vasoactive intestinal peptide (VIP), as in the case of the inhibitory population, or acetylcholine (Ach) and substance P, as in the case of the excitatory population [160].

In the myenteric plexus, we found that NaV1.9 was expressed in a small population of nNos+ neurons, while being expressed much more broadly in ChAT+ neurons, as there

+ were very few ChAT neurons that did not express NaV1.9 (Fig. 3-18). We also found

+ about 50% of TRPV2 neurons also expressed NaV1.9. Additionally, we found most neurons within the submucosal plexus also expressed NaV1.9 (Fig. 3-18).

-/- Gut motility in NaV1.9 mice

To determine if the loss of NaV1.9 lead to any changes in gross gut motility, we measured whole gut (WGTT) and duodenocaecal transit times (DCTT). We also measured stool content in terms of wet and dry weight. Although we found no differences in the whole gut transit times, we determined that there was a significant

-/- reduction in the DCTT of NaV1.9 mice when compared to littermate controls (Fig. 3-

19). Additionally, we also measured the total stool weight, as well as the wet and dry

-/- weights, and found that NaV1.9 mice had both a significantly higher wet and dry stool weight, although the total stool weights did not seem to show any difference (Fig. 3-19).

127 -/- Metabolic changes in NaV1.9 mice

A large portion of IBS patients report visceral hypersensitivity and many other patients also report changes in metabolism, either in weight gain or loss. Since NaV1.9 is known to be involved in visceral hypersensitivity, and we previously observed slower colon

-/- transit times and the increased wet and dry stool weights in the NaV1.9 mice, we wanted to determine if there were any changes to gross metabolic functions of the

-/- NaV1.9 mice. Therefore, we performed EchoMRI in conjunction with comprehensive lab animal monitoring system (CLAMS) to measure several metabolic parameters. We observed no changes in either total water, body weight, percent body fat, perfect lean

-/- muscle, O2 volume, CO2 volume, or energy expenditure in NaV1.9 when compared to littermate controls (Fig. 3-20). Although we did see significantly higher free water

-/- content in the NaV1.9 mice, the fact that no other changes were seen strongly indicated that this was most likely due to random variations in the EchoMRI experiments rather than to a real physiological effect.

128 Discussion

We and others have shown that NaV1.9 is expressed throughout both the

+ myenteric and submucosal plexi. NaV1.9 is expressed in a majority of ChAT and

TRPV2+ neurons, and in a smaller subpopulation of nNOS+ neurons. We also find that it is expressed in neurons of the submucosal plexus, but a more comprehensive analysis is needed to determine in what cell types it is expressed.

Additionally, NaV1.9 seems to be important for gut motility. In particular, the colon transit time was significantly faster, which could have implications for proper absorption of nutrients, and ultimately alter metabolic function. However, we found no differences in

-/- basal metabolism between NaV1.9 mice and controls. However, future experiments will need to assess the contribution of NaV1.9 to metabolism and visceral hypersensitivity in the CVH models. NaV1.9 is known to be potentiated by inflammatory mediators administered through the gut, so it’s feasible that NaV1.9 contributes to visceral pain.

However, it would also be interesting to see if changes in metabolism or in other

-/- measures used for mouse models of IBS are effected in NaV1.9 mice since human patients with mutations in NaV1.9 consistently complain of gut issues, such as diarrhea and constipation.

129 Methods and Materials

Gut Transit Times

Mice 8-12 weeks of age were used for measuring gut transit times. For whole gut transit time (WGTT), 0.25mL of red dye was inserted into the stomach with a gavage needle and total transit time was measured as the time it took for the red dye to appear in the feces of the mouse. For duodenocaecal transit time (DCTT), a bead was inserted via the anus to a distance of about 2.5 inches. The time it took for the bead to exit from the mouse was recorded as the DCTT. Mice were isolated into containers the entire time during the recording of either WGTT or DCTT. The same mice were used for WGTT and DCTT one week apart.

CLAMS

Indirect calorimetry and additional measures were performed with a 24-chamber, open- circuit, indirect calorimeter (Comprehensive Lab Animal Monitoring System, Columbus

Instruments). Mice were monitored continually and individually in the CLAMS for 4 days:

3 days to monitor adaptation to the novel cage environment, and a fourth day for data to report. The CLAMS measured periodically for each chamber rates of O2 consumption

(VO2) and CO2 production (VCO2), from which respiratory exchange rates were calculated (RER=VCO2/VO2). Software (Oxymax, Columbus Instruments) further utilizes VO2 and RER in an established formula to estimate rates of energy expenditure

(EE), with EE = (3.815 + (1.232 * RER)) * VO2 (Lusk, 1928). The CLAMS also measured food intake (Teklad 2018, 3.1 kcal/gram), water intake, and physical activity

(infrared beam array). CLAMS data were processed in Excel in time segments (daily 24-

130 hr, and 12-hr dark and light). Averages of each measure were calculated for each mouse, for each segment. VO2, VCO2, and EE were analyzed per kg of body weight, and also renormalized per kg of lean mass, by using body composition data (EchoMRI-

100) acquired the week prior to indirect calorimetry.

EchoMRI.

Scans are performed by placing the mouse into a thin walled plastic cylinder (1.7mm thick- 4.7 cm inner diameter) with a plastic cylindrical insert added to limit movement.

The tube is then inserted into a bore to the left side of the analyzer. The bore is surrounded by a resistive electro magnet which generate a static low intensity field

(<0.05 Tesla). The animal’s hydrogen nuclei are stimulated by safe radio frequency pulses. The system generate a signal that modifies the spin pattern of hydrogen atoms within the subject and uses an algorithm to evaluate the resulting T1 and T2 relaxation curves specific to each of the four components measured (lean, fat, free water and total water). Each scan takes 92-96 seconds.

Quantification and statistical analysis

Data are presented as mean ± SEM. ‘n’ represents the number of samples analyzed.

Statistical analyses performed as indicated in figure legends. No data were excluded.

Differences were considered statistically significant if p<0.05. Representative data are from experiments that were replicated biologically at least three times with similar results. Detailed statistical analyses are mentioned in Fig. legends. Statistical analysis was done with GraphPad (Prism 7.04, USA) and Microsoft Excel (USA).

131 Acknowledgements

This work was supported by a Department of Defense (DoD) National Defense Science and Engineering (NDSEG) graduate fellowship (JS). We would like to thank Susan Aja for help with the CLAMS experiments, Nadine for performing the EchoMRI, and Qian for helping during the gut motility assays.

132

Figure 4-1. Expression of NaV1.9 in the enteric nervous system. (A) Whole-mount myenteric sections from sfGFP-NaV1.9 mice stained against GFP showing overlap with nNOS, TRPV2, and ChAT. (B) Whole-mount submucosal sections from sfGFP-NaV1.9 mice stained against GFP showing overlap with PGP9.5.

133

-/- Figure 4-2. Gut transit time and stool content in NaV1.9 mice compared to

-/- littermate controls. (A) Duodenocaecal transit time in NaV1.9 mice is lower when

-/- compared to littermate controls (WT: 242.2  32.9 sec, n = 13; NaV1.9 : 164.6  13.4 sec, n = 13, p = 0.04). (B) Whole gut transit time measured in NaV1.9-/- is not different

-/- from littermate controls (WT: 121.0  9.1 min, N = 7; NaV1.9 : 115.7  7.22 min, n = 6, p

= 0.78). (C) The wet stool weight measured over the course of two hours was heavier in

-/- -/- the NaV1.9 mice than in littermate controls (WT: 0.19  0.14 g, n = 7; NaV1.9 : 0.35 

0.045 g, n = 6, p = 0.014). (D) The dry stool weight measured over the course of two

-/- hours was heavier in the NaV1.9 mice than in littermate controls (WT: 0.08  0.008, n =

-/- 7; NaV1.9 : 0.13  0.017, n = 6, p = 0.03). (E) However, the percentage of water measured over the course of two hours was not significantly different between the

134 -/- -/- NaV1.9 and WT controls with respect to the stool (WT: 57.9  1.4%, n = 7; NaV1.9 :

62.3  3.0 %, n = 6, p = 0.22). Two-tailed unpaired and paired Student’s t test was used for all analyses. All data presented as Mean  SEM.

135

-/- Figure 4-3. No effects on metabolism are observed in NaV1.9 mice. A whole-body

-/- composition analysis was done on NaV1.9 mice compared to their littermate controls using EchoMRI. There were no statistically significant differences observed for either the total water (B), body weight (C), % body fat (D), or % lean muscle (E). Although

-/- there was a significantly greater weight of free water (A) in the NaV1.9 , this was not considered physiologically relevant, as none of the other parameters showed any differences. With the same cohort of mice, a (CLAMS) analysis was also performed.

There no were no statistically significant differences in the volume of oxygen taken in

-/- (F) or the volume of carbon dioxide expelled (G) from NaV1.9 mice when compared to controls. (H) There was also no significant difference seen in the amount of energy expenditure during the measurement period. Two-tailed unpaired and paired Student’s t test was used for all analyses.

136 THE ROLE OF NaV1.9 AND NaV1.1 IN SOMATOSENSORY PERCEPTION

Chapter 5. Conclusions

137 Summary of findings

The findings shown here demonstrate a critical and unappreciated role of NaV1.9 and NaV1.1 for itch and visceral hypersensitivity, respectively. With the help from our collaborators Dr. Xinzhong Dong and Jimmy Meixiong, we showed a novel and important role of NaV1.9 in the transmission of histamine-dependent and histamine- independent itch. In addition, with the help from Dr. Brierley’s, Dr. Pasricha’s, and Dr.

Basbaum’s group we helped substantiate the findings that NaV1.1 is involved in the transmission of mechanical pain, specifically that NaV1.1 is critical for the development of visceral hypersensitivity that occurs frequently in abdominal pain associated with irritable bowel syndromes.

NaV1.9 is important for the coding of itch in the periphery.

Previously, several groups reported that gain-of-function mutations in NaV1.7 and

NaV1.8 lead to severe pruritus [60]. Additionally, inhibiting NaV1.7, a critical contributor to the initiation of action potentials, lead to a dramatic reduction in histamine-induced itch [59]. However, until now, no group has done a thorough analysis on the contribution of NaV channels to the transmission of pruritic stimuli. With the use of our novel GFP- tagged NaV1.9 mouse line, we were able to determine that NaV1.9 is expressed in a majority of MrgprA3+ and MrgprC11+ neurons. We further went on to show that BAM8-

22 (MrgprC11 agonist) and histamine-induced itch are almost completely absent in our

-/- NaV1.9 mouse model, while CQ-induced itch had roughly a 50% reduction in bouts of

-/- scratching. This was surprising since previous NaV1.9 mouse models show this

138 channel having only a moderate contribution to the maintenance of inflammatory- induced mechanical and thermal hypersensitivity. Although this channel was hypothesized to be critical for setting the resting membrane potential and amplifying subthreshold stimuli, it was predicted that inhibiting this channel would lead to dramatic reductions in pain-related behaviors. However, here we found that NaV1.9 was critical for BAM8-22 and histamine-induced itch, and very important for CQ-induced itch.

In addition, we selectively recorded from MrgprA3+ neurons and determined effects on channel behavior of both NaV1.9 and NaV1.8 caused by activation of histamine, MrgprA3, and MrgprC11 receptors. We discovered that activation of all three receptor types lead to a potentiation of NaV1.9 and to a hyperpolarizing shift in activation in TTX-R current, which we believed was mostly due to an effect on NaV1.9.

We were not able to exclude the possibility that the activation of these receptors also lead to a hyperpolarizing shift in the activation of NaV1.8 in our DRG recordings.

However, heterologous expression of MrgprA3 and MrgprC11 along with mouse NaV1.8 in ND7/23 cells, in this case a mouse DRG derived cell line, we showed that the activation of MrgprA3 and MrgprC11 did not significantly shift the activation of NaV1.8.

Paradoxically, we found that both in our DRG and ND7/23 recordings that NaV1.8 current was drastically reduced after the activation of MrgprA3.

Although NaV1.7 and NaV1.8 are also predicted to be important for itch sensation, it quite surprising that NaV1.9 is such a critical player. Since NaV1.9 is the least homologous protein in the NaV channel protein, this could have important implications for future drug design looking at selective inhibitors of itch.

139 L799P/WT The NaV1.9 mice phenocopy the itch observed in human patients with the orthologous mutation.

After determining the important contribution of NaV1.9 to itch, we wanted to explore how the gain-of-function NaV1.9 mutation described previously could be involved in the spontaneous scratching reported in several of the patients. Leipold et al. previously determined how this mutation lead to a gain-of-function of the channel kinetics, but how paradoxically it lead to a conduction block of neurons expressing the channel [38]. This mutation is located at the distal end of the sixth transmembrane segment of domain II and therefore is predicted to have an important contribution to channel inactivation. Indeed, this mutation leads to slower deactivation kinetics and to a large hyperpolarizing shift in steady-state inactivation of NaV1.9. This leads to a massive influx of sodium ions and to the eventual inactivation of other important ion channels even at resting conditions. As such, this would help explain the inability of human patients to fell pain due to the conduction block suffered by nociceptors expressing this channel. However, although these patients either had higher pain thresholds or experienced no pain, they still complained of severe pruritus. Therefore, the question remains, how then does this channel, which is presumable expressed in both pain and itch neurons, lead to loss of pain but a gain in itch phenotype? We wanted to address this question by investigating if mice with the orthologous mutation also showed a gain- of-itch phenotype like what was seen with the patients, and if the itch neurons that expressed this channel had conduction block or if at least a subset of them were hyper

L799P/WT excitable. After the generation of the sfGFP-NaV1.9 mice, we looked to see if the basal scratching in these mice was any different from their littermate controls. Although

140 the degree of scratching was likely not as severe as what was seen with the human patients, since we did not see self-inflicted wounds from the excessive scratching, we did find that there was a significantly higher amount of scratching in the mutant mice.

Inhibition of NaV1.1 reduces mechanical hypersensitivity in multiple chronic pain and IBS mouse models.

Previous research has demonstrated that NaV1.1 is important for the development of mechanical pain. However, here we have further substantiated this claim by showing that in a mouse model of IBS, the total Na current is decreased from

DRGs innervating the colon when compound B is administered. Since we have previously shown that compound B selectively inhibits NaV1.1, this effect is unlikely due to the inhibition of other NaV channels expressed in DRGs. In addition, we also showed that we could completely reverse the mechanical hypersensitivity in both mouse models of IBS using Compound B, which likely indicates a necessary contribution of NaV1.1 to the development of this visceral hypersensitivity. In line with these initial findings, another compound - Compound F - similar in selectivity as Compound B, was also found to attenuate the mechanical hypersensitivity in the acetic-acid evoked mouse model of IBS.

However, even though NaV1.1 is important for the development of the mechanical hypersensitivity in these mouse models, caution should be used when trying to extrapolate these findings to the visceral pain experienced by IBS patients. Apart from NaV1.5 [70], no other NaV channel has been functionally implicated in IBS. And although there are several IBS mouse models, no model wholly mimics the human

141 disease and drugs that are efficacious in mouse models don’t always turn out to be useful when administered to human patients [161].

NaV1.9 is expressed in the enteric nervous system and could be involved in gut motility.

Supporting prior work, we demonstrated that NaV1.9 is indeed expressed in a subset of neurons within both the myenteric plexus and submucosal plexus. We have also shown that the loss of NaV1.9 leads to changes in colon transit time and in stool

-/- content. Although we did not see changes in basal metabolism in our NaV1.9 mice, it

-/- would be interesting to explore possible changes in metabolism in NaV1.9 mice using similar IBS mouse models used for NaV1.1. This is particularly interesting due to previous reports of patients with gain-of-function mutations in NaV1.9 experiencing gastrointestinal symptoms, such as diarrhea, constipation, blotting, pain, and many other symptoms experienced by individuals with IBS. NaV1.9 is expressed throughout the ENS, and no doubt makes an important contribution to the proper functioning of the

ENS.

142 Future Directions

IBS is currently a major health concern. Unfortunately, IBS is a multifaceted condition with a vast array of symptoms that are difficult to treat, especially when it comes to the visceral pain experienced by a large portion of IBS patients [81]. Currently, there are few options to help treat the pain associated with IBS. However, our discovery that NaV1.1 is critical to the development of mechanical hypersensitivity in mouse models of IBS sheds some light on the potential underlying mechanisms of this visceral pain. We have identified a potential drug target to help treat the pain experienced in at least a fraction of IBS patients. However, future research will be needed to determine the translatability of inhibiting NaV1.1 to attenuate the visceral pain associated with IBS.

Additionally, since NaV1.1 is expressed in the brain, and inhibiting this channel centrally could have dire consequences, it will be important to develop drugs that cannot pass the blood brain barrier.

We have also shown that NaV1.9 is expressed throughout the ENS and the loss of NaV1.9 affects gut motility. However, more research is needed to determine to what extent NaV1.9 is important for proper ENS function. Although targeting NaV1.1 could help alleviate gut pain, inhibiting NaV1.9 could help with the other symptoms experienced by patients with IBS, such as the bloating, constipation, diarrhea, or any of the other major symptoms these patients suffer.

As for itch, it is a rapidly expanding field, as new proteins and neuronal subtypes are discovered that are important for the sensation of itch. For example, protease- activating receptor 2 (PAR2) has been implicated in triggering the itch sensation that occurs in atopic dermatitis [162, 163]. In addition, toll-like receptors (TLRs) which are

143 transmembrane glycoproteins involved in the innate immune response, and have been shown to be important for signaling pain, as also important for itch. Several groups have shown that TLR3, TLR4, and TLR7 act via distinct mechanisms to transmit itch sensations [164-166]. And it’s starting to be appreciated that there could be various subtypes of these putative itch neurons.

NaV channels, which are critical for the propagation of any neuronal signals, especially for the transmission of sensory stimuli, would be important to consider for future investigations of itch. Although we have shown that NaV1.9 is important for histamine-dependent and Mrgpr-dependent itch, would it also be the case for PAR2- or

TLR-dependent itch? While we only focused on one NaV channel, there are several others that are expressed in DRGs and TGs that could also be important for coding itch signals, such as the obvious candidates NaV1.7 and NaV1.8. However, research has also demonstrated that NaV1.1, NaV1.6, and NaV1.3 are important for pain, and it would be interesting to investigate if they are functionally important for the transmission of itch stimuli, as well.

However, a major public concern now is finding treatments for chronic itch conditions, such as the itch that develops from atopic dermatitis, eczema, disease- related itch, and as a side effect of common prescription drugs. Current medications are ineffective at treating certain kinds of itch that develop from a variety of different medical conditions. Therefore, future work in NaV channels and itch should focus on which subtypes are important for the development and maintenance of chronic itch. In fact, Qu L et al. has reported a significant increase in TTX-R current in a mouse model of allergic contact dermatitis [167]. And since many chronic itch conditions lead to

144 enhanced excitability of itch neurons, it would not be surprising to find NaV channels as key to that hyperexcitability. Therefore, future research should focus on what NaV subtypes are important for chronic itch. NaV1.7 has been a major focus on pharmaceutical companies, with several NaV1.7 selective inhibitors showing promise in reducing certain kinds of pain. Although these NaV1.7 inhibitors could be used to treat itch, NaV1.7 in known to be expressed in olfactory neurons and in the central nervous system, making side effects more likely. Other NaV channels, such as NaV1.8 and

NaV1.9, are known to have a more restricted expression pattern in the peripheral nervous system. So, it would be important to determine to what extend each NaV channel subtype is important for itch for future drug development efforts focused on treating itch.

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164 Curriculum Vitae Department of Physiology, Johns Hopkins University School of Medicine 725 N. Wolfe Street, Baltimore MD 21205

Education and Training May 2011 BS (Biochemistry) BA (Psychology) Mercer University (Macon GA)

2011—2013 NIH-funded PREP (Postbaccalaureate Research Education Program) Johns Hopkins University School of Medicine (Baltimore MD)

2013— 2018 PhD student, Johns Hopkins University School of Medicine (graduation August 2018) BCMB (Biochemistry, Cellular and Molecular Biology) program Mentor: Prof. Frank Bosmans (Department of Physiology)

Publications and Manuscripts

Walid MS, Sanoufa M and Salvatierra J (2010) Syringomyelia: a complication of an underlying pathology. J. Clinical Medical Research 2:102-4.

Lee DA, Salvatierra J, Velarde E, Wong J, Ford E and Blackshaw S (2013) Functional interrogation of adult hypothalamic neurogenesis with focal radiological inhibition. Journal of Visual Experiments 81, e50716

Lee DA, Yoo S, Pak T, Salvatierra J, Velarde E, Aja S and Blackshaw S (2014) Dietary and sex-specific factors regulate hypothalamic neurogenesis in young adult mice. Frontiers in Neuroscience 8:157.

Kalia J, Milescu M, Salvatierra J, Wagner J, Klint JK, King GF, Olivera BM and Bosmans F (2014) From Foe to Friend: Using Animal Toxins to Investigate Ion Channel Function. Journal of Molecular Biology 427:158-175

Salvatierra J, Lee DA, Wang H, Newman EA, Bedont J, Demelo J and Blackshaw S (2014) The LIM homeodomain factor Lhx2 is required for hypothalamic tanycyte specification and differentiation. Journal of Neuroscience 34:16809-20

Matin-Eauclaire MF, Salvatierra J, Bosmans F and Bougis PE (2016) The scorpion toxin Bot IX is a potent member of the -like family and has a unique N-terminal sequence extension. FEBS Letters 590:3221-32

165 Salvatierra J, Castro J, Erickson A, Li Q, Braz J, Gilchrist J, Grundy L, Rychkov GY, Deiteren A, Rais R, King GF, Slusher BS, Basbaum A, Pasricha P, Brierley S and Bosmans F. Nav1.1 inhibition can reduce visceral hypersensitivity. JCI Insight 3, e121000

Salvatierra J, Bustamante MD, Meixiong J, Xinzhong D and Bosmans F. A disease mutation reveals a role for Nav1.9 in acute itch. JCI (in review)

Honors and Awards

2008-2010 SMART Scholarship ($12,000) Department of Defense

2010- Gamma Sigma Epsilon (Chemistry Honors Society)

(honorable mention) NSF Graduate Research Fellowship

2015-2018 NDSEG Graduate Fellowship ($112,000, for three years) Department of Defense

(declined) Ford Foundation Graduate Fellowship The National Academies of Sciences, Engineering, and Medicine

2017 Trainee Professional Development Award ($1000) Society for Neuroscience

Teaching and Leadership

2008-09 General Chemistry Teaching Assistant Mercer University, Macon GA

2009-10 Organic Chemistry Teaching Assistant Mercer University, Macon GA

2009-10 Treasurer, American Chemical Society Mercer University, Macon GA

2010-11 President, American Chemical Society Mercer University, Macon GA

2011 Master at Arms, Gamma Sigma Epsilon (Chemistry Honors Society) Mercer University, Macon GA

166 2011 Biochemistry Teaching Assistant Mercer University, Macon GA

Presentations

2011 Talk on “Cogmed: A cognitive rehabilitation intervention to improve working memory in older adults.” 22nd Annual Student Mentoring Conference in Gerontology and Geriatrics Tybee Island, GA.

2012 Talk on “Lhx2 regulation of hypothalamic development is critical for homeostatic regulation of weight and metabolism.” Tri-PREP conference University of Pennsylvania

2013 Talk on “Hypothalamic-specific disruption of Lhx2 function affects tanycyte differentiation and body weight.” Mid Atlantic PREP & IMSD Research Symposium Virginia Tech

2017 Poster on “The role of Nav1.9 in somatosensory perception.” Society for Neuroscience Annual Meeting Washington D.C.

2018 Poster on “Modulation of Nav1.9 by pruritic (itch) stimuli.” Biophysical Society Annual Meeting San Francisco, CA

2018 Poster on “A disease mutation reveals a role for NaV1.9 in acute itch.” Channelopathy 2018 Chicago, IL

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