Studies Into the Mechanistic Basis of Local Anesthetic Action on Transient Receptor Potential Cation Channel Subfamily V Member 1 in Vitro

STUDIES INTO THE MECHANISTIC BASIS OF LOCAL ANESTHETIC ACTION ON TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL SUBFAMILY V MEMBER 1 IN VITRO

RICARDO ENRIQUE RIVERA

BSc., The University of Puerto Rico-Cayey, 2008

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

The Faculty of Graduate Studies

(Pharmacology)

THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)

April 2013

Transient receptor potential subfamily V, member 1 (TRPV1) channels are important integrators of noxious stimuli with pronounced expression in nociceptive neurons. Local anesthetics have been shown to modulate these nonselective cation channels in vivo and in vitro.

However, little is known about the specific interactions between local anesthetic molecules and

TRPV1 channels. This thesis therefore was dedicated to examining the mechanistic basis by which local anesthetic compounds act on TRPV1 channels from a perspective of neuronal manipulation for nociceptive blockade.

The experimental approach involved a series of in vitro laboratory studies where wild- type TRPV1, TRPV4, and mutant TRPV1 channels were expressed in Xenopus leavis oocytes and cation currents recorded using the two-electrode voltage clamp technique. QX-314 and lidocaine activated TRPV1 channels at millimolar concentrations, but not TRPV4 channels. The

TRPV1 antagonist, capsazepine, blocked QX-314- and lidocaine-evoked inward currents through a vanilloid-dependent pathway. At sub-activating concentrations (< 1 mM), QX-314 potently inhibited capsaicin-evoked TRPV1 currents. This thesis’ main results establish that the quaternary lidocaine derivative, QX-314, exerts biphasic effects on TRPV1 channels, inhibiting capsaicin-evoked TRPV1 currents at lower (micromolar) concentrations and activating TRPV1 channels at higher (millimolar) concentrations.

Further pharmacological characterization of amino-amide inhibition showed that QX-314 and lidocaine inhibit vanilloid- and proton-evoked currents in TRPV1 channels. Studies defining the molecular determinants of blockade revealed that lidocaine inhibits TRPV1 channels with nanomolar affinity, while the neutral derivative, benzocaine, does not, indicating that a titratable amine mediates the high-affinity block. Consistent with this hypothesis, extracellular

ii tetraethylammonium (TEA) and tetramethylammonium (TMA) application produced potent, voltage-dependent pore block.

The overall conclusion is that local anesthetics, previously reported to be able to enter cells through the activated TRPV1 pore, also act as multi-agonist permeant pore blockers. These findings provide an elementary structural model for the molecular interactions between established nerve blocking compounds and the TRPV1 nociceptor. At a fundamental level, this work introduces a novel use for these compounds as molecular probes for the study of TRP channels, whereas at a clinical level, the present results represent a step forward in the development of long-lasting, nociceptive-specific agents for the treatment of pain.

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Preface

The author, Ricardo E Rivera-Acevedo, performed the majority of the research and analysis leading to the results included in this dissertation which is comprised of work that has been published, awaiting publication and currently unpublished. Chapters 2, 4, and 5 represent articles that have been published, and are presented in their original forms, except for the reduction and modification of the introduction and methods sections to preserve continuity. The background for each of these chapters is laid out in the broad introductory section of this thesis. Chapter 3 contains currently unpublished work as of this writing. All the material is presented in the most up-to-date form at the time of completion of this thesis. Additionally, to avoid redundancy, a comprehensive general methods section was included. Xenopus laevis oocytes were used and isolated using methods approved by the UBC Animal Care Committee, certificate #A10-0074. The relative contributions of R.E. Rivera-Acevedo and fellow researchers are summarized below for each chapter:

Chapter 2: Rivera-Acevedo, R.E., Pless, S.A., Ahern C.A., Schwarz, S.K.W. The quaternary lidocaine derivative, QX-314, exerts biphasic effects on the transient receptor potential vanilloid subtype 1 channels in vitro. Published: Anesthesiology 114 (2011) pp. 1425-34. Reproduced with permission.

R.E. Rivera-Acevedo was responsible for approximately 80% of the work in this Chapter; including designing and performing all experiments, analyzing data, and writing the first draft of the manuscript, as well as re-writing portions of the manuscript requested by journal reviewers. S.A Pless contributed to the overall project design and the manuscript revision. C.A. Ahern was involved in developing the project and revising the manuscript. S.K.W. Schwarz was involved in developing the project and supervising the writing and revising of the manuscript as corresponding author.

Chapter 3: Rivera-Acevedo, R.E. Local anesthetics exhibit pH-dependent inhibition of transient receptor potential vanilloid subtype 1 channels in Xenopus oocytes.

Chapter 4: Rivera-Acevedo, R.E., Pless, S.A., Schwarz, S.K.W., Ahern C.A. Extracellular quaternary ammonium blockade of transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes. Published: Mol Pharm 82 (2012) pp. 1129-35.

R.E. Rivera-Acevedo was responsible for approximately 80% of the work in this Chapter; including designing and performing all experiments, analyzing data, and writing the manuscript, as well as performing additional experiments requested by journal reviewers. S.A Pless contributed to the overall project design and the manuscript revision. S.K.W. Schwarz was involved in developing the project and revising the manuscript. C.A. Ahern was involved in developing the project and supervising the writing and revising of the manuscript as corresponding author.

Chapter 5: Rivera-Acevedo, R.E., Pless, S.A., Schwarz, S.K.W., Ahern C.A. Expression- dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes. Channels 7 (2013) pp. 47-50.

R.E. Rivera-Acevedo was responsible for approximately 60% of the work in this Chapter; including designing and performing all experiments, analyzing data, and writing the manuscript. S.A Pless contributed to the overall project design and the manuscript writing. S.K.W. Schwarz was involved in developing the project and revising the manuscript. C.A. Ahern was involved in developing the project and supervising the writing and revising of the manuscript as corresponding author.

Table of Contents

Abstract ...... ii Preface ...... iv Table of Contents ...... vi List of Tables ...... ix List of Figures ...... x List of Abbreviations ...... xi List of Symbols ...... xiii Acknowledgements ...... xiv Chapter 1: General introduction ...... 1 SCOPE OF TOPIC AND APPROACH TO RESEARCH ...... 1 OVERVIEW ...... 4 HISTORY OF LOCAL ANESTHESIA ...... 6 Cocaine: the first local anesthetic ...... 6 Rise of modern local anesthetics ...... 8 LOCAL ANESTHETIC STRUCTURE ACTIVITY RELATIONSHIP (SAR) ...... 10 Aryl group ...... 12 X function ...... 12 Aminoalkyl group ...... 13 PHYSIOLOGY AND PHARMACOLOGY OF NERVE BLOCK ...... 13 Basic nerve function ...... 13 Molecular mechanisms of local anesthesia ...... 15 Importance of fiber size on function ...... 17 DIFFERENTIAL FUNCTIONAL NERVE BLOCK ...... 18 Subarachnoid and epidural anesthesia ...... 18 Uncovering the mechanisms of differential block ...... 22 The new size principle ...... 24 Decremental block...... 25 Pharmacodynamic variations in local anesthetic compounds ...... 28 Anatomy and differential block ...... 30 Summary...... 32 MOLECULAR INTEGRATORS OF PAIN ...... 33 Nociception ...... 33 Nociceptor ion channels ...... 34 The transient receptor potential channel superfamily ...... 39 TRPV1: The “Capsaicin Receptor” ...... 43 Pathophysiology of mammalian TRPV1 channels ...... 45 TRPV1 channel regulation ...... 46 THE EVOLUTION OF LOCAL ANESTHETIC RESEARCH ...... 48 TRPV1 as a target for analgesic drugs ...... 49 The quest for the “Holy Grail” of local anesthesia ...... 50

General Materials and Methods ...... 52 CHEMICALS AND SOLUTIONS ...... 53 MOLECULAR BIOLOGY ...... 53 OOCYTE PREPARATION AND INJECTION ...... 54 ELECTROPHYSIOLOGY...... 55 CALCIUM IMAGING ...... 56 STATISTICAL ANALYSIS ...... 57 Chapter 2: The quaternary lidocaine derivative, QX-314, exerts biphasic effects on TRPV1 channels in vitro ...... 58 INTRODUCTION ...... 58 RESULTS ...... 60 DISCUSSION ...... 70 Chapter 3: pH-dependent local anesthetic inhibition of the transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes ...... 76 INTRODUCTION ...... 76 RESULTS ...... 78 QX-314 inhibits proton-induced TRPV1 currents in Xenopus laevis oocytes ...... 78 Lidocaine inhibits capsaicin and proton-induced TRPV1 currents in oocytes ...... 80 DISCUSSION ...... 83 Chapter 4: Extracellular quaternary ammonium blockade of transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes ...... 87 INTRODUCTION ...... 87 RESULTS ...... 89 Lidocaine inhibition of TRPV1 channels in Xenopus laevis oocytes ...... 89 The neutral LA benzocaine does not strongly inhibit TRPV1 channels...... 91 Quaternary ammonium compounds potently inhibit capsaicin-induced TRPV1 currents in oocytes ...... 92 TRPV1 inhibition by quaternary ammonium compounds is voltage-dependent ...... 93 Mutations near the selectivity filter weaken TEA inhibition...... 96 DISCUSSION ...... 100 Chapter 5: Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes ...... 105 INTRODUCTION ...... 105 RESULTS ...... 107 QX-314 exhibits expression-dependent block of TRPV1 channels ...... 107 TEA does not exhibit expression-dependent inhibition of TRPV1 channels ...... 109 Both QX-314 and TEA block of TRPV1demonstrate voltage dependence ...... 111 DISCUSSION ...... 113 Chapter 6: General discussion ...... 117 TRPV1 CHANNEL ACTIVATION BY LOCAL ANESTHETICS ...... 118 Direct insight into local anesthetic-mediated toxicity in vitro ...... 118 Structure-activity relation of local anesthetic TRPV1 agonism ...... 122 The aromatic “A-region” ...... 124 The amide bond “B-region” ...... 125

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The hydrophilic “C-region” ...... 126 Local anesthetic structure-activity relationship overview ...... 126 MOLECULAR BASIS FOR TRPV1 CHANNEL INHIBITION BY LOCAL ANESTHETICS ...... 131 Local anesthetics are multimodal TRPV1 channel inhibitors ...... 132 Quaternary ammonium determines local anesthetics inhibition ...... 134 Properties of the extracellular TRPV1 pore illuminated by local anesthetics ...... 137 Strengths and limitations of the study ...... 139 FUTURE DIRECTIONS FOR LOCAL ANESTHETIC TRPV1 RESEARCH ...... 140 References ...... 143

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List of Tables

Table 1. Physico-chemical properties of local anesthetics ...... 30

List of Figures

Figure 1. General structural characteristics found in all clinically available local anesthetic compounds ...... 10 Figure 2. Molecular structures for common clinically available local anesthetic compounds ..... 11 Figure 3. Illustration exposing the gross anatomy of the spinal cord...... 20 Figure 4. Subarachnoid anatomy ...... 21 Figure 5. Decremental conduction determined using a mathematical model of the myelinated axon...... 26 Figure 6. Pharmacodynamic changes in local anesthetics alter concentration-dependent selective neuronal inhibition ...... 29 Figure 7. Local anesthetic concentration after injection ...... 31 Figure 8. Conductance behavior ...... 33 Figure 9. Ion channels expressed in the peripheral terminals of primary afferent neurons ...... 39 Figure 10. Predicted topology of a TRP channel family subunit ...... 41 Figure 11. Partial rat TRP channel sequences utilizing multiple alignment tool COBALT (constraint-based multiple alignment tool) of the pore forming region ...... 42 Figure 12. Membrane topology and key residues involved in TRPV1 channel regulation ...... 45 Figure 13. Capsaicin activation of TRPV1 channels expressed in Xenopus laevis oocytes ...... 61 Figure 14. QX-314 activation of TRPV1 channels expressed in Xenopus laevis oocytes ...... 64 Figure 15. QX-314 does not activate TRPV4 channels ...... 65 Figure 16. The competitive TRPV1 antagonist, capsazepine, blocks QX-314-evoked currents in Xenopus oocytes and mammalian tsA201 cells ...... 67 Figure 17. QX-314 inhibits capsaicin-evoked TRPV1 currents ...... 69 Figure 18. QX-314 concentration-dependently inhibits proton-evoked TRPV1 currents in Xenopus laevis oocytes ...... 79 Figure 19. Lidocaine concentration-dependently inhibits capsaicin- and proton-evoked TRPV1 currents in Xenopus laevis oocytes ...... 81 Figure 20. Lidocaine inhibits capsaicin-evoked TRPV1 currents in Xenopus laevis oocytes in a concentration-dependent and reversible manner ...... 90 Figure 21. Benzocaine does not strongly inhibit capsaicin-evoked TRPV1 currents in Xenopus laevis oocytes ...... 92 Figure 22. The quaternary ammonium compounds, tetraethylammonium and tetramethylammonium, potently inhibit capsaicin-evoked TRPV1 currents ...... 95 Figure 23. Capsaicin activation of TRPV1 channel mutants in Xenopus laevis oocytes ...... 97 Figure 24. Mutation of residues near the selectivity filter ameliorates TRPV1 inhibition by tetraethylammonium ...... 98 Figure 25. QX-314 inhibition dependent on TRPV1 expression levels in Xenopus oocytes ..... 108 Figure 26. TEA inhibition of TRPV1 channels does not demonstrate expression-dependence in Xenopus laevis oocytes ...... 110 Figure 27. QX-314 and TEA mediate inhibition of TRPV1 channels through open channel pore block ...... 112 Figure 28. Structural composition of capsaicin and local anesthetic compounds used in this study...... 123 Figure 29. Functional interactions between amino-amide local anesthetic molecules and TRPV1 residues associated with vanilloid binding ...... 128

List of Abbreviations

Amino Acid One Letter Code A Ala alanine C Cys cysteine D Asp aspartate/aspartic acid E Glu glutamate/glutamic acid F Phe phenylalanine G Gly glycine H His histidine I Ile isoleucine K Lys lysine L Leu leucine M Met methionine N Asn asparagine P Pro proline Q Gln glutamine R Arg arginine S Ser serine T Thr threonine V Val valine W Trp tryptophan Y Tyr tyrosine

Other abbreviations Å angstrom (1e-10 m) ASIC acid-sensing ion channel Ca2+ calcium ion Cav voltage-gated calcium channel CGRP calcitonin gene-related peptide Cm minimum blocking concentration Erev reversal potential HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid K+ potassium ion K2P two pore potassium channel Kv voltage-gated potassium channel LA local anesthetic Na+ sodium ion Nav voltage-gated sodium channel NMDG N-methyl-D-glucamine P2X2 ATP-gated purinergic P2X receptor PIP2 phosphatidylinositol 4,5-bisphosphate PKA cAMP-dependent protein kinase pKa acid dissociation constant PLC phospholipase C QA quaternary ammonium

TEA tetraethylammonium TMA tetramethylammonium TRP transient receptor potential TRPV1 transient receptor potential vanilloid sub-type 1 TRPV4 transient receptor potential vanilloid sub-type 4 TRPA1 transient receptor potential ankyrin sub-type 1 V1/2 half-activation voltage (in mV) WT wild-type

xii

List of Symbols

α membrane spanning protein subunit F fluorescence ΔF change in fluorescence δ electrical distance z valence λ wavelength (nM)

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Acknowledgements

I would like to begin by expressing my sincere gratitude to both of my co-supervisors, Dr. Stephan K.W. Schwarz and Dr. Christopher A. Ahern, for their guidance, mentorship, and patience throughout these years. They have each imparted a great deal of their knowledge to my personal and professional formation. For always showing me their support during the best and worst of times, without them this would not have been the fulfilling life-changing experience it has turned out to be. I would also like to recognize my supervisory committee, Dr. Stephanie Borgland, and Dr. Bernard MacLeod. Their unshakable demands for higher standards have helped me become a better scientist. Drs. Ana Niciforovic and Stephan Pless, for the indispensable guidance in all things related to molecular biology and ion channel pharmacology. I am grateful to the emeritus professors, Drs. Michael Walker and Richard Wall, for providing very insightful and often humorous perspectives into the multi-faceted aspects of pharmacological research. I must not forget Drs. Harley Kurata, Eric Accili, and Filip van Petegem for the additional mentoring during times of doubt and for being supportive when most needed. In addition, I would also like to express my appreciation to all friends and family who motivated me not to give up during the dark lonely days in the lab. In particular, Drs. Maen Sarhan and Koen Raedschelders, whose guidance helped tremendously during times when I doubted my path in life. Finally, I wish to thank my mother Doris J. Acevedo, father Lucas E. Rivera, and aunt Ana L. Acevedo, for the support and encouragement throughout my studies and for being exceptional role models throughout my life.

“Oye yo vivo contento y sigo en el vacilón, pues si me apuro me muero y si no me muero también, tu ve”

-Hector Lavoe-

“Listen I live my life happily and always in the party, cause if I’m in a rush I’ll die, and if I don’t I’ll die as well, you see”

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Chapter 1: General introduction

SCOPE OF TOPIC AND APPROACH TO RESEARCH

Pain is defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” according to the

International Association for the Study of Pain (IASP, 1994). Pain serves as an important physiological mechanism to make us aware of noxious injurious situations and to quickly remove ourselves from the insult preventing further harm. Problems arise when the acute pain experienced from localized tissue damage (during a surgical intervention for example) becomes persistent, unremitting, and unmanageable. To this day, pathological pain has been an under treated global health problem, especially for patients suffering from advanced diabetes, neurological disorders, and cancer, diseases which inflict chronic pain (Pizzo, 2012). Over 100 million people in North America and many millions more worldwide are thought to be affected

(Pizzo, 2012). Pain management issues strain the health systems of many Western countries where the medical costs for treating these conditions are in the billions of dollars (Roy, 2002).

Conservative reports estimate that the personal and financial burden from unmanageable pain in the US reaches $635 billion (Pizzo, 2012). In the US, the market for neuropathic pain analgesics alone is expected to double from the current $2.6 billion to $5 billion by 2018 (Szallasi and Sheta,

2012). Hence, our research is motivated by the clear global demand for new targets to effectively treat and prevent pathological pain.

The abolishment of pain has long been one of the central goals for all practitioners of the medical profession. Safe and effective pain control is a critical concern in the realm of modern medicine for perioperative care to this day, but it is not uncommon for patients to still experience severe pain even under ideal medical treatment. Poor post-operative pain management may lead

1 to the development of chronic or neuropathic pain conditions which often require a cocktail of multiple daily medications, including opioids, non-steroidal anti-inflammatory drugs (NSAIDS), corticosteroids, anti-depressants, and local anesthetics (LAs) (Katz and Barkin, 2008). Most of the drugs available either provide incomplete pain relief or exhibit a robust spectrum of dangerous adverse events and associated contraindications, ranging from addiction and tolerance

(opioids), systemic metabolic de-regulation and weight gain (corticosteroids), to gastrointestinal and liver damage (NSAIDS). Of the available compounds, LAs are currently the most effective method of achieving nociceptive inhibition without impairment of consciousness. These molecules directly target and silence the source of the pain response, which appears to be the sensitization and ectopic activation of nociceptive neurons, through the intracellular inhibition of voltage-gated sodium channels (Omana-Zapata et al., 1997). Regrettably, at present LA administration for the treatment of extended post-operative pain is still highly impractical as the effects are relatively short acting (a few hours), exhibit undesirable concomitant block of motor function, and effective continuous administration requires insertion of indwelling catheters connected to infusion pumps.

Recently, alternative pharmacological strategies have been developed to explore the possibility of producing long-lasting nociceptive-specific analgesia in humans by targeting the transient receptor potential cation channel, subfamily V, member 1 (TRPV1). TRPV1 channels are polymodal mammalian nociceptive integrators abundantly expressed in primary pain sensing

(Aδ- and C-fibers) with a pore large enough to allow entry of macromolecules at least 400 Da

(Meyers et al., 2003). The traditionally employed method to achieve partial nociceptive specific anesthesia involves the careful titration of LAs, such as lidocaine, to produce a phenomenon known as differential nerve block. This technique exploits the intrinsic property for smaller

(pain-serving/nociceptive) nerve fibers to be electrically silenced before larger sensory and motor neurons. Although reasonably effective, the administration of millimolar concentrations required to induce nerve block of conventional LAs may lead to potentially life-threatening systemic toxicity (Scott, 1986). Within the last 5 years the quaternary lidocaine derivative, QX-

314, has risen from relative obscurity to become a promising candidate compound for nociceptive-selective block. Whereas QX-314 traditionally has been considered relatively membrane-impermeant due to its permanent positive charge, peripheral administration of QX-

314 alone has recently been demonstrated to produce prolonged (>12 h) motor, sensory, and nociceptive blockade in animal models in vivo (Lim et al., 2007). Coupled with the TRPV1 agonist, capsaicin, QX-314 has been reported to produce potent nociceptive-specific analgesia

(Binshtok et al., 2007). Presumably, QX-314 is able to selectively inhibit nociceptive fibers by entering through the activated pore of the TRPV1 channel. However, little knowledge exists on the mechanistic basis of interaction between LAs (including QX-314) and TRPV1 channels, and in-depth in vitro pharmacological characterization is required to accurately model the LA-

TRPV1 complex.

This thesis aims to fill this void and answer these questions by directly investigating the interactions between LAs and the mammalian TRPV1 channel in a stable, reproducible, isolated in vitro environment. Using the well-established Xenopus laevis oocyte expression system, this work will qualitatively and quantitatively characterize the pharmacological spectrum of QX-314 and lidocaine activity on TRPV1. The present investigations have identified novel molecular mechanisms and interactions that occur between both clinically available LAs as well as QX-314 and the TRPV1 channel. At the same time, the ligand-binding assays necessary to effectively investigate these dynamic polymodal membrane proteins have been optimized. This research

3 introduces an additional element to the repertoire of relevant molecular targets in analgesic pharmacology and expands upon the foundation of the quest for one of the “Holy Grails” of anesthesiology – a safe, long-lasting, non-addictive, and truly nociceptive-selective local anesthetic.

OVERVIEW

Throughout recorded history, humans have sought efficacious methods to abolish pain.

Prior to the development of anesthetics, it was necessary to tie down and subdue patients during surgical procedures in order to prevent involuntary reactions that could potentially harm them further. The skilled surgeons of the day were those that carried out operations swiftly limiting the suffering to the individual. The few analgesics available, such as opium and ethanol, were not potent or effective enough to mitigate the intense pain associated with surgery. Early 19th century writer Fanny Burney (1752–1840) eloquently described the excruciating pain she felt during a mastectomy (performed on the 30th of September 1811 by Napoleon’s chief surgeon) in a letter written to her sister Esther:

“This resolution once taken, was firmly adhered to, in defiance of a terror that surpasses all description, & the most torturing pain. The dreadful steel was plunged into the breast – cutting through veins – arteries – flesh – nerves – I needed no injunction not to restrain my cries.

I began a scream that lasted unintermittently during the whole time of the incision – & I almost marvel that it rings not in my Ears still! So excruciating was the agony. Oh Heaven!–I then felt the knife racking against the breast bone–scraping it! This performed while I yet remained in utterly speechless torture. My dearest Esther, not for days, not for weeks, but for months I could not speak of this terrible business without nearly again going through it!”(Hemlow, 1986).

In the late 19th century, European chemists began re-discovering compounds to deal with this problem: general anesthetics in the form of N2O and diethyl ether, and the Andean local anesthetic, cocaine. Interestingly, it was dentists, not physicians, who were responsible for the introduction of anesthesia into the operating room, as physicians of the time were more focused on treating the prevalence of deadly infections (Calatayud and Gonzalez, 2003). General anesthetics, which as the term indicates, produce a general depression of the body’s systems, was in its infancy crude, dangerous, and impractical for minor surgeries. Cocaine, on the other hand, produced complete block of pain and motor function in a localized area with effects that were readily reversible after only a few minutes (the definition of local anesthesia). With a wider margin of safety than centrally acting agents, experimentation with LAs led physicians to discover the first steps in achieving nociceptive-specific anesthesia through a phenomenon known today as differential nerve block.

In the last decade, exploration with experimental LAs has uncovered novel molecular interactions targeting the mammalian nociceptive integrator, the transient receptor potential vanilloid subtype-1 channel (TRPV1). The research for this thesis has expanded on the fact that

LAs dynamically regulate these nociceptors. Through molecular pharmacology and site-directed mutagenesis, the present work will characterize the interactions between LAs and these polymodal membrane proteins, while providing a simple model to describe these effects. As a whole, this research aims to add to the fundamental principles of TRPV1 pharmacology and introduce a novel but important target in the search for a safe, effective, long-lasting, and nociceptive-specific anesthetic.

HISTORY OF LOCAL ANESTHESIA

Cocaine: the first local anesthetic

In step with the evolution of techniques to block pain was the development of modern

LA compounds from cocaine. Coca leaves come from a bush of the genus Erythroxylum, a member of the Erythroxylaceae family which grows abundantly in Nicaragua, Venezuela,

Bolivia, and Peru since pre-Columbian times (Calatayud and Gonzalez, 2003). Of all the species in this genus, Erythroxylum coca contains the highest concentration of the cocaine alkaloid in its leaves, about 0.7–1.8% by weight (Van Dyke and Byck, 1982). The coca leaf is an integral part of Bolivian and Andean culture, with its earliest use and cultivation records dating back to 700

BC. (Calatayud and Gonzalez, 2003). Native populations of Peru believed the plant to be divine and a vital part of the socio-economic structure (Ruetsch et al., 2001). In the sixteenth century,

Spanish friars who accompanied the Conquistadors reported that coca leaves were mixed with chalk (calcium carbonate), rolled into small balls, and chewed to alleviate hunger, thirst, and to sustain strenuous activity. The first reference to coca’s anesthetic effects come from a Spanish

Jesuit friar, Bernabe Cobo (1582–1657), who, in his 1653 manuscript on the New World, mentions the alleviation of a toothache by chewing coca leaves (Calatayud and Gonzalez, 2003).

The birth of modern local anesthesia began in 1859 from a PhD project by the German chemist, Albert Niemann (1834–1861) (Cousins and Bridenbaugh, 1997). The Austrian naturalist,

Carl Von Scherzer (1821–1903), during his stop in Peru collected a large sample of coca leaves which he gifted to Niemann for further analysis (Calatayud and Gonzalez, 2003). Niemann, working in the Friedrich Wöhler Laboratory in Göttingen, managed to successfully isolate the coca leaves’ main alkaloid, naming it cocaine. Unfortunately, Niemann would pass away the following year, but his work was to be carried forth by his colleague, Wilhelm Lossen (1838–

1906), who in 1865 also determined cocaine’s correct molecular formula –C17H21NO4–

(Calatayud and Gonzalez, 2003). From its inception, Niemann clearly documented that this new compound produced a pronounced numbness of the tongue. The first experimental study on cocaine was actually conducted in 1868 by the Peruvian physician, Thomas Moreno y Maiz, as part of his doctoral thesis in Paris. Although he did not mention its use for surgery, he accurately described cocaine’s LA effects following injection in rats, guinea pigs, and frogs (Calatayud and

Gonzalez, 2003).

Twenty five years after first being isolated, the final step in the clinical use of cocaine was taken by Viennese ophthalmologist, Carl Koller (1857–1944) (Liljestrand, 1967). Through personal experimentation with his close friend (the renowned psychoanalyst, Sigmund Freud),

Koller noted a deadening effect on his tongue when he swallowed cocaine, and grasping the magnitude of this discovery, he pursued the observation further with experiments on dog and guinea pig corneas. On September 11, 1884, Dr. Koller would become the first person to perform surgery on a patient using an LA (Liljestrand, 1967). Through a series of successive and serendipitous events, news of this milestone spread quickly. Unable to attend the German

Ophthalmologist Society Congress set to meet in Heidelberg a few days later on September 15,

Koller asked his friend and colleague, Dr. Joseph Brettauer, to read his paper at the conference

(Fink, 1985). The impact of this seminal work was enormous. Coupled with the contemporary technological development of the hypodermic needle invented in 1852 by Charles Gabriel Pravaz

(1791–1853) and perfected by Alexander Wood (1817–1884) allowing precise local drug administration, Koller’s findings would very quickly spark a deluge of research on the mechanisms of local and regional anesthesia (Calatayud and Gonzalez, 2003; Ruetsch et al.,

2001).

Not long after the introduction of cocaine as a clinical LA, the first accurate accounts of differential block were recorded. In 1898, the renowned German surgeon, August Bier (1861–

1949), began injecting dilute concentrations of cocaine into his patient’s dural sac in hopes of producing the maximal area of anesthesia with the least amount of drug possible (Cousins and

Bridenbaugh, 1997). During the procedure, he recorded an interesting sequential loss of function leading to complete neural block, and a sequential return to complete normalcy (Raymond, 1992).

From Bier’s work, the technique of spinal anesthesia spread quickly, as well as this peculiar observation he witnessed.

The LA properties of cocaine constituted a huge leap forward for the field of medicine, but the compound was not without serious drawbacks. Nearly simultaneously with the first applications, reports began to surface of systemic, central, and cardiovascular toxicity resulting in many deaths of both patients and addicted medical staff (Ruetsch et al., 2001). As the popularity of cocaine use increased, so did the frequency of these central nervous and cardiovascular system toxic reactions (Ruetsch et al., 2001). This prompted the pharmaceutical industry to commence development of new and less toxic alternative LA compounds.

Rise of modern local anesthetics

The necessity for new and safer LA drugs to replace cocaine coincided closely with the nascent field of modern organic chemistry (Ruetsch et al., 2001). In 1898, the same year the structural formula for cocaine was resolved by Richard Willstätter (1872–1942), Alfred Einhorn

(1856–1917) would synthesize the first amino-amide LA, nirvaquine. Nirvaquine proved to be a tissue irritant and its use was quickly halted. Einhorn refocused his efforts towards the development of amino-ester LA compounds. First creating benzocaine in 1900, it would be his

8 second compound, Novocaine (procaine; first introduced in 1905), which would revolutionize the field of pain control (Calatayud and Gonzalez, 2003; Ruetsch et al., 2001). With relatively few serious side effects, procaine became the standard LA during the first half of the 20th century.

While representing a great leap forward compared with cocaine, the anesthetic effects of the compound were weak and short-acting, requiring high concentrations of the drug applied together with epinephrine, and some patients proved to be highly allergic to this agent

(Calatayud and Gonzalez, 2003). The last of the ester type LA developed would be tetracaine in

1930, often used as a substitute for cocaine as a topical airway anesthetic (Ruetsch et al., 2001).

As surgeries became longer the search for a more effective longer lasting substitute compound continued.

In 1944 the Swedish chemist, Nils Löfgren (1913–1967), and his colleague, Bengt

Lundqvist, created a xylidine derivative they named lidocaine (ω-diethylamino-2,6- dimethylacetanilide) (Calatayud and Gonzalez, 2003; Löfgren and Wilson, 1948). This new amino-amide compound became a huge success, due to its relative safety, potency, rapid onset of effects, and relative paucity of allergenic reactions (Calatayud and Gonzalez, 2003; Ruetsch et al.,

2001). During the synthesis of lidocaine, Löfgren discovered that treatment of the base anilide with excess ethyl chloride or bromide creates the strongly hydrophilic quaternary ammonium [N-

(2,6-dimethylphenylcarbamoylmethyl)triethylammonium chloride or bromide] known now as

QX-314 (Löfgren and Wilson, 1948). Following the creation of lidocaine, all new LAs would incorporate the amide linker. In step with lidocaine, even longer-lasting LAs were being created, such as mepivacaine (1957), bupivacaine (1963), etidocaine (1972), articaine (1976), and the S- enantiomeric ropivacaine in 1996 (Ekenstam, 1957; Garcia, 1982; Löfgren and Tegner, 1960;

McClure, 1996; Ruetsch et al., 2001).

LOCAL ANESTHETIC STRUCTURE ACTIVITY RELATIONSHIP (SAR)

The approximately 30 modern clinically available LAs are based exclusively on two distinctly unique alkaloids, cocaine and isogramine (Wilson et al., 1991). Cocaine is an aminoalkyl ester of benzoic acid; isogramine is a 2-(aminoalkyl)indole. The structures of clinically useful LAs all contain a centrally located sp2-hybridized carbon atom to which alkyl or aryl groups are attached directly or through an organic non-carbon heteroatom [Figure 1 & 2].

Although similar in structure, due to the current prevalence of use and the direct application of amino-amide compounds in this work, focus will be placed on the structure of lidocaine (amino- amide) derivatives. As alluded to above, amino-amide LAs consist of a phenyl group attached to an amine function by an amide linker. These compounds are remarkably stable, resisting hydrolysis even under prolonged autoclaving. As a result, they are more potent, have a lower prevalence of side effects, are effective with or without additional vasoconstrictors (such as epinephrine), and induce less local irritation than benzoic acid derivatives. Unlike benzoic acid derivatives, which undergo enzymatic degradation in plasma, amino-amide degradation occurs primarily in the liver through amide bond cleaving (Williams et al., 2002; Wilson et al., 1991).

Figure 1. General structural characteristics found in all clinically available local anesthetic compounds. Adapted from (Wilson et al., 1991).

Figure 2. Molecular structures for common clinically available local anesthetic compounds. 1) Cocaine [(1R,2R,3S,5S)-methyl 3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane-2- carboxylate] 2) Procaine [2-(diethylamino)ethyl 4-aminobenzoate] 3) Benzocaine [ethyl 4- aminobenzoate] 4) Tetracaine [2-(dimethylamino)ethyl 4-(butylamino)-2-chlorobenzoate] 5) Lidocaine [2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide] 6) Mepivacaine [N-(2,6- dimethylphenyl)-1-methylpiperidine-2-carboxamide] 7) Prilocaine [N-(2,6-dimethylphenyl)-2- (propylamino)propanamide] 8) Bupivacaine [1-butyl-N-(2,6-dimethylphenyl)piperidine-2- carboxamide] 9) Etidocaine [N-(2,6-dimethylphenyl)-2-(ethyl(propyl)amino)butanamide] 10) Articaine [methyl 4-methyl-3-(2-(propylamino)propanamido)thiophene-2-carboxylate] 11) S- Ropivacaine [(S)-N-(2,6-dimethylphenyl)-1-propylpiperidine-2-carboxamide].

Aryl group

The aryl group or lipophilic portion of the molecule is an essential component found in all clinically useful LAs. A phenyl group is attached to a carbonyl (amino-ester benzoic acid derivatives) or a 2,6-dimethylphenyl group is attached to the sp2 carbon atom through a nitrogen bridge (amino-amide lidocaine derivatives). Substitution of the phenyl group with methyl side chains in the 2- or 2- and 6- positions enhances LA activity (Löfgren and Wilson, 1948). The amide bond is significantly more stable to hydrolysis than the ester bond. Moreover, o,o- dimethyl groups increase lipophilicity in the aromatic region and through steric hindrance decrease access to amide hydrolysis, increasing duration of action. Aromatic groups for both amino-ester and amino-amide compounds are highly lipophilic and are suggested to play a partial role in LA binding to Na+ channels (Williams et al., 2002; Wilson et al., 1991).

X function

This intermediate linker region almost always contains a short alkylene chain of one to three carbons in length linked to the phenyl group via several possible organic compounds. X may be a carbon, oxygen, nitrogen, or sulfur (benzoic acid derivatives). The most desirable LA activity and toxicity profile is achieved with oxygen substitutions in place. The intermediate linker region determines the drugs chemical stability, duration of action, and relative toxicity

(Wilson et al., 1991). Amino-amides are generally more resistant to metabolic degradation than amino-esters, resulting in a longer duration of action. Addition of small alkyl groups, especially around the amide moiety (with etidocaine for example), inhibits hydrolysis and increases duration of action. However, increasing duration of action also increases systemic toxicity

(Garcia, 1982; Williams et al., 2002; Wilson et al., 1991).

Aminoalkyl group

The hydrophilic amino function found in both amino-ester and amino-amide compounds facilitates the formation of water-soluble salts. Most LAs are tertiary amines because primary and secondary amines exhibit increased tissue irritation (Löfgren and Wilson, 1948). The necessity for the insertion of aminoalkyl groups in order to induce LA activity is also an ardently debated topic, as benzocaine is still able to produce anesthesia without the inclusion of this moiety (Williams et al., 2002). For lidocaine derivatives, lengthening the alkylene chain attached to the amine from one to two to three increases pKa of the tertiary amino group from 7.7 to 9.0 to

9.5. In other words, increasing the length of the intermediate chain reduces LA potency as a result of a reduction of onium ions under physiologic conditions. Formation of onium ions

(cations derived from the protonation of a mononuclear parent hydride) is necessary for binding to residues in the Na+ channel inner vestibule (Frazier et al., 1970; Strichartz, 1973) [cf.

Molecular mechanisms of local anesthesia]. LAs having higher lipid solubility and lower pKa values exhibit more rapid onset and lower toxicity (Williams et al., 2002).

PHYSIOLOGY AND PHARMACOLOGY OF NERVE BLOCK

Basic nerve function

Transient electrical conduction across the surface membrane of excitable neuronal tissues allows for rapid transmission of efferent and afferent information throughout the body. The fundamental regulation of electrical activity occurs through pore forming membrane proteins know as ion channels (Hille, 2001). At rest, a peripheral neuron exhibits an intracellular membrane potential of approximately −90 mV. This negative potential inside the fiber is maintained by electrogenic ion channel pumps (such as Na+/K+ ATPase), continually

13 transporting Na+ to the outside of the cell and K+ to the inside (three Na+ ions for every two K+ ions) (Eisner and Lederer, 1980; Skou, 1989; Thomas, 1969), effectively establishing a concentration gradient for Na+ and K+ across the membrane. This, coupled with K+ “leak” channels, sets the level of the resting membrane potential to the reversal potential of K+ ions

(Guyton, 1991). The principal mechanisms that underlie the generation and propagation of neuronal action potentials were established in a series of seminal publications by Hodgkin,

Huxley, and Katz studying Loligo pealii squid giant axons (Hodgkin and Huxley, 1952a;

Hodgkin and Huxley, 1952b; Hodgkin et al., 1952). This process is initiated when the resting membrane potential begins to depolarize through activation of Ca+ and Na+ permeable metabotropic or ionotropic membrane proteins [c.f. Nociceptor ion channels]. Upon reaching a threshold level (between −70 mV and −50 mV), a sudden conformational change in voltage-

+ gated Na (Nav) channels is tightly coupled to ‘activation’ and opening of the pore domain which allows a surge of extracellular Na+ ions along the electro-chemical gradient into the cytoplasm, and the ensuing rapid membrane depolarization initiates the opening of Nav channels in adjacent neural compartments establishing a self-propagating current wave across an axon (Hodgkin et al.,

1952). Activation gating of the Nav channel pore (1–2 ms) is followed by rapid inactivation, a process which limits Na+ ion conductance, continued neuronal excitation and allows for the initiation of tissue repolarization (Hodgkin and Huxley, 1952b). Depolarization also initiates the

+ + opening of the slower voltage-gated K (Kv) channels whose outward flow of K ions, together

with Nav channel inactivation aids in the repolarization of the neural membrane to resting levels priming neurons for subsequent impulse generations (Hodgkin and Huxley, 1952a).

Molecular mechanisms of local anesthesia

Local anesthetics prevent action potential generation and conduction by blocking individual Nav channels in neuronal membranes thereby preventing the sudden up-stroke in action potential (Hille, 1966; Taylor, 1959). First demonstrated in the landmark papers of

Narahashi and colleagues studying giant squid axons in the summers of 1968 and 1969, discovered that LAs exert their action potential blocking action by binding to the intracellular pore of Nav channels (Narahashi et al., 1970). Once injected, the LA molecules exist in a dynamic equilibrium between charged and uncharged species, the charged form being the active channel blocking form (Frazier et al., 1970; Narahashi et al., 1970). However, because the charged cation is relatively membrane-impermeant, the molecule has to first cross the neuron’s lipid bilayer as an uncharged neutral base (verified with pH changes and quaternary compounds)

(Frazier et al., 1970; Narahashi et al., 1970). This notion has supported a scientific corroboration of many clinical observations of LA ineffectiveness in inflamed tissues, where the pH is relatively acidic, thus increasing the charged, impermeant form of the drug (Frazier et al., 1970).

Nav channels exist in distinct conformational states (resting, closed, open, and inactivated) with each state having a different affinity for the LA molecule (Aldrich et al., 1983; Strichartz, 1973;

Vandenberg and Horn, 1984). It has been shown that LAs have the highest affinity for the open, inactivated, and the transitional closed conformations of the Nav channel over the resting state

(Butterworth and Strichartz, 1990). The high affinity exhibited by the LA molecules to these particular states restricts the conformational changes necessary for a drug-bound channel to undergo full pore aperture and ion conduction. Although LA binding occurs in the channels intracellular pore region, pore obstruction seems to be one of numerous mechanisms invoked to explain LA inhibition of Nav activity. Experimental data, computational analysis and site-

15 directed mutagenesis on the putative binding site for LAs in the human cardiac Nav channel isoform Nav 1.5 (Phe1760) suggests that LAs prevent Nav channel conductance by introducing a positive charge that electrostatically impedes Na+ ions, rather than blocking them (McNulty et al.,

2007; Pless et al., 2011). A LA molecule bound to the phenylalanine via a cation-π reaction

(noncovalent interaction between the face of an electron-rich aromatic π system and a cationic molecule) raises the energy barrier for Na+ ions traversing the pore two-fold (Pless et al., 2011).

The significantly increased energy barrier combined with steric hindrance to ion movement provides a basis for the observed block in current (McNulty et al., 2007). In order for the drug- bound channel to reopen, the LA molecule must dissociate from the channel, allowing the channel to return to its resting state. As a result, LAs extend the neurons refractory period by delaying the inactivated Nav channel’s return to a resting conformation by a factor of 50–100 fold more (Mani and Strichartz, 2005). At sufficiently high concentrations, enough LAs molecules bind to a critical amount of Nav channels that impulse propagation is inhibited in an affected area.

LAs block the Na+ current via two methods: tonic and phasic inhibition (Courtney, 1975;

Strichartz, 1973). Tonic inhibition occurs when the time between incoming action potentials is long enough to allow the dissociation of the LA from the Nav channel (< 0.5 Hz in most cases)

(Courtney, 1975; Strichartz, 1973). For example, when a LA solution is introduced into an inactive nerve system, these molecules are only able to bind to a select few spontaneously activated Nav channels. Depending on the intrinsic properties of the LA (pKa, lipophilicity, and protein binding), equilibrium between baseline drug-bound and unbound channels is established.

During infrequent nerve stimulation, some of the channels become bound to the LA molecules.

However, there is sufficient time between impulses allowing the LA-Nav channel complexes to

16 dissociate once again re-establishing the baseline equilibrium. As long as the action potentials occur with this periodicity the same exact effect will be observed (Mani and Strichartz, 2005).

Phasic (“use-dependent”) inhibition occurs when there is not enough time between action potentials for this equilibrium to be re-established, and rapidly incoming action potentials cause resting channels to open and then inactivate. During these impulses, LA molecules enter and bind to the open states of the Nav channels. However, without sufficiently long intervals allowing the newly formed LA-Nav channel complexes to dissociate, only a fraction of the channels can return to a resting state and with each successive action potential, more and more channels are blocked until nerve conduction dissipates below the transmission threshold. Therefore, action potential conduction is increasingly inhibited at higher frequencies of impulses (Hille, 1977).

Importance of fiber size on function

In terms of neural classification, fiber size and diameter appear to be the most important parameters for nerve function, determining conduction velocity, excitability, and internodal spacing (de Jong, 1994). The diameter and myelenization, in particular, seem to be critical factors for fiber function. Beginning with the work of Gasser and Erlanger, nerves have been categorized into three major classes based on conduction velocity, threshold, and after-potential

(de Jong, 1994). The largest group are the myelinated somatic nerves, better known as A-fibers, comprised of four sub-groups according to decreasing size: A, A, A, and A; the intermodal length ranges from approximately 20 to 3 µm. The largest fiber type, Aα, conducts impulses at

80–120 m/s and has been related to motor function and reflex activity. The second largest, Aβ, transmits touch and pressure signals between 48–90 m/s, while the third largest, Aγ, transmits muscle spindle tone at 24–48 m/s. The smallest A-fiber type, Aδ, transmits temperature and

17 nociceptive signals at around 6–24 m/s. The other two nerve classes, myelinated preganglionic autonomic nerves (B-fibers), and non-myelinated axons (C-fibers) are more uniform in their function and activity. B-fibers transmit autonomic information to the postganglionic C-fibers at speeds between 10–15 m/s. “Nonmyelinated” C-fibers, like the Aδ-fibers, subserve pain and temperature transmission, but are thinner and have much lower conduction velocities - usually between 1–2 m/s (de Jong, 1994). Of particular interest for this project are the neural mechanisms that underlie the transduction of painful stimuli by these subservient nociceptive fibers.

DIFFERENTIAL FUNCTIONAL NERVE BLOCK

Subarachnoid and epidural anesthesia

The observation of the stepwise loss of function following a spinal injection of cocaine meticulously recorded by August Bier would set the precedent for the now established technique of differential nerve block. Functional blockade or differential block is the effect produced in a peripheral nerve fiber bundle, whereby careful titration of a LA blocks nociceptive signals, but spares motor and sympathetic function (de Jong, 1994; Fink and Cairns, 1983). Nerve block with

LAs is often considered an all-or-non-phenomenon; all nerve bundle types are rendered non- conductive (touch, temperature, and movement disappear), or remain completely functional (de

Jong, 1994). This “black and white” picture of anesthesia is not always possible or desired. There are many clinical situations where block of some nerves over others would prove more amenable.

Two methods that produce the most visible effects of differential block are epidural and subarachnoid (spinal) anesthesia (Kocarev et al., 2010; White et al., 1998). During a “walking epidural”, for example, it is possible to abolish pain, but keep partial muscle activity and

18 sensation active for a patient. In spinal anesthesia, although all the senses become abolished below the waist, differentiation between the dermatomes is observed cephalad (toward the head or anterior section) from the point of injection (Greene, 1958; Raymond, 1992).

Spinal anesthesia (or subarachnoid block) has become standard in the repertoire of the modern anesthesiologist. A temporary interruption of neural signals across a localized region of the spinal cord is produced through the administration of a LA solution into the cerebrospinal fluid (CSF) [Figure 3 & 4] (Cousins and Bridenbaugh, 1997). Spinal anesthesia is the best clinical example of an in vitro nerve bath preparation. Once administered, LAs can be contained inside the dural sac while the bare nerve roots float inside (Raymond, 1992). Following an injection, the feet will become warm and numb before the leg muscles are paralyzed. During recovery, motor block disappears long before the sense of touch returns to the feet. Spinal anesthesia has many advantages over general anesthesia, especially for surgical procedures involving the lower abdomen, the perineum, and the lower extremities, such as hernia repairs, and gynecological and urological operations; or patients whose preexisting comorbid conditions render them poor candidates for general anesthetics (Cousins and Bridenbaugh, 1997).

Figure 3. Illustration exposing the gross anatomy of the spinal cord. Adapted (Cousins and Bridenbaugh, 1997)*.

* Any additional use of this material including promotional or commercial use in either print, digital or mobile device format is prohibited without the permission of the publisher. Please contact the Wolters Kluwer Health/Lippincott Williams & Wilkins Book Permissions group at [email protected].

Figure 4. Subarachnoid anatomy. Adapted from (Cousins and Bridenbaugh, 1997)*.

Presently, the most adaptable and most extensively used technique for producing nociceptive-specific differential block is epidural anesthesia (Bromage, 1967; Cousins and

Bridenbaugh, 1997). First developed in 1901 by Fernand Cathelin (1831–1920), its usefulness in obstetrics would not be fully appreciated until the 1940’s (Cousins and Bridenbaugh, 1997). The real advances of differential block research can be seen with epidural anesthesia, where it has helped increase the efficacy and safety of the technique. It is now possible (to an extent) to block pain, while leaving sensation, motor, and sympathetic function relatively unchanged (Cousins and Bridenbaugh, 1997). The epidural space is not as voluminous as the subarachnoid space, and extends from the base of the skull to the sacrococcygeal membrane (Cousins and Bridenbaugh,

1997). It is widest posteriorly, from 1 mm at C5 to 6 mm at the L2 vertebra. Like spinal

21 anesthesia, anesthetics administered epidurally can be injected throughout a wider range of the spinal column (C7- T2) interspace (Cousins and Bridenbaugh, 1997). It produces differential block that can be easily modified by titrating the LA concentration (de Jong, 1994). However, the local concentration is not the only factor affecting clinical differential block: the compound used also plays a significant role, (see Pharmacodynamic variations in local anesthetic compounds). During delivery of a child, for example, it is imperative that the anesthetic concentration be slowly administered to the optimal level that decreases pain without blocking uterine contractions. Compared with epidural anesthesia, spinal anesthesia produces faster onset of effects and is not as controllable, one of the reasons it is not a preferred method in labor analgesia (Cousins and Bridenbaugh, 1997). On the other hand, epidural anesthesia requires larger volumes and concentrations of LAs, which can result in pharmacologically active plasma concentrations. This can lead to many unwanted systemic effects, which do not occur with the low doses administered for spinal anesthesia (Cousins and Bridenbaugh, 1997).

Uncovering the mechanisms of differential block

Researchers studying differential block and nerve physiology before 1929 were working blindly. That is, while they knew that nerves carried current and varied in size anatomically, they lacked tools to study molecular function and pharmacology. A key piece of technology developed in 1929, the cathode ray oscilloscope, allowed Herbert S. Gasser and Joseph Erlanger to produce the first long-standing theories on the mechanisms of differential block. Using the benchmark LA of the time, cocaine, they were able to view and tease out the compound action potentials of the different nerve fibers (Gasser and Erlanger, 1929). They made accurate measurements of conduction velocities in different fiber types, discovering that conduction was a

22 linear function of the diameter of the fiber. They suggested that fibers of different velocities could be identified on account of their temporal dispersion: blocking small fibers first would shorten the potential wave from behind while large fiber block first would shorten the wave from the front (Gasser and Erlanger, 1929). They categorized nerves into three main classes: myelinated somatic A-fibers, myelinated autonomic B-fibers, and non-myelinated C-fibers.

Testing the saphenous nerves of dogs, differential action to cocaine was visible in every single fiber studied. Not only that, but it followed the same trend: Small fibers were blocked before large ones. The experiments were conducted in two series. The first series depended on the wave function of action potentials, which noted time to disappearance for each of the different , , , and  wave components (Gasser and Erlanger, 1929). The second series of experiments took a closer look at the more persistent and ambiguous  complex (Gasser and Erlanger, 1929).

Erlanger and Gasser concluded that the order of block was C, B, and then A-fibers, with the lowest concentration of cocaine blocking C-fibers first. The model was summarized on a fiber size basis, small blocked before large, their argument being smaller fibers had thinner myelin sheaths permitting easier access to the axial protoplasm. They also assumed that cocaine acted by a chemical combination with the protoplasm, stating that because surface per unit volume increases directly as the diameter decreases, the smaller the fiber the greater the accessibility

(Gasser and Erlanger, 1929). This so-called “protoplasm” theory later lost validity when it was discovered that membrane channels, rather than axoplasm, are the actual seat of conduction block, but the size principle remains textbook dogma (Hodgkin and Huxley, 1952a). Appearing to solve one problem, many other experimental observations remained unexplained, such as why larger fibers are occasionally blocked before smaller ones and why block was anteceded by a great retardation in velocity (Gasser and Erlanger, 1929).

The new size principle

From Gasser and Erlanger’s seminal work, the general teaching developed that the larger the diameter of a nerve fiber, the more resistant it was to LA blockade (de Jong, 1980). In 1961,

Nathan and Sears introduced a new dimension to clinical nerve block (Nathan and Sears, 1961).

Trying to consolidate the inconsistencies concerning blocking of fibers of different sizes, they tested individual myelinated and non-myelinated fibers of a cat in vivo. Their work corroborated many of the observations made by Gasser and Erlanger, but the most important contribution was what they termed absolute differential block, later known as Cm (minimum blocking concentration). They observed that application of LAs at variable concentrations produced selective block of small non-myelinated fibers without affecting larger myelinated fibers. Based on an assumption that different fiber types have different safety factors for conduction, the smaller the fiber, the lower the concentration of LA needed to render it non-conducting (Nathan and Sears, 1961). Observations like these further cemented the Gasser-Erlanger size theory. By the time Franz and Perry (1974) tackled the problem, they worked around the idea that it was not different Cm’s blocking specific fiber sizes, but the critical axon lengths. Based on the saltatory conduction models first proposed by Ichiji Tasaki, which in 1939 demonstrated that the impulse in a myelinated nerve could usually jump one, occasionally two, but never three completely blocked nodes (Tasaki, 1939). Extrapolating from that model, Franz and Perry argued that the bigger the axon, the longer the internodal length, and the longer the length a LA had to bath a fiber to block it. They demonstrated that nonmyelinated C-fibers and myelinated Aδ-fibers were, in general, blocked at the same time with the same LA concentration, and that although difficult to quantify, there was only a small margin between the Cm of larger axons (Franz and Perry,

1974). Although they dismissed Cm as the reason for differential block, it was unknowingly

24 validated in their argument. They suggested that a minimal blocking concentration would reach the shorter critical length of small axons before reaching the longer critical length of large axons.

This implies that the larger the axon the larger the apparent (or functional) Cm, a term now commonly used by clinicians (Franz and Perry, 1974).

Decremental block

A sub-blocking concentration (sub-Cm) of LA covering a nerve fiber may in fact block conduction if sufficient length of fiber is exposed to the anesthetic through a mechanism known as decremental block. Although now an established mechanism, decremental block was once regarded with great skepticism. Noted as a “great retardation in velocity” by Gasser and Erlanger, it was dismissed by many physiologists as an erroneous observation (No and Condouris, 1959).

The physiologists had argued, based on the established dogma of the day, that nerve conduction was an all-or-nothing law (No and Condouris, 1959). The behavior, now known as decremental conduction, was revisited again in 1959 by Lorente de No and Condouris. Testing some of the novel LAs of the day, prilocaine and lidocaine, they showed decremental conduction to be a qualifiable property of axons (No and Condouris, 1959). Using an oscilloscope, they observed a decrement in height from a standardized action potential traveling through a frog’s sciatic nerve to a LA treated segment. In the segment the current underwent an intense decrease before being completely extinguished upon reaching a target cathode. The observations promoted decremental conduction from an artifact to a pharmacological fact, although the instrumental limitations of their time inhibited them from proposing a satisfactory theory behind what had actually occurred.

In 1976, Condouris would again revisit decremental block. Using computer simulations, his team tested models of LA binding, as well as the powerful tetrodotoxin, on nerve block [Figure 5].

The phenomenon was shown to fit perfectly with the Hodgkin and Huxley model of a myelinated axon, and required up to 10 sub-Cm bathed nodes of Ranvier to block an impulse (Condouris et al., 1976). From these models of threshold blocking events it was predicted that the neural membrane becomes progressively less excitable as increasingly more Nav channels are rendered non-conducting by an increasing number of LA molecules (de Jong, 1994).

Figure 5. Decremental conduction determined using a mathematical model of the myelinated axon. A 15-node segment of the axon was exposed to lidocaine. The curves show the membrane potential at successive nodes as two 50-Hz impulses traveled down the axon (Raymond et al., 1989).

Presently, decremental block seems to be a more prominent mechanism in differential block than previously thought (Raymond and Strichartz, 1989). The current theory proposes the likely basis for differential block to be intrinsic differences in conduction safety over a length of fiber, rather than the probability of 3 adjacent nodes being blocked (Raymond et al., 1989).

Exposing a 5 mm length of axon to a dilute LA solution produces solid block while reducing the potential for systemic toxicity. While on the other hand short exposure lengths would dramatically increase the dose of anesthetic required to block fiber conduction, increasing the possibility of toxicity (Raymond et al., 1989). Low concentrations are ideal because systemic LA toxicity has dangerous, far-reaching effects: As LA plasma concentrations increase, perioral numbness and dizziness are followed by tinnitus, muscular twitching, convulsions, coma, cardiorespiratory arrest, and death (Cousins and Bridenbaugh, 1997). Dilute solutions produce differential block by exposing a series of identical nodes to sub-blocking concentrations of LA.

This results in a progressive decrement of the action potential because each successive node is driven by a weaker action potential, generating an incrementally weaker response to drive the next node. Conduction fails when the attenuated response is too weak to depolarize the next node, and the local response falls nearly to 0 over the next three to five internodal lengths

(Raymond et al., 1989). The experimental evidence also suggested that a region of decrement could extend beyond 20 successive internodes, exceeding the range of 10 nodes proposed by

Condouris et al (Raymond et al., 1989). This work has been proven to correlate directly with gross clinical data (Raymond, 1992). Linking clinical observations from spinal anesthesia to anatomical findings and in vitro measurements on isolated nerves, it has been well recognized that the effective bathed length decreases progressively along the spinal cord from sacral to thoracic segments (Fink, 1989). Thus, in the thoracic region, where nerve root lengths are in the range where exposure length is an important factor for blocking concentration, the larger fibers will have fewer nodes exposed to LA and might therefore preserve conduction longer than small fibers where more nodes would be exposed (Raymond and Strichartz, 1989).

Pharmacodynamic variations in local anesthetic compounds

So far, the clinical differential block model has been based on pharmacokinetic observations of neuronal conduction under the influence of LAs. However, up until the early

1970s, a detailed pharmacodynamic model for these compounds had not yet been developed (No and Condouris, 1959). The list of clinical LAs had grown considerably since the discovery of cocaine; although these compounds share the same basic molecular structure, the slight differences in atomic composition play a notable role in the three most important physiochemical properties of LAs: lipid solubility, protein binding, and pKa [Table 1] (Wildsmith et al., 1987). It was soon found that the intrinsic properties of LA molecules also had an effect on the rate of differential block.

Procaine for example, is rarely used for peripheral nerve or epidural block because of its low potency, slow onset, short duration, and limited tissue penetrating ability (Ruetsch et al.,

2001). On the other hand, due to its moderate potency and rapid onset, lidocaine has become widely employed for infiltration, peripheral nerve block and both epidural and spinal anesthesia

(Ruetsch et al., 2001). Using in vivo experiments, all LAs tested (lidocaine, etidocaine, bupivacaine, and 2-chloroprocaine) produce differential block, albeit not all of them to the same degree [Figure 6] (Ford et al., 1984). For example, myelinated B-fibers appear more sensitive than C-fibers to lidocaine, 2-chloroprocaine, and bupivacaine (Rosenberg and Heinonen, 1983).

Bupivacaine, about 3 times as potent as the lipid-soluble 2-chloroprocaine in vitro; at 22°C is equipotent to the much more lipid-soluble etidocaine, in rabbit A-fibers in vivo (Gissen et al.,

1980; Rosenberg and Heinonen, 1983). However, clinically, they affect motor nerve fibers differently; etidocaine produces more profound motor block (Rosenberg and Heinonen, 1983).

Bupivacaine, on the other hand, has a preference for unmyelinated C-fibers at lower

28 concentrations, contributing to its clinical effectiveness in blocking “slow” burning pain (Fink,

1989).

Compounds with an overall moderate degree of lipophilicity, such as lidocaine, appeared to have the greatest differential effect on fibers of different conduction velocities [Figure 6]

(Gissen et al., 1980). Meanwhile, tetracaine a more lipophilic (hence more potent) compound, had the smallest differential effect between A- and C-fibers (Gissen et al., 1980). This implies that highly lipid-soluble drugs are able to penetrate considerable lipoprotein coverings rapidly, while with less lipophilic compounds A-fiber diffusion barriers delay the onset of block

(Wildsmith et al., 1987).

Figure 6. Pharmacodynamic changes in local anesthetics alter concentration-dependent selective neuronal inhibition. Linear regression curves from vagus (B- and C-fibers) and sciatic nerves (A-fibers) of action potential amplitudes with increasing concentrations of local anesthetic agents. Adapted from (Gissen et al., 1980).

Table 1. Physico-chemical properties of local anesthetics. Highlighted in grey are the primary LAs used to induce differential block. Adapted from (Ruetsch et al., 2001).

Anatomy and differential block

The complex mechanics of inward diffusion are greatly affected by the extradural nerve fibers and the surrounding tissue (de Jong, 1994). An injected LA solution can be spread or contained depending on the anatomical obstacles present (Fink, 1989). Clinical LAs greatly exceed the minimum blocking concentrations (Cm) to ensure adequate regional block (de Jong,

1994). From the point of injection, a supra-Cm can be expected to spread along the nerve in either direction. Wherever nerve fibers become covered with fibrous tissue, diffusion barriers become a substantial obstacle for drug penetration leading to sub-blocking concentrations inside nerve bundles. When this occurs, bathed nerve length is a major consideration of whether block is produced or not (de Jong, 1994).

In subarachnoid blockade [Figure 7A], the safety of conduction is decreased the same amount in the short internode fibers of segment T4 as in the long internode fibers of segment T8, because the proportional node number and anesthetic concentration is the same (Fink, 1989).

Although only the short-internode fibers will undergo decremental block at the T4 level, both long and short fibers will be blocked at T8 (Fink, 1989). For epidural blockade [Figure 7B], the concentration of LA is approximately the same throughout the affected portion of the epidural

30 space. In this case, long and short internode fibers can be represented in the same segment because the length of exposed axons between the end of the dural cuff and exit of the intervertebral foramen is approximately the same (Fink, 1989). The number of nodes bathed in the intervertebral canal is not sufficient to establish conduction block of the long internode fibers, but enough for short internode fiber block (Fink, 1989). Due to the lack of barriers, spinal anesthesia requires a much smaller amount of drug than many other sites. LA injections in other regions encounter numerous obstacles, such as bone and tissue, before the drug is able to reach the neural target.

Figure 7. Local anesthetic concentration after injection. The figure above demonstrates the axial differential block mechanism in the thoracic region. In A (subarachnoid) and B (epidural), the two crenated lines represent long and short internode myelinated fibers, intersected by dashed lines representing the critical bathed lengths. The concentration gradient of anesthetic in the cerebral spinal fluid is symbolized by the density of stippling. Adapted from (Fink, 1989).

Summary

Differential block research has revealed certain key factors necessary for the successful and effective production of nociceptive-specific nerve block with LAs. Fiber size remains as one of the most important determinants of selective block by LAs. The smaller and thinner the nerve fiber the easier it is for a LA to diffuse into the cytoplasm to block the intracellular pore of Nav channels. The next key factor involved in selective neural blockade is the critical axonal length necessary to completely inhibit current propagation. Due to the ability of nerve impulses to jump one, even two completely blocked nodes of Ranvier, a three node minimum blocking length is required to halt conduction. Hence, the bigger the axon, the longer the internodal length, and the longer the length a LA solution needs to bath a nerve in order to reach the three node minimum

[Figure 8]. An important mechanism of differential block, related to the length of exposed axons, is decremental conduction. Dilute solutions produce differential block, by exposing a series of identical nodes to sub-blocking concentrations of LA. The result is a progressive decrement of the action potential because each successive node is driven by a weaker action potential, generating an incrementally weaker response to drive the next node. The use of specific LA compounds also plays a role in the level of differential block observed. Compounds with an overall moderate degree of lipophilicity (eg. lidocaine) have the greatest differential effect, while more lipophilic compounds (eg. tetracaine) have the smallest differential effects between A- and

C- fibers. The last key factor affecting differential block with LAs is anatomy, as it is often not possible to introduce the necessary minimum blocking concentration in all areas of a fiber bundle.

An effective concentration of LA solution is thus dependent on physical barriers that reduce or promote diffusion from a site of injection.

Figure 8. Conductance behavior in a) large myelinated nerves, b) small myelinated nerves, and c) unmyelinated nerves. Arrows represent the progression of membrane current in nerves of different sizes and velocities (Gissen et al., 1980).

MOLECULAR INTEGRATORS OF PAIN

Nociception

Peripheral nociception is the complex neural process of encoding and managing noxious

(painful) stimuli (IASP, 1994). In the 17th century, René Descartes (1596–1650) would become the first analytical neurophysiologist to develop a theory linking pain to nervous system function

(Oh, 2006). He proposed that nerves conduct sensations through tubes containing a fluid messenger system, controlled by valves that opened or closed in order to direct the activity of the brain, forming the precursor for the modern ion channel concept. Today, acute pain can be considered as one of the basic senses, similar to taste, hearing, or vision, where stimuli of varying intensities are detected through specialized cells of lightly myelinated Aδ- and unmyelinated C-fibers, whose cell bodies originate from the trigeminal (head) and dorsal root

33 ganglia (body) and are tuned to the specific properties of the stimulus (Julius and Basbaum,

2001). These small to medium diameter primary afferent neurons (named so because they conduct signals toward the CNS) are comprised functionally of thermal sensors activated at temperatures above 39°C (C-fibers) or below 5°C (Aδ-fibers), high-threshold mechanical sensors that only transmit information indicating injurious force (Aδ), and polymodal sensors activated by thermal, chemical, and mechanical stimuli (Aδ- and C-fibers) (Mani and Strichartz, 2005).

Due to their faster conduction speeds, it is believed that Aδ-fibers mediate the initial sharp, acute, localized response to injury, while C-fibers parley a delayed, diffuse, dull, poorly localized pain response (Julius and Basbaum, 2001). Requiring a weaker stimulus for excitation than do C- fibers, Aδ-fiber density is highest on the fingertips, face, and lips, but low on the back (Mani and

Strichartz, 2005). These examples of nociceptors apply to peripheral and central nociception, as the mechanisms that underlie visceral, teeth, corneal, and vascular nociception are still to be completely resolved (Julius and Basbaum, 2001). Activation of high-threshold thermal, mechanical, or chemical sensing ion channels expressed in the peripheral sensory neurons elicits inward cationic transduction currents. Inward cationic flux elevates the resting membrane

potential towards a depolarized threshold, where widespread Nav channel activation occurs initiating a train of action potential waves carrying the noxious information towards the CNS

(Woolf and Ma, 2007).

Nociceptor ion channels

In the last decade, advances in molecular genetics have revealed an array of ion channels expressed in primary afferent sensory neurons that play key roles in the detection and transduction of nociceptive stimuli (Alexander et al., 2011). Among these are Nav channel

+ isoforms (Nav 1.7–1.9), two-pore domain K (K2P) channels, ATP-gated P2X (P2X1–7) receptors, acid sensing ion channels (ASIC1–3), and transient receptor potential (TRP) channels

[Figure 9] (Hwang and Oh, 2007; Mathie, 2010; Woolf and Ma, 2007). As previously mentioned,

Nav channel activation initiates action potential generation and propagation in excitable tissue.

Nav channels are composed of four domains (I–IV) attached to each other by extended cytoplasmic linkers. Each domain contains six transmembrane (TM) spanning α-helixes (S1-S6), with a pore forming loop between S5 and S6, and modulator β-subunit associations (Catterall et al., 2005; Noda et al., 1984). Nine isoforms (Nav 1.1–1.9) of this channel have so far been identified in humans, each with specific expression patterns throughout excitable tissues. Three of the Nav channel isoforms (Nav 1.7, 1.8, and 1.9) have been isolated specifically in peripheral sensory neurons and are directly linked to physiological and pathophysiological pain transmission (Dib-Hajj et al., 2009). In humans, clinical phenotyping and genetic assessment have reveled nonsense mutations in the SCN9A gene which code faulty Nav 1.7 α-subunits (Cox et al., 2006; Fertleman et al., 2006; Yang et al., 2004). Mutations in this gene lead to hypo or hyper conducting channels establishing three rare human pain syndromes (Dib-Hajj et al., 2009).

The autosomal dominant gain-of-function mutations related to inherited erythromelalgia and paroxysmal extreme pain disorder, create hyperexcitable Nav 1.7 channels, producing episodes of extreme pain and burning in patients afflicted with these conditions (Dib-Hajj et al., 2007;

Fertleman et al., 2006; Yang et al., 2004). On the other hand, the autosomal recessive loss-of- function mutations in individuals with channelopathy-associated congenital indifference to pain

(CIP) create hypoexcitable Nav 1.7 channels (Cox et al., 2006). As a result, individuals with CIP are generally insensitive to pain and smell, but display otherwise normal sensory faculties (Dib-

Hajj et al., 2007).

The 4TM family of K+ channels or K2P are also expressed throughout excitable tissues.

The 15 members of the K2P channel family are divided into six eclectically named subfamilies:

TWIK, TREK, TASK, TALK, THIK, and TRESK (Alexander et al., 2011). These channels mediate the K+ “leak” current, essential in regulation of cellular resting membrane potentials, which are modulated by a variety of environmental, physiologic, and pharmacologic mediators, additional to voltage (Enyedi and Czirjak, 2010). The first K2P channel, TWIK-1, was cloned in silico (Lesage et al., 1996) and contains the characteristic structural features of four TM spanning segments assembled into dimers, with two pore forming loops between each segment pair. Gene knockout studies have implicated K2P channels, particularly TREK1 and TRESK1, in controlling nociceptive and sensory neuron excitability and impulse transduction (Bayliss and

Barrett, 2008). Quantitative real time-PCR has revealed that human TREK1 and TRESK1 channels expression occurs almost exclusively in peptidergic and non-peptidergic DRG neurons

(Alloui et al., 2006; Dobler et al., 2007). Electrophysiological assays testing C-fiber responses to noxious heat, demonstrated a decreased threshold for thermal-evoked firing in TREK1-/- mice compared to wild-type (Alloui et al., 2006). Furthermore, significant increases of nocifensive responses to noxious mechanical, osmotic and inflammatory stimuli were detected in TREK1-/-

KO mice in vivo. Activation of TREK1 by inhalational anesthetics (halothane, isoflurane, diethyl ether, and chloroform) in whole-cell and excised patch-clamp experiments provides a plausible mechanism for the decreased neuronal excitability via K2P induced hyperpolarization (Patel et al., 1999). The TRESK1 channel is thought to exert a significant role in maintaining the standing

+ outward K current (IKso) of primary DRG neurons, as IKso in TRESK[G339R] functional KO mice was significantly reduced compared to wild-type (Dobler et al., 2007). Also, similar to

TREK-/- KO models, whole-cell recordings of TRESK[G339R] functional KO in DRG neurons

36 show significant increases in excitability compared with wild-type (Dobler et al., 2007). The abolishment of IKso by bupivacaine administration in TRESK1 expressing DRG neurons, without altering resting membrane potential, points towards an additional mechanism for local anesthetic-induced electrical silencing via K2P channel modulation (Dobler et al., 2007). Shown to be a promising therapeutic target, many of the physiologic and pharmacologic properties of

K2P remain to be elucidated.

Extracellular acidosis during ischemia, inflammation, and anaerobic activity elicits a nociceptive response through the activation of TRPV1 [cf. TRPV1: the “Capsaicin receptor”] and the acid-sensing ion channel (ASIC) family widely expressed throughout the peripheral nervous system (Caterina and Julius, 2001; Waldmann and Lazdunski, 1998). ASIC channels are members of the amiloride-sensitive Na+ channel and degenerin (NaC/DEG) channel superfamily

(Sakai et al., 1999). The structure of assembled ASIC channels revealed through x-ray crystallography shows each subunit contains two TM domains that assemble to form homo- or heterotrimers (Gonzales et al., 2009; Jasti et al., 2007). Five distinct ASIC channel isoforms have so far been identified in mammals (ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3) each with distinct physiological and pharmacologic properties (Alexander et al., 2011). Endogenous ASIC channel activation occurs exclusively through extracellular proton ligation, with each assembled

ASIC homomer detecting a narrow range of moderately acidic conditions: ASIC1a ~6.2–6.8,

ASIC1b ~5.1–6.2, ASIC2a ~4.1–5.0, and ASIC3 ~6.2–6.7 (ASIC2b homotrimers are not proton- gated) (Alexander et al., 2011; Holzer, 2009). The activation of ASIC channels in peripheral neurons forms one of the primary components for acid-evoked nociception in humans (Ugawa et al., 2002). Pharmacologic analysis of intradermal proton-induced pain in humans supports a specific role for ASIC channels in the initiation and maintenance of acid-induced nociception

(Ugawa et al., 2002). A significant analgesic effect in these patients was demonstrated following application of the non-selective ASIC inhibitor amiloride, providing a therapeutic basis for ASIC channel targeting during ischemic and inflammatory pain.

P2X (P2X1–7) receptors are ATP-gated non-selective cation channels with pronounced expression in central and peripheral neurons, as well as non-neuronal tissue (Burnstock and

Kennedy, 2011; Collingridge et al., 2009). First cloned in 1994, P2X receptors share a high degree of structural homology with ASIC channels, each subunit composed of two TM domains that assemble into homo- or heterotrimers. (Brake et al., 1994; Gonzales et al., 2009; Valera et al.,

1994). Among the distinct P2X receptors isoforms, the homomeric P2X3 and heteromeric P2X3/2 are thought to play a significant role in chronic and neuropathic pain via ATP-evoked neuronal excitability (Wirkner et al., 2007). This has been supported by significant antinociceptive effects produced by intrathecal administration of P2X3 and P2X2/3 selective receptor antagonist A-

317491 in animal pain models (McGaraughty et al., 2003). Studies performed on P2X receptors have helped to identify ATP as a nociceptive ligand and established a role for these ion channels in ATP-mediated neuronal excitability (Jarvis, 2010). Although P2X receptors may one day be valuable therapeutic targets for antinociception, clinically useful drugs for these channels remain to be discovered.

Figure 9. Ion channels expressed in the peripheral terminals of primary afferent neurons. Noxious stimuli induce (or inhibit in the case of K2Ps) high-threshold transducer ion channel gating, evoking an inward Ca2+ mediated transduction potential leading to peripheral terminal depolarization via Nav channel activation. Figure adapted from (Mathie, 2010; Woolf and Ma, 2007).

The transient receptor potential channel superfamily

The remaining nociceptor ion channel family to be described here is also the most functionally and pharmacologically diverse. Transient receptor potential (TRP) channels are a superfamily of membrane proteins, widely expressed in fish, insects, and mammals, as well as other members of the animal kingdom (Ramsey et al., 2006). In mammals, each TRP gene codes for six subfamilies: TRPC (canonicle), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin),

TRPML (mucolipin), TRPP (polycysteine). There exists two additional families TRPN

(NOMPC), present in C. elegans, and TRPY, present in yeast (Clapham, 2003). Most TRP channels are non-selective cationic channels, displaying distinct permeability’s for Ca2+, Na+, K+,

39 and Mg2+ ions. They are expressed in diverse range of tissues, with local densities in the free terminals of nociceptors nerve fibers and the skin. Activation of all TRPs in sensory fibers elicits inward cationic currents that initiate membrane depolarization (Tominaga et al., 1998). Their functions are related to various physiological processes, including the detection of stimuli associated with the senses and the maintenance of ionic homeostasis. Consistent with this physiological role, a diverse range of mechanical, chemical, and thermal stimuli are able to promote the activation of the TRP family members (Brauchi and Orio, 2011), yet the precise molecular mechanisms by which these polymodal stimuli integrate is still unknown.

Each TRP channel assembles into a tetramer formed by four subunits, usually identical, organized symmetrically around a solvent exposed central pore (Latorre et al., 2009). Each of these four subunits is composed of six transmembrane spanning domains (S1–S6). The basic organizational structure, as well as their selectivity for cations, likens TRP to other homotetrameric proteins, particularly those of the voltage-gated K+ channels (i.e. KcsA, MthK,

Kv1.2) whose crystal structures have been used to speculate on TRP organization (Gaudet, 2009;

Long et al., 2005a; Long et al., 2007). Low resolution structural data obtained for TRPV1 via cryo-electro-microscopy (CEM) support the generalities of the predicted models (Latorre et al.,

2009; Moiseenkova-Bell et al., 2008). The channels’ N- and C-termini, both long and distinct between each TRP channel subtype, are found in the intracellular portion of the protein [Figure

10]. These intracellular regions of the protein contain various characteristic sequence motifs that participate, not only in channel assembly, but also by modulating channel activation.

Figure 10. Predicted topology of a TRP channel family subunit. Each TRP channel subunit is composed of six transmembrane spanning segments (S1–S6). The S5–S6 linker contains an extracellular loop forming the pore domain and selectivity filter. Each subunit assembles to form a tetrameric structure similar to voltage-gated K+ channels. Adapted from (Clapham et al., 2003).

According to local sequence alignments, it is possible to identify a common structural element shared by these proteins: the pore-forming region [Figure 10]. From the terminal portion, this structure begins in the S5 transmembrane section, the turret pore (displaced toward the extracellular side), the pore helix (holding the pore selectivity filter) (Long et al., 2005b;

Owsianik et al., 2006), the selectivity filter (allowing the channel to discern what ions are able to pass the pore) (Owsianik et al., 2006), and the S6 transmembrane section [Figure 11]. The carboxy-termini segments found in the internal pore helix (S6) of each subunit link, forming a

41 bundle crossing in the cytoplasmic face of the pore, are believed to constitute the channel gate

(Susankova et al., 2007; Valente et al., 2008).

TRP channel pore region Residue Num rTRPM1 --ILWYIRVLDIFGVNKYLGPYVMMIGKMMI-DMLYFVVIMLVVLMSFGVARQAILHPEE 1038 rTRPV1 ------QMGIYAVMIEKMILRDLCRFMFVYLVFLFGFSTAVVTLIEDGK 604 rTRPV3 ------NMLYYTRGFQSMGMYSVMIQKVILHDVLKFLFVYILFLLGFGVALASLIEKCS 614 rTRPM8 ------LRLIHIFTVSRNLGPKIIMLQRMLI-DVFFFLFLFAVWMVAFGVARQGILRQNE 894 rTRPC6 ----DTLKDLTKVTLGDNVKYYNLARIKWDPTDPQIISEGLYAIAVVLSFSRIAYILPAN 617 rTRPA1 AIFFYWMNFLLYLQRFENCGIFIVMLEVIFK-TLLRSTGVFIFLLLAFGLSFYVLLNFQD 900 rTRPM1 KPSWKLARNIFYMP-----YWMIYGEVFA--DQID------LYAMEINPPCGENLYDEEG 1085 rTRPV1 NNSLPMESTPHKCRGSACKPGNSYNSLYS--TCLE------LFKFTIG------MGD 647 rTRPV3 KD------KKDCS------SYGSFSD--AVLE------LFKLTIG------LGG 642 rTRPM8 QRWRWIFRSVIYEP-----YLAMFGQVPS--DVDS--TTYDFSHCTFSGNESKPLCVELD 945 rTRPC6 ESFGPLQISLGRTVKDIFKFMVIFIMVFV--AFMIG--MFNLYSYYIGAKQNEAFTTVEE 673 rTRPA1 AFSTPLLSLIQTFS------MMLGDINYRDAFLEP-----LFRN------933 rTRPM1 KRLP------PCIPGAWLTPALMACYLLVANILLVNLLIAVFNNTFFE 1127 rTRPV1 LEFT------ENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNK 689 rTRPV3 LNIQ------QNSTYPILFLFLLITYVILTFVLLLNMLIALMGETAEN 684 rTRPM8 EYNL------PRFP-EWITIPLVCIYMLSTNILLVNLLVAMFGYTVGI 986 rTRPC6 SFKTLFWAIFGLSEVKSVVINYNHKFIENIGYVLYGVYNVTMVIVLLNMLIAMINSSFQE 733 rTRPA1 ------ELAYPVLTFGQLIAFTMFVPIVLMNLLIGLAVGDIAE 970

Figure 11. Partial rat TRP channel sequences utilizing multiple alignment tool COBALT (constraint-based multiple alignment tool) of the pore forming region. Highlighted in sky blue is the predicted S5 transmembrane helix sequence and in green the predicted S6 transmembrane helix sequence (based on predictions obtained from the TMHMM server 2.0). The residues highlighted in yellow represent the selectivity filter for rTRPV1 (donated by Ximena Steinberg).

One characteristic shared by the TRPC, TRPV, and TRPM subfamilies is the presence of a 25 amino acid sequence located immediately adjacent to the S6 helix, nicknamed the TRP domain (Latorre et al., 2009). This TRP domain is considered a signature for these types of channels. It contains two highly conserved so-called “TRP boxes”. Box 1, in the extreme N- terminal side of the TRP domain, and Box 2, that is less common, found in the extreme C- terminal side of the TRP domain that are believed to mediate temperature sensation and channel gating (Montell, 2005). It has been demonstrated that positively charged residues located in the first TRP box are involved in the sensitization of the channel by PIP2 and possibly other modulatory cytosolic lipids (Brauchi et al., 2007; Rohacs, 2007; Rohacs et al., 2008). This TRP domain has also been directly associated with the movement of the gate, suggesting that this is a 42 multifunctional domain essential for the activity of these channels (Ferrer-Montiel et al., 2004;

Vlachova et al., 2003).

TRPV1: The “Capsaicin Receptor”

The transient receptor potential subfamily V, member 1 (TRPV1) channel, otherwise known as the “Capsaicin Receptor”, is the most well studied member of the TRP cation channel superfamily. This vanilloid subfamily contains 5 other members, in addition to TRPV1,

(TRPV2–TRPV6) exhibiting distinct roles in environmental sensation. The gene for human

TRPV1 is located at 17p13.2, with the associate peptide spanning 839 amino acids (838 for rat

TRPV1) and a predicted molecular mass of 95,000 Da (Caterina et al., 1997; Hayes et al., 2000).

The TRPV1 channel is widely distributed throughout the body, with particularly high expression in afferent pain sensing neurons (Aδ- and C-fibers), dorsal root, nodose and trigeminal ganglia (Caterina et al., 1997; Szallasi et al., 1995). Expression of TRPV1 has also been identified in the central nervous system (Sanchez et al., 2001), skin (Southall et al., 2003), bladder smooth muscle (Daly et al., 2007), liver (Avraham et al., 2008), and airway epithelial cells (Reilly et al., 2003), as well as ~60% of spinal afferents throughout the gastrointestinal tract and colon (Christianson et al., 2006). In nociceptive neurons, TRPV1 is mainly expressed postsynaptically on the cell bodies found in the lamina I and II of the lumbar L4–L6 of the dorsal horn (Guo et al., 1999; Valtschanoff et al., 2001). Activation of capsaicin-sensitive sensory nerve fibers has two main effects. First, it initiates the generation of action potentials that travel up the nociceptive signaling pathways. Second, activation elicits the release of neurotransmitters which, in turn, initiate the biochemical cascade that underlies neurogenic inflammation (Szallasi et al.,

2007). Following tissue injury, some of these transmitters contribute to the process of neurogenic

43 inflammation by activation of proalgesic second messenger proteins including calcitonin gene- related peptide (CGRP), somatostatin, and substance P (Holzer and Maggi, 1998; Maggi, 1995).

The peptide cascade in turn excites inhibitory neurons in the laminae I, II, and IV, enhancing

GABA and glycinergic inhibitory postsynaptic currents (Ferrini et al., 2007; Ueda et al., 1994;

Zhou et al., 2008; Zhou et al., 2001). TRPV1 expression has also been identified in the lateral collateral path, the region where most visceral afferents terminate (Hwang and Valtschanoff,

2003). Robust TRPV1 expression appears in the brain regions associated with pain transmission and stimuli integration, such as the rostral ventromedial medulla (RVM), periaqueductal grey

(PAG), amygdala, solitary tract nucleus, hypothalamus, somatosensory cortex, anterior cingulated cortex, and insular cortex (Millan, 2002; Palazzo et al., 2012; Palazzo et al., 2008;

Zschenderlein et al., 2011). As such, TRPV1 is established as an important integrator of nociceptive information (Caterina et al., 1997; Tominaga et al., 1998). TRPV1 is unique in that it is a polymodal nociceptor exhibiting a dynamic threshold of activation [Figure 12] (Caterina et al., 1997). Agents involved in inflammation can act synergistically to lower the activation threshold for this channel (Jordt et al., 2000; Tominaga et al., 1998).

Figure 12. Membrane topology and key residues involved in TRPV1 channel regulation. Predicted six transmembrane spanning subunit (shown in green), including the extracellular pore-forming loop region between the S5 and S6. Vanilloid-binding sites are in red (R114, Y511, S512, Y550, E761). Residues involved in extracellular proton-mediated activation are in yellow (V538, E600, T633, E648). Sites for TRPV1 phosphorylation in orange: PKA-mediated (S116, T144, T370, S502), PKC-mediated (S502, S800), and CaMKII-mediated phosphorylation (S502, T704). Figure adapted from (Pingle et al., 2007; Szallasi and Sheta, 2012).

Pathophysiology of mammalian TRPV1 channels

TRPV1 channels play a significant role in the pathophysiology of various diseases found in humans. TRPV1 dysregulation appears to an important factor for the development of neuropathic pain conditions associated with trigeminal neuralgia, diabetic neuropathy, post- herpetic neuralgia, late-stage cancer, amputation, and physical nerve damage (Nilius et al., 2005).

Reciprocally, upregulation of TRPV1 channels due to chronic inflammation has been directly linked to the pathogenesis of various chronic diseases such as Crohn’s disease, ulcerative colitis, irritable bowel syndrome, gastroesophageal reflux disease, mastalgia, prurigo nodularis, and

45 vulvar allodynia (Birder, 2007; Gopinath et al., 2005; Nilius et al., 2005; Tympanidis et al.,

2004; Yiangou et al., 2001). In addition, TRPV1 overstimulation forms significant components of inflammation leading to asthma, allergic dermatitis, and pancreatitis through the downstream recruitment of inflammatory peptides substance P, calcitonin gene-related peptide, and neuropeptide Y (Nilius et al., 2005). As a result, it has been well established that TRPV1 channels play an important function in the initiation and maintenance of inflammatory diseases.

Therefore, it is not surprising that most major pharmaceutical companies have directed extensive resources to the discovery of small molecule TRPV1 antagonists.

TRPV1 channel regulation

As a multimodal nociceptor, TRPV1 is activated and/or sensitized by a wide range of proinflammatory and proalgesic mediators (Holzer, 2008; Szallasi et al., 2007). These include vanilloids (Caterina et al., 1997), temperature > 43°C (Caterina et al., 1997), acidic and basic pH

(Dhaka et al., 2009; Jordt et al., 2000), bradykinin (Cesare et al., 1999; Chuang et al., 2001), nerve growth factor (Chuang et al., 2001), anandamide (Zygmunt et al., 1999), arachidonic acid metabolites (Huang et al., 2002), lipoxygenase products (Hwang et al., 2000), leukotriene B

(Shin et al., 2002), prostaglandins (Moriyama et al., 2005), adenosine triphosphate (Tominaga et al., 2001), jelly fish and spider venoms (Cuypers et al., 2006; Siemens et al., 2006), volatile anesthetics (Cornett et al., 2008), propofol (Fischer et al., 2010), and LAs (Leffler et al., 2008;

Rivera-Acevedo et al., 2011). It has been demonstrated that phosphorylation plays an important role in channel regulation as well. Arachidonic acid metabolites such as prostaglandins initiate the activation of cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and

Ca2+/calmodulin-dependent kinase II (CaMKII)-mediated pathways that modulate neuronal

TRPV1 activity (Bhave et al., 2002; Jung et al., 2004; Premkumar and Ahern, 2000; Rathee et al.,

2002). The recruitment of these isoenzymes is associated with the increased responses to endovanilloids, capsaicin, protons, and thermal stimuli, as well as decreased Ca2+-induced

TRPV1 desensitization (Bhave et al., 2002; De Petrocellis et al., 2001; Jung et al., 2004;

Lopshire and Nicol, 1998; Vellani et al., 2001). It appears that these effects are mainly mediated through the phosphorylation of residues found in the intracellular face of TRPV1, and the N- and

C- termini (Jung et al., 2004; Mohapatra and Nau, 2003; Mohapatra et al., 2003; Numazaki et al.,

2002; Rathee et al., 2002). The endogenous regulatory lipid, phosphatidylinositol 4,5- bisphosphate (PIP2), also plays an important role in channel regulation via simultaneous sensitization and desensitization depending on the environmental conditions (Brauchi et al.,

2007; Chuang et al., 2001). The regulatory mechanisms that mediate the effects produced by

PIP2 have yet to be resolved.

Its upregulation under conditions of inflammation and established role in nociceptive transduction have made this channel an attractive target for novel analgesic agents (Voight and

Kort, 2010). TRPV1 channel function can be controlled pharmacologically by two ways: desensitization using TRPV1 agonists or inhibition by TRPV1 antagonists (Baraldi et al., 2010;

Gunthorpe and Szallasi, 2008). Unlike TRPV1 antagonism though, TRPV1 desensitization by agonists such as capsaicin or other potent TRPV1 ligands abolishes the functionality of the whole neuron expressing TRPV1 for an extended period, until normal cellular function is recovered. As mentioned previously, pharmaceutical companies have positioned substantial resources into the development of compounds that block TRPV1 activation in a competitive and non-competitive manner (Kym et al., 2009; Voight and Kort, 2010; Wong and Gavva, 2009).

Although a select few small molecule TRPV1 antagonists are currently undergoing clinical trials,

47 most induce unwanted side-effects such as mild hyperthermia (> 39 °C), limiting their clinical usefulness (Gavva et al., 2008; Romanovsky et al., 2009; Wong and Gavva, 2009).

THE EVOLUTION OF LOCAL ANESTHETIC RESEARCH

Significant progress has been made in our understanding of the mechanisms that mediate the physiology and pharmacology of pain inhibition using LAs, but there is also a clear need for improvement. The development of new techniques to produce long-lasting and nociceptive- specific local anesthesia continues to be investigated, particularly through the use of novel drug delivery systems (Grant et al., 2004). We are still far from making the ideal LA, which should produce reversible blockade of sensory neurons with minimal effects on motor neurons. It should also possess a rapid onset and have sufficiently long duration of analgesic action without toxicity.

Finding the ideal LA however can only be attained through further structure-activity relationship

(SAR) studies, particularly with regards to selective inhibition of nociceptor targets. Leads for the design of ideal agents will likely stem from a more systemic pharmacological and toxicological analysis of the currently available LA catalogue.

LAs are currently being investigated for uses other than those associated with perioperative anesthesia and analgesia. New therapeutic LA treatments are being examined for use in chronic and neuropathic pain management, post-herpetic neuralgia, burns, cancer, and strokes (Mani and Strichartz, 2005). These positive therapeutic effects occur at concentrations much lower than those required for regional anesthesia. For example, the IC50 for Nav channel inhibition by lidocaine is ~100 µM, whereas this compound extracellularly inhibits 1 muscarinic receptors with an IC50 of 20 nM in Xenopus oocytes (Hollmann and Durieux, 2000;

Hollmann et al., 2000). Preliminary studies have found that LAs affect several steps in the

48 inflammatory process, including inhibition of polymorphonuclear granulocytes, macrophage, monocyte, and the release of cytokines from peripheral nerves (Cassuto et al., 2006; Hollmann and Durieux, 2000). Recently, the therapeutic potential of LAs is now being tested on the relatively new class of membrane proteins, the transient receptor potential (TRP) ion channel family, directly involved in acute nociception, and the initiation and maintenance of persistent inflammatory states (Binshtok et al., 2007; Leffler et al., 2008; Leffler et al., 2011).

TRPV1 as a target for analgesic drugs

Less than 15 years after its cloning, the TRPV1 channel as an essential component of nociceptive signal transduction has become one of the most extensively studied analgesic targets, as TRPV1 inhibition has the potential to represent a mechanism for novel nociceptive-selective analgesics without the side effects associated with currently available pain killers. In the last few years, a number of promising selective TRPV1 antagonists have reached Phase I and II clinical trials for the treatment of inflammatory, neuropathic, and cancer-related pain (Szallasi et al.,

2007; Szallasi and Sheta, 2012). One of the first successful agents to reach Phase I trials was SB-

705498 (Chizh et al., 2007). Developed by GlaxoSmithKline, SB-705498 is a potent and selective TRPV1 antagonist that blocks capsaicin, proton, and thermal activation (Rami et al.,

2006). This orally available compound was effective at reducing heat- and pain-evoked changes due to capsaicin application on the skin without serious side effects. Currently, Phase II clinical trials are underway to evaluate the efficacy of SB-705498 at reducing post-surgical pain following wisdom teeth extraction (Szallasi and Sheta, 2012). AstraZeneca’s AZD-1386 also underwent Phase II trials in 2008 for treatment of gastroesophageal reflux disease. A placebo- controlled, randomized double blind trial found significant pain relief to heat, mechanical, and

49 chemical nociceptive responses in the esophagus, although a mild increase in body temperature was observed (~0.4°C) (Krarup et al., 2011). PharmaEste developed PHE377, a compound for the potential treatment of diabetic neuropathic pain and post-herpetic neuralgia that recently commenced Phase I trials. Reported to inhibit all forms of TRPV1 activation, it did not produce hyperthermia in rat and dog models (Szallasi and Sheta, 2012). In late 2011, Abbott Laboratories reported the development of a novel class of chroman-substituted isoquinoline ureas, whose lead molecule, A-1165442, demonstrated a significant reduction in osteoarthritic pain in rat models as well as bone cancer pain in mice models, without producing detectable increases in core body temperature (Reilly et al., 2012). As they are already well established as a target for novel analgesic compounds, TRPV1 antagonists have the potential to become a widely used, safe, and effective alternative to induce nociceptive-selective analgesia, particularly for conditions associated with neuropathic pain and inflammation (Szallasi and Sheta, 2012). Collectively, these results have spurred on the present thesis’ interests in the pursuit of novel methods to effectively block TRPV1 channels with established small molecular ion channel inhibitors, such as LAs.

The quest for the “Holy Grail” of local anesthesia

The more than 100-year history of local anesthesia research has substantially furthered knowledge of the inner workings of the nervous system and pain sensation. Regrettably, this has yet to produce a definitive molecular model for the design of long-lasting and nociceptive- selective LAs. The ever-evolving advances in technology continue to improve the safety of clinical anesthesia and have introduced novel approaches to achieve differential nerve selectivity.

In the last few years, studies have raised the possibility that QX-314 (N-[2,6-

50 dimethylphenylcarbamoylmethyl] triethylammonium or lidocaine N-ethyl chloride), a permanently charged lidocaine derivative traditionally deemed relatively membrane-impermeant, can be selectively navigated into nociceptive neurons (A- and C-fibers) through activation of transient receptor potential vanilloid 1 (TRPV1) channels (Binshtok et al., 2007; Binshtok et al.,

2009). Shortly before this was first reported, it was independently discovered that QX-314

(previously believed to possess no clinically useful effects) all by itself concentration- dependently and reversibly produces robust long-lasting local anesthesia in vivo, up to 12 times longer than lidocaine and in both nociceptive, sensory and motor fibers (Lim et al., 2007). It was reported that when capsaicin is co-applied with QX-314 in vivo, responses to noxious mechanical and thermal stimuli can be abolished without motor or tactile deficits (Binshtok et al., 2007).

Furthermore, capsaicin-QX-314 co-application significantly accelerated the onset of motor and sensory blockade in mice, effects which were inhibited using the competitive TRPV1 channel antagonist capsazepine (Ries et al., 2009). Although QX-314 entry into nociceptive neurons has been attributed to TRPV1 activation by capsaicin, direct mechanistic insight into the QX-314-

TRPV1 complex, and for that matter amino-amide LAs, has not been extensively pursued. We consider the possibility that QX-314, as well as conventional amino-amide LAs, directly gate and permeate TRPV1 channels by themselves, with capsaicin co-application facilitating the conformational changes necessary for this interaction to take place. In order to fill this knowledge gap, and undertake in-depth in vitro pharmacological characterization to accurately model the LA-TRPV1 complex, the aim for this thesis hence was to pharmacologically define and quantify amino-amide LA activity on TRPV1-channels using a stable and reproducible experimental system, and provide a molecular narrative for the polymodal actions of LA molecules on TRPV1 channels.

General Materials and Methods

The present study employed an electrophysiological approach using the two-electrode voltage technique (TEVC), supported by Ca2+ imaging and site-directed mutagenesis to elucidate the pharmacological mechanisms mediating LA interactions with TRP channels. The most ideal and direct assay for TRP channel activity is electrophysiology. A few approaches exist with varying degrees of difficulty and accessibility. Although patch-clamp techniques are more sensitive and offer higher resolution of biophysical phenomena, the Xenopus oocyte two- electrode voltage clamp conformation allows higher throughput pharmacological analysis of channel activity. The technique, first developed by Miledi and colleagues, has been widely employed to identify the behavior of ion channels in a stable, easily controlled environment

(Barnard et al., 1982; Gundersen et al., 1984). A significant advantage of using the Xenopus oocyte system over other electrophysiological modalities is its suitability for expressing a diverse range of membrane proteins and associated mutations (Goldin, 2006). As a result, oocyte TEVC has become an indispensable tool for structure-functional studies involving ion channels and remains one of the gold standard for ligand-gated pharmacology and physiology assays (Mathie,

2010). Additionally, the ability of TRP channels to preferentially conduct Ca2+ ions means they are highly suitable for Ca2+ based fluorometric imaging techniques as a rapid assay for channel activity. This imaging technique is indispensable to strengthen the data acquired electrophysiologically using TEVC. Cell fluorescence was detected using laser scanning confocal microscopy. When significant, the values of the effect on [Ca2+] in non-transfected cells were taken as baseline and subtracted from values obtained from transfected cells. Drug response behavior was evaluated using capsaicin (10 μM) on TRPV1. Detailed descriptions of material and methods are included in the proceeding pages.

CHEMICALS AND SOLUTIONS

All drugs were obtained from Sigma-Aldrich Canada (Oakville, ON, Canada). Capsaicin (8- methyl-N-vanillyl-6-nonenamide) was dissolved in absolute ethanol to produce a stock solution of 100 mM and stored at -20°C. Capsazepine was dissolved in absolute ethanol to produce a stock solution of 100 mM. GSK1016790A (GSK101; [N-((1S)-1-[[4-((2S)-2-[[(2,4- dichlorophenyl)sulfonyl]amino-3-hydroxypropanoyl)-1-piperazinyl]carbonyl]-3-methylbutyl]-1- benzothiophene-2-carboxamide) was dissolved in dimethyl sulfoxide for a stock solution of 1 mM. Lidocaine hydrochloride, QX-314 chloride, QX-314 bromide, benzocaine, tetraethylammonium chloride (TEA), tetramethylammonium chloride (TMA) and N-methyl-D- glucamine chloride (NMDG) were directly dissolved in the extracellular solution [cf.

Electrophysiology]. The solutions were buffered using 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) and pH values corrected to pH 7.4 with NaOH. Solutions to study pH-dependence of inhibition were prepared using the external solution buffered with sodium citrate and adjusted to a pH 5.5 with HCl. Control and test solutions were applied with an automated fast-switching multibarrel perfusion system (ValveBank8; AutoMate Scientific, Inc.,

Berkeley, CA, USA).

MOLECULAR BIOLOGY

The rat TRPV1 and human TRPV4 cDNA (provided by Dr. Sebastian Brauchi, Universidad

Austral de Chile) were expressed in Xenopus oocytes and tsA201 cells ligated to pcDNA3 expression vector. Xenopus laevis oocytes were injected with rat TRPV1 capped mRNA (~1

µg/µl), which was synthesized using the T7 mMessage mMachine kit (Ambion Canada,

Streetsville, ON, Canada). Mutant channels, L647A, E648A, and F649A, were generated using

53 site-directed mutagenesis. Post-injection, oocytes were incubated for 1–2 days to obtain expression levels between 0.1 and 20 μA. For experiments described in chapter 4, only oocytes expressing < 3 µA were used. TRPV clones were confirmed by sequencing the constructs using the Genewiz core facility at The University of British Columbia. All TRPV-pcDNA3 plasmids were linearized with Not1 enzyme (New England BioLabs Inc., Pickering, ON, Canada) and purified via ethanol precipitation. Messenger RNA was synthesized from linear cDNA using mMessage mMachine T7 transcription kit (Ambion, Austin, TX, USA) and purified using

RNeasy column purification kit (Qiagen Inc., Toronto, ON, Canada).

OOCYTE PREPARATION AND INJECTION

Xenopus laevis frogs were terminally anesthetized by immersion in 2 mg/ml tricaine methanesulphonate (Sigma-Aldrich, Oakville, ON, Canada). Stage V–VI oocytes were isolated and defolliculation was performed using a collagenase treatment, involving mild agitation with 1 mg/mL collagenase type 1a in Oocyte Ringer’s 2 solution: (mM) 82.5 NaCl, 2.5 KCl, 1 MgCl2,

5 HEPES, adjusted to pH 7.6 using NaOH, and sterile filtered, for approximately 1 hr. Between defolliculation and injection, oocytes were incubated at 18°C for ~1 h in Oocyte Ringer’s 3 solution, which contained 500 mL Leibovit’s L-15 Medium (Gibco, Life Technologies, Inc.,

Burlington, ON, Canada), 15 mM HEPES, 5 mL glutamine, 5 mL gentamycin, adjusted to pH

7.6 using NaOH, and sterile filtered. Injections of 30–50 nL (~1 μg/μL) mRNA encoding the channel of interest was carried out using a Nanoliter 2000 digital microdispenser (World

Precision Instruments Inc, Sarasota, FL, USA). Following injection oocytes were placed in an orbital shaking incubator for 24–72 h in Oocyte Ringer’s 3 solution at 18°C. All animal

54 protocols were approved by and performed in accordance with The University of British

Columbia Animal Care Committee.

ELECTROPHYSIOLOGY

We recorded voltage-clamped TRPV1 and TRPV4 currents from oocytes with the two-electrode voltage-clamp technique using an oocyte clamp amplifier (model OC-725C; Warner Instrument

Corp., Hamden, CT, USA). Microelectrodes were pulled from borosilicate glass tubes (Frogy

1001; Harvard Apparatus, Holliston, MA, USA) and backfilled with 3 M KCl. Ringer’s solution

(116 mM NaCl, 2 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, and 5 mM HEPES; adjusted to pH 7.4 with NaOH) was used as the standard external solution. In some experiments, NaCl was added to the standard Ringer’s solution to produce an equiosmolar control solution compared to an applied drug-containing solution to rule out hyperosmolarity as a cause of TRPV1 activation. To test whether our compounds exert intrinsic activity on TRPV1, cells were superfused with separate increasing drug containing extracellular solutions. Compound modulation of TRP channel activity was determined through measurements of non-selective inward cationic currents at a standard holding potential of –60 mV unless otherwise stated [cf. Chapters 4 and 5]. The effects from drug perfusion were normalized to a near maximally activating concentration of capsaicin (50 μM) or an approximate EC50 of capsaicin (15 μM) or pH (5.5) depending on the specific ligand-binding assay. All control and test solutions were applied for 10 s with a 2 min washout period between applications using an automated fast-switching perfusion system

(ValveBank8, AutoMate Scientific, Inc., Berkeley, CA, USA) in a custom-made 30-µl bath chamber. We standardized drug applications to 10 s with a 2 min washout period to minimize

Ca2+-induced desensitization (Koplas et al., 1997) as well as potentiation associated with

55 extended capsaicin administration due to direct channel phosphorylation by cAMP-dependent protein kinase (PKA) or indirect activation and phosphorylation of TRPV1 interacting enzymes recruited by phospholipase C (PLC) (Bhave et al., 2002; Vellani et al., 2001). In chapters 2 and 3, compound application was performed with serially increasing drug concentrations on a single oocyte. To further avoid sensitization which could occlude our drug-response analysis, in chapters 4 and 5 controls were performed followed by a single test compound application on a single oocyte. The pCLAMP 10.0 software suite (Molecular Devices, Sunnyvale, CA, USA) was used for data acquisition and offline analysis.

CALCIUM IMAGING

Experiments were performed on mammalian tsA201 cells cultured in Dulbecco’s modified

Eagles medium supplemented with 10% fetal bovine serum, 4 mM L – glutamine and 1% penicillin-streptomycin in a humidified 5% CO2, 95% O2 incubator at 37°C. Cells employed in

Ca2+ imaging were transfected with TRPV1 using the Ca2+ phosphate precipitations method and incubated in 14 mm single glass bottom plates. TRPV1 transient transfected tsA201 cells were loaded with 1 µM acetoxymethyl ester form of fluo-4 (Molecular Probes, Eugene, OR, USA) for

15 min and washed for an additional 10 min before recording. The dye was excited with an argon laser (excitation λ = 488 nm; emission λ = 516 nm) using an Olympus FV1000 confocal microscope (Olympus, Markham, ON, Canada). Analyses were performed offline using ImageJ software (Wayne S. Rasband, Special Volunteer, National Institutes of Health, Bethesda, MD,

USA; software available at http://rsb.info.nih.gov/ij; accessed January 27, 2011). Drugs were applied via a manual pressure ejection system positioned 1 mm from the cells of interest. Data

56 are expressed as fluorescence (F) or change in fluorescence (ΔF), divided by basal fluorescence

(F0).

STATISTICAL ANALYSIS

Calculations for statistical comparisons were performed with Prism 5 (GraphPad, San

Diego, CA, USA) and Sigma Plot 10 (Systat Software Inc., Chicago, IL, USA) software. Serial dose-response data were analyzed with repeated ANOVA (with Dunn’s Multiple Comparison

Test for post hoc comparisons between individual groups) or paired Student’s t test as appropriate. In single drug application experiments, baseline capsaicin activation was calculated from the mean of the control and recovery inward currents recorded in response to an approximate EC50 concentration of capsaicin (15 μM). Concentration-response data was fit using the Hill four-parameter equation, a nonlinear regression model commonly used to characterize concentration dependence of pharmacological responses.

In this equation, E represents the drug response, E0 is baseline response in the absence of drug,

Emax is maximal drug response, C is the drug concentration, EC50 is the half-maximal effective concentration, and h is the Hill coefficient (Goutelle et al., 2008). The fitting of the Hill equation was also used to calculate the half-maximal inhibitory concentrations (IC50) of the compounds tested. Statistical tests were two-tailed, and we considered a P value 0.05 statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001. Data are given as mean SEM where appropriate.

Chapter 2: The quaternary lidocaine derivative, QX-314, exerts biphasic effects on TRPV1 channels in vitro

INTRODUCTION

As described in the previous chapter, the TRPV1 channel has recently garnered substantial interest as a potential target for novel analgesic compounds (Gomtsyan et al., 2005;

Patapoutian et al., 2009; Premkumar, 2010; Roberts and Connor, 2006; Veronesi and Oortgiesen,

2006; Wang et al., 2007). Among the investigated candidate molecules is the quaternary lidocaine derivative, QX-314, which features an additional N-ethyl group attached to the amine function. As a result, the traditional view has been that QX-314 is membrane-impermeable and blocks Na+-dependent action potentials only when administered intracellularly, rendering the agent devoid of clinically useful LA activity. However, it has recently been shown that QX-314, concentration-dependently and reversibly produces robust long-lasting nociceptive, sensory, and motor blockade with a slow onset in animal models in vivo (Lim et al., 2007). A subsequent report raised the intriguing possibility that QX-314 can be selectively navigated into nociceptive neurons through the pore of activated TRPV1 channels, providing a potential mechanism for achieving nociceptive-specific blockade (Binshtok et al., 2007). Further pharmacological exploration of this possibility confirmed that co-application of QX-314 with the TRPV1 agonist, capsaicin, significantly accelerates the onset of sensory blockade due to QX-314 (Ries et al.,

2009). However, direct mechanistic insight into the interaction of QX-314 with TRPV1 has been lacking.

In addition, it has been suggested that TRPV1 channel agonism may represent a possible mechanism for LA toxicity (Leffler et al., 2008). It has recently been found that intrathecal QX-

314 administration at concentrations as low as 0.5 mM produces unacceptable adverse effects in

58 mice (including death at ≥ 5 mM) (Schwarz et al., 2010). These effects occurred at lower concentrations than those associated with robust motor blockade, suggesting a low therapeutic index for use in spinal anesthesia. The findings also alluded to a distinct neurotoxic action compared to lidocaine (Schwarz et al., 2010): Whereas lidocaine neurotoxicity mainly manifests as irreversible conduction blockade, intrathecal QX-314 acutely produced marked irritable, nocifensive-type behavior in the animals. While QX-314 holds promise as a LA agent to produce long-lasting peripheral antinociception, it is imperative on the basis of these observations that more preclinical studies take place to better define the mechanisms underlying its therapeutic and adverse effects prior to testing in humans. In this chapter, the possibility is considered that QX-

314, at millimolar concentrations, might produce direct TRPV1 activation as a mechanism for acute irritation. To test this hypothesis directly, an in vitro study was conducted to examine the mechanistic basis by which QX-314 acts on TRPV1 channels. The results demonstrate that QX-

314 exerts biphasic effects on expressed TRPV1 channels, where micromolar application potently inhibits capsaicin-induced cation currents while millimolar concentrations produce robust and reproducible channel activation.

RESULTS

To test the hypothesis that QX-314 directly activates TRPV1 channels, Xenopus laevis oocytes expressing rat TRPV1 channels were examined in a two-electrode voltage-clamp setup, calibrated using the prototypical TRPV1 agonist, capsaicin [Figure 13]. Capsaicin produced no effects in oocytes injected with saline control solution (data not shown). In oocytes injected with

TRPV1 mRNA, administration of capsaicin concentration-dependently produced reversible inward cationic currents consistent with TRPV1 channel expression and activation [inset in

Figure 13]. All cells injected with TRPV1 mRNA responded to capsaicin (n = 15). In the experimental system, capsaicin demonstrated an activation range between 1 and 100 µM, with maximal activation occurring at ~100 µM [Figure 13]. The EC50 for capsaicin activation of

TRPV1 was 19.2 ± 0.5 µM; consistent with previous findings (Jordt and Julius, 2002; Jordt et al.,

2000), the Hill coefficient was 2.1 ± 0.1 (Hill four parameter non-linear regression, P < 0.001), indicating cooperativity between binding sites to open the TRPV1 ionic pore (Yifrach, 2004).

Whereas activation occurred at higher concentrations than in some previous studies, it is possible that the observed differences in capsaicin sensitivity are attributable to differences in the expression system used (e.g., oocytes vs. HEK293t cells), the TRPV1 isoforms studied (e.g., rat vs. human), or the procedure of drug application (e.g., we employed an automated fast-switching perfusion system [cf. General Materials and Methods, Drugs and Chemicals] and standardized drug applications to 10 s, ensuring a plateau would be reached while limiting desensitization by extended capsaicin administration) (Mohapatra and Nau, 2003; Mohapatra et al., 2003).

Figure 13. Capsaicin activation of TRPV1 channels expressed in Xenopus laevis oocytes. Concentration-response curve for capsaicin-evoked TRPV1 currents in oocytes. Increasing capsaicin concentrations were applied to each cell, until saturation was observed. The data were fitted using the Hill equation; error bars denote SEM. The inset shows a representative experiment showing increasing inward currents with increasing capsaicin concentrations in Xenopus oocytes expressing TRPV1 channels. Cells were held at −60 mV. Each cell was sequentially perfused for 10 s with capsaicin-containing solution, with a washout of 2 min between applications.

To investigate TRPV1 activation by QX-314, increasing concentrations were applied to

Xenopus laevis oocytes injected with TRPV1 mRNA. Before and after QX-314 administration, capsaicin at a concentration producing a near-maximal response (50 µM) was applied as a positive control and reference. Both QX-314 salts (bromide and chloride applied at 1−60 mM; see below) directly activated TRPV1 in a concentration-dependent fashion. QX-314 chloride produced inward currents consistent with TRPV1 activation at 10–60 mM, with the largest effect occurring at 60 mM [Figure 14A & C]. Application of QX-314 bromide (≥ 30 mM) produced oocyte membrane blackening and cell death on contact with the solution (30 mM, n = 6/10 cells tested; not shown). Whereas the inward currents produced by QX-314 chloride generally were completely abolished following washout, at 60 mM some cells (n = 6/18) exhibited evidence of membrane disintegration upon administration, leading to cell death. For this reason, and in order to avoid possible non-specific activation due to hyperosmolarity, the range of QX-314 concentrations was limited to 60 mM [cf. Chapter 2, Discussion] (Ciura and Bourque, 2006;

Dhaka et al., 2009). However, in experiments where NaCl was added to the Ringer’s control solution to render it equiosmolar to the solutions containing 60 mM QX-314 or lidocaine [cf.

General Materials and Methods, Electrophysiology], no differences were observed compared to standard Ringer’s solution or effects due to hyperosmolarity in this range (data not shown).

Since QX-314 produced activation of TRPV1, a generic organic cation, NMDG, was also tested to determine if the activation is unique to quaternary (i.e., permanently charged) LAs such as QX-314 or if it represents a generic effect produced by high concentrations of permanently charged organic compounds. As shown in Figure 13A, NMDG at a concentration equimolar to the highest QX-314 and lidocaine concentrations (60 mM) produced negligible TRPV1

62 activation. Higher concentrations of NMDG (150 mM and 300 mM) evoked small inward currents (data not shown).

Consistent with a previous study (Leffler et al., 2008), lidocaine (1–60 mM) similarly produced concentration-dependent TRPV1 activation. The experiment was performed in the same fashion as with QX-314, with applications of 50 µM capsaicin before and after lidocaine administration. Compared to QX-314 chloride, lidocaine produced larger inward currents at 10 and 30 mM, but comparable effects at 60 mM [Figure 14B & C]. All cells perfused with lidocaine exhibited complete recovery, with no evidence of oocyte disintegration.

Figure 14. QX-314 activation of TRPV1 channels expressed in Xenopus laevis oocytes.

Figure 14. QX-314 activation of TRPV1 channels expressed in Xenopus laevis oocytes. (A) QX-314 (10–60 mM) concentration-dependently produced inward currents in an oocyte expressing TRPV1 channels. Each concentration was applied for 10 s with a 2 min washout period; the holding potential was −60 mV. For comparison and as a positive control, each experiment was flanked by capsaicin application at a concentration producing a near-maximal response (50 µM). The organic cation, N-methyl-D-glucamine (NMDG; 60 mM), applied as a negative control, produced negligible activation. (B) Traces from a similar experiment performed with lidocaine. Lidocaine (10–60 mM) concentration-dependently produced TRPV1-mediated inward currents whereas NMDG (60 mM) was without significant effect. (C) Mean peak current amplitudes ± SEM of experiments described in A and B, normalized to responses produced by 50 µM capsaicin. QX-314 produced 0.5 ± 0.1% (10 mM), 3.5 ± 1.0% (30 mM), and 21.5 ± 7.0% (60 mM) of the maximal capsaicin-evoked current (n = 12); lidocaine produced 2.5 ± 0.4% (10 mM), 8.8 ± 2.0% (30 mM), and 21.1 ± 5.0% (60 mM) of the maximal capsaicin-evoked current (n = 6). NMDG produced 0.6 ± 0.4% of the responses evoked by 60 mM QX-314 (n = 9). *** P < 0.001.

In order to test if the observed effects of QX-314 are exclusive to TRPV1 or also apply to other TRP isoforms, a series of experiments was conducted to study its actions on TRPV4, the closest TRPV1 homologue [cf. Discussion, fourth paragraph]. Using the potent TRPV4 agonist,

GSK101 (500 nM) as control, QX-314 chloride produced no activation of TRPV4 channels [60 mM; n = 7; Figure 15].

Figure 15. QX-314 does not activate TRPV4 channels. QX-314 (60 mM; n = 7) did not elicit responses in Xenopus laevis oocytes expressing TRPV4 channels, compared to control currents elicited by 500 nM GSK101 [cf. General Materials and Methods, Drugs and Chemicals]; for details, see Results.

To investigate if QX-314 activates TRPV1 channels through similar mechanisms as capsaicin, the effects on QX-314- and lidocaine-evoked currents of the well-known and well- characterized competitive TRPV1 antagonist, capsazepine, was subsequently examined [Figure

16A]. The experiments were carried out in a similar fashion as those in Figure 13, with 50 µM capsaicin applied at the beginning and end of each experiment. As shown in Figure 16A & B,

QX-314-evoked currents were effectively blocked by capsazepine. Similarly, capsazepine produced near complete blockade of TRPV1 activation by lidocaine [100 µM; Figure 16C & D].

To rule out the possibility that the observed TRPV1 channel activation by QX-314 was specific to the Xenopus laevis oocyte expression system, experiments were conceived to verify our results on TRPV1 channels transiently transfected in the mammalian tsA201 cell line. Here, expressed TRPV1 activity was determined by measuring Fluo-4 AM Ca2+ transients with laser- scanning confocal microscopy. In these studies using 10 µM capsaicin as control, fluorescence from Ca2+ entry into the cytosol was readily apparent in tsA201 cells (data not shown).

Application of 60 mM QX-314 chloride produced a robust increase in fluorescence compared to background fluorescence levels [Figure 16E, left and middle panel]. The increase in fluorescence was similar for 10 µM capsaicin and 60 mM QX-314 (data not shown). The transient rise in cytoplasmic Ca2+ evoked by QX-314 (or capsaicin) was nearly completely inhibited by capsazepine [Figure 16E, right panel].

Figure 16. The competitive TRPV1 antagonist, capsazepine, blocks QX-314-evoked currents in Xenopus oocytes and mammalian tsA201 cells.

Figure 16. The competitive TRPV1 antagonist, capsazepine, blocks QX-314-evoked currents in Xenopus oocytes and mammalian tsA201 cells. (A) Capsazepine (100 µM) blocked TRPV1 activation by QX-314 (60 mM; n = 6; holding potential, –60 mV). Capsaicin at a concentration producing a near-maximal response (50 µM) was applied before and after the experiment for comparison and control. (B) Mean current amplitudes ± SEM measured from experiments described in A. Capsazepine produced near complete blockade (96.8 ± 0.8%; n = 6) of QX-314-evoked TRPV1 activation. (C) Representative traces showing lidocaine-evoked inward currents. (D) Mean current amplitudes ± SEM measured from the experiment shown in C, demonstrating near complete blockade (98.4 ± 0.9%; n = 5) by capsazepine of TRPV1 activation by lidocaine. (E) 60 mM QX-314 evokes a robust Ca2+ increase in tsA201 cells expressing the TRPV1 channel (n = 10). Application of capsazepine (100 µM) inhibited QX-314-evoked responses (n = 4). ΔF was similar for 60 mM QX-314 and 10 µM capsaicin (n = 10; data not shown). * P < 0.05; I norm = normalized current; cap = capsaicin; czp = capsazepine; bkgd = background (control).

A previous study had suggested that QX-314, like lidocaine, can inhibit capsaicin-evoked

TRPV1 currents (Leffler et al., 2008). However, the concentrations used in these experiments were comparatively high (30 mM), possibly underscoring the inhibitory potency of QX-314, as the results in this thesis clearly show direct activation of TRPV1 by QX-314 in the millimolar range (see above). Consequently, experiments were conducted to determine if QX-314 might also inhibit TRPV1 currents at sub-activating concentrations. To test this possibility, the effects of micromolar QX-314 concentrations on currents activated by capsaicin at a concentration near its EC50 (17 µM) were investigated. As illustrated in Figure 16, the data demonstrate that QX-

314 blocked inward currents in a concentration-dependent and reversible manner with 79 ± 9% of current blocked at 30 µM and 93 ± 4% blocked at 300 µM (n = 10). The calculated IC50 was

8.0 ± 0.4 µM, indicating that QX-314 is a potent inhibitor of TRPV1 channels at sub-activating concentrations.

Figure 17. QX-314 inhibits capsaicin-evoked TRPV1 currents. (A) QX-314 concentration- dependently blocked capsaicin-evoked inward currents in oocytes expressing wild-type (WT) TRPV1 channels (holding potential, −60 mV). Each solution containing a particular drug concentration was applied for 10 s with a 2 min washout period. (B) Remaining normalized peak currents induced by capsaicin (17 µM) following application of QX-314 (mean ± SEM; each data point, n = 10).

DISCUSSION

This chapter’s results show that the quaternary lidocaine derivative, QX-314, exerts biphasic effects on TRPV1 channels, inhibiting capsaicin-evoked TRPV1 currents at lower (micromolar) concentrations and activating TRPV1 channels at higher

(millimolar) concentrations. From the data, it is apparent that QX-314, administered in the millimolar range, activates TRPV1 channels in a concentration-dependent fashion.

Although it was not possible in the experiments to reach saturation with QX-314 and construct a full concentration-response curve on TRPV1 activation, the results indicate that QX-314 is slightly less potent (and/or less efficacious) a TRPV1 activator than lidocaine, previously found in human embryonic kidney (HEK293t) cells to produce

TRPV1 activation with an EC50 of 12 mM (Leffler et al., 2008). Of note, the authors found evidence that 100 mM lidocaine (a maximally effective concentration) induced seal breaks in the cells. Somewhat similarly, the present experiments found that QX-314 concentrations ≥ 60 mM produced oocyte disintegration and cell death, raising the possibility of a cytotoxic effect of these high concentrations.

The observed QX-314-evoked currents may provide a basis for a change of the current molecular model for LA activation of TRPV1, thought to occur through a hydrophobic pathway to activate a domain in the cytosol (Leffler et al., 2008). It has previously been suggested that LAs interact with a domain that is similar, but not identical, to the vanilloid-binding region composed of transmembrane domains 3 and 4 and the respective cytosolic interfaces (Gavva et al., 2004; Jordt and Julius, 2002; Leffler et al., 2008). It is noteworthy then that the permanently charged QX-314 produced similar effects as lidocaine on TRPV1. A possible explanation for this observation is that the

70 other regions of the channels may be involved in LA binding and that binding domains for vanilloids and lipophilic LAs are distinct (Dhaka et al., 2009; Leffler et al., 2008).

Previous investigations with HEK293t cells found that TRPV1 was not activated by 30 mM QX-314, compared with robust lidocaine activation at an equimolar concentration

(Leffler et al., 2008). The present studies, however, demonstrate that 60 mM QX-314 activates TRPV1 channels expressed in Xenopus laevis oocytes as well as in tsA201 cells.

Importantly, in both cell types, activation was inhibited by capsazepine, indicating that both LAs use an activation pathway that is similar to each other and the prototypical agonist, capsaicin. Hence, it would appear that QX-314 produces activation through a molecular mechanism similar to that used by vanilloids such as capsaicin (Bevan et al.,

1992; Walpole et al., 1994).

If QX-314 evokes TRPV1-mediated currents in oocytes and tsA201 cells, why did a previous study not observe any QX-314-dependent activation in HEK293t cells

(although QX-314 inhibited TPRV1-evoked currents)? Firstly, the previous study only used a single concentration of both lidocaine and QX-314 (30 mM each). This precluded a more thorough concentration-response analysis. A biphasic action of QX-314 may have therefore been missed. Secondly, it is a possibility that TRPV1 channels adopt a different molecular confirmation in oocytes and tsA201 cells compared to HEK293 cells, allowing the LA binding domain to be exposed to a highly hydrophilic compound such a QX-314.

However, this is unlikely because the present study recorded a significant increase in cytoplasmic Ca2+ levels in TRPV1-transfected tsA201 cells when exposed to 60 mM QX-

314 [cf. Figure 16].

Mammalian tissues differentially express a variety of TRP channel isoforms

(Garcia and Schilling, 1997; Kunert-Keil et al., 2006). The present experiments sought to investigate if QX-314 would evoke currents in TRP isoforms closely related to TRPV1, the closest homologue being TRPV4. The TRPV4 channel has ~40% homology with

TRPV1, particularly in the area of the ion pore and selectivity filter (Guler et al., 2002;

Liedtke et al., 2000). Like TRPV1, it is activated by a wide range of physical and chemical stimuli, including synthetic phorbol derivative 4-phorbol-12,13-didecanoate

(4-PDD) (Watanabe et al., 2002), arachidonic acid metabolites (Watanabe et al., 2003), moderate warmth (> 27 Cº) (Watanabe et al., 2002), hypotonic cell swelling (Strotmann et al., 2000), pressure (Suzuki et al., 2003), membrane stretch (Thodeti et al., 2009), and sheer stress (Gao et al., 2003). Using the novel TRPV4 agonist, GSK1016790A, as a positive control (Thorneloe et al., 2008). QX-314 produced no activation in these channels. This observation suggests that QX-314 and lidocaine may be activating TRPV1 channels through a TRPV1-specific mechanism, possibly involving a region similar to or near the vanilloid-binding domains. However, one cannot exclude the possibility that the conformation TRPV4 presents in Xenopus laevis oocytes does not allow QX-314 access to bind and activate the channel.

The findings of the present study might help explain the recent in vivo observations of severe irritation associated with intrathecal QX-314 administration in mice (Schwarz et al., 2010). In general, pain upon injection of LAs is a well-known phenomenon and often a source of discomfort for patients undergoing local anesthesia

(Arndt, 1992). One possible mechanism may be the low pH of some LA formulations (de

Jong and Cullen, 1963), as TRPV1 is considered the main transducing receptor system

72 for proton-induced excitation of nociceptive sensory neurons (Caterina et al., 2000;

Leffler et al., 2008; Leffler et al., 2006). However, the drug-containing solutions in the present study were titrated to a pH of 7.4, and previous studies have suggested that LAs themselves might produce pain upon injection by directly activating TRPV1 (Leffler et al., 2008). Whereas both QX-314 and lidocaine activated TRPV1 at clinically relevant concentrations, lidocaine-evoked pain in humans rapidly ceases as nerve conduction block sets in, a feature that is unlikely shared by QX-314 due to its significantly longer

LA onset time (Lim et al., 2007). This may serve to explain why intrathecal lidocaine does not produce the sustained severe irritation acutely associated with QX-314 (Schwarz et al., 2010). That said, lidocaine itself is well known to be neurotoxic, and on the basis of the present study it would seem possible that Ca2+ influx mediated by TRPV1 activation

(Amantini et al., 2007; Friederich and Schmitz, 2002; Shin et al., 2003; Thomas et al.,

2007) contributes to LA-induced cell death by inducing necrotic and apoptotic mechanisms (Friederich and Schmitz, 2002; Johnson et al., 2004). The cellular degeneration due to QX-314 chloride (60 mM) and bromide (≥ 30 mM) in this study may well relate to the adverse intrathecal effects observed in vivo. In this regard, QX-314 appears to share features of the toxicity profile of other quaternary ammonium compounds, e.g., tetraethylammonium-C12 (Seitz et al., 1989), N- phenylethylamitriptyline, N-propylamitriptyline, N-propyldoxepin (Gerner et al., 2005;

Gerner et al., 2002; Sudoh et al., 2004), and tonicaine (N-phenylethtyl lidocaine) (Gerner et al., 2000; Khan et al., 2002; Wang et al., 1995); all of which act as LAs, but produce severe neurotoxicity in vivo (Schwarz et al., 2010). Collectively, the available data raise the possibility that the quaternary ammonium function and associated permanent positive

73 charge confers specific structure-activity properties that give rise to an adverse effect profile distinct from that of amphipathic LAs. At a molecular level, the topic of structure- activity relationships involved in the interaction of LAs with TRPV1 channels will be subject of chapter 4 later in this thesis.

In contrast to the effects at millimolar concentrations, QX-314 at lower, sub- activating (micromolar) concentrations potently inhibited capsaicin activation of TRPV1 in vitro, with an IC50 of 8 µM and near complete blockade reached at 300 µM. Lidocaine has previously been shown to block rat TRPV1 channels expressed in HEK293t cells, albeit with a substantially lower potency (IC50, 45 mM) (Leffler et al., 2008). In addition,

TRPV1 inhibition from the intracellular pore has been reported with other quaternary ammoniums, including tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium (Jara-Oseguera et al., 2008). Whereas the specific molecular mechanisms for the complex effects of extracellularly applied QX-

314 on TRPV1 channels remain to be elucidated, the present observations provide novel insight into the interactions between this quaternary lidocaine derivative and TRPV1 channels. As TRPV1 channels play a central role in peripheral nociceptive transduction, they represent attractive potential therapeutic targets for the management of acute pain and chronic pain syndromes.

In conclusion, the results in this chapter involving experiments on Xenopus laevis oocytes as well as tsA201 cells show that the quaternary lidocaine derivative, QX-314, behaves as a biphasic regulator of expressed rat TRPV1 channels. At low, micromolar concentrations, QX-314 inhibits TRPV1 activation in the presence of capsaicin, while at higher concentrations in the millimolar range more relevant to clinical local anesthesia,

QX-314 is a TRPV1 channel agonist similar to lidocaine (Leffler et al., 2008). These results not only describe a novel property of quaternary LAs, but also might provide a new molecular explanation for LA adverse events when used for spinal and regional anesthesia.

Chapter 3: pH-dependent local anesthetic inhibition of the transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes

INTRODUCTION

As previously discussed, the list of chemical ligands that can activate and/or sensitize TRPV1 channels is extensive, and includes protons (pH < 6) (Holzer, 2004).

Indeed, the detection of acidic conditions is an important characteristic for these channels in primary afferent neurons (Holzer, 2009). Protons are able to regulate TRPV1 activity via two distinct mechanisms. For instance, above the threshold for direct pH activation, proton-induced TRPV1 sensitization occurs. During mild tissue acidosis, for example

(pH 7–6), capsaicin and temperature activation are potentiated through an overlapping mechanism, increasing the current activation rate and producing a reciprocal reduction in deactivation (Ryu et al., 2003; Ryu et al., 2007). In the presence of acute acidification

(pH < 6), the TRPV1 channel undergoes a unique conformational change, independent from vanilloid or heat activation, to induce a pore opening (Ryu et al., 2007).

Among the acid sensing ion channels, TRPV1 is the only one to activate during severe tissue acidosis (pH < 6) (Holzer, 2009). Gene-knockout models have established the polymodal nociceptor TRPV1 as an essential target for sensation and development of hyperalgesia due to localized extracellular tissue acidosis during inflammation (Ryu et al.,

2003). Following tissue injury, an overabundance of extracellular protons form a significant component of the local nociceptive response, lowering physiological pH levels and modulating pH sensitive nociceptor activity (Jordt et al., 2000). The release of neuropeptides such as substance P and CGRP, together with proton sensitization contribute to the process of neurogenic inflammation by activation of proalgesic second

76 messenger proteins and initiating membrane depolarization (Holzer and Maggi, 1998;

Maggi, 1995).

The previous chapter described the findings that lidocaine and QX-314 produce biphasic TRPV1 channel activity, exhibiting both activating and inhibiting properties. In particular, micromolar QX-314 concentrations potently inhibited TRPV1 activation in

Xenopus laevis oocyte preparations in the presence of capsaicin. TRPV1 antagonist competition assays have suggested that proton activation does not modify the capsaicin- binding pocket (Gavva et al., 2005). In light of this, this chapter is dedicated to a series of experiments aimed at determining whether QX-314 and lidocaine inhibition are modality- specific for capsaicin, or if block by these compounds is conserved during TRPV1 proton-activation.

RESULTS

QX-314 inhibits proton-induced TRPV1 currents in Xenopus laevis oocytes. In the previous chapter, results were shown that demonstrated that QX-314 inhibits capsaicin- activated TRPV1 channels at pH 7.4 (Rivera-Acevedo et al., 2011). To determine whether this effect is a general property of the compound, or an agonist-specific interaction, QX-314 inhibition was tested here under conditions of proton-evoked TRPV1 channel activation. Expressing rat TRPV1 in Xenopus laevis oocytes, a pH 5.5-containing solution (producing a sub-maximal response; data not shown) was co-applied for this purpose with increasing concentrations of QX-314, flanked by two control applications of pH 5.5 alone. As shown in [Figure 18B], micromolar applications of QX-314 produced a concentration-dependent, but reversible decrease in proton-evoked inward currents. QX-

314 demonstrated increased potency for current inhibition under proton-evoked TRPV1 channel activation, compared with capsaicin activation [Figure 18C & D]. The resulting

IC50 for proton-evoked QX-314 inhibition was 900 ± 200 nM, with a Hill coefficient of

1.2 ± 0.8 [Figure 18D].

Figure 18. QX-314 concentration-dependently inhibits proton-evoked TRPV1 currents in Xenopus laevis oocytes. The serial co-application of pH 5.5 in solution containing different concentrations of QX-314 was flanked by two control applications of external solution only at a pH 5.5. Each application was performed for 10 s and 2 min washout periods were used between all applications. Proton-evoked currents were not observed in uninjected oocytes (data not shown). (A) Electrostatic potential map of the quaternary QX-314 molecule, highlighting the permanent positive charge. (B) Repre- sentative proton-evoked current traces after the co-application of pH 5.5 with increasing micromolar concentrations of QX-314 at a holding potential of −60 mV. Calibration bars: horizontal = 1 min; vertical = 4 µA. (C) Concentration-response data comparing the inhibition of pH 5.5 proton-evoked inward currents by QX-314, to capsaicin-evoked current inhibition at pH 7.4. Data points at each concentration represent mean ± SEM of remaining current, normalized to the mean peak current. Data was fit with the Hill equation (here and in all following fits: Hill four-parameter, non-linear regression). (D) Bar graph comparing the change in the log IC50 of QX-314 observed with capsaicin (8.2 µM) vs. proton activation (900 nM).

Lidocaine inhibits capsaicin and proton-induced TRPV1 currents in oocytes. On the basis of the finding that QX-314 inhibits both capsaicin and proton-evoked inward currents in TRPV1 channels, a similar set of experiments was performed here with the amphipathic LA, lidocaine. When co-applied with capsaicin at pH 7.4, lidocaine reduced

TRPV1 peak current amplitudes at micromolar concentrations, albeit incompletely

[Figure 19A]. The resulting IC50 for capsaicin-evoked lidocaine inhibition was 17.2 ± 2

µM, with a Hill coefficient of 2.1 ± 0.4 [Figure 19C]. Co-application of TRPV1- activating pH 5.5 solutions with lidocaine also produced TRPV1 inhibition [Figure 18B].

The IC50 for proton-evoked lidocaine inhibition was 23.1 ± 9 µM, with a Hill coefficient of 0.9 ± 3 [Figure 19C]. Compared with the effects at pH 7.4, a significant increase in the efficacy of lidocaine inhibition was observed at pH 5.5 [Figure 19C & E]. The results clearly show that extracellular lidocaine, similar to QX-314, produces concentration- dependent, but reversible inhibition of multi-agonist TRPV1-induced inward currents in vitro.

Figure 19. Lidocaine concentration-dependently inhibits capsaicin- and proton- evoked TRPV1 currents in Xenopus laevis oocytes.

Figure 19. Lidocaine concentration-dependently inhibits capsaicin- and proton- evoked TRPV1 currents in Xenopus laevis oocytes. The serial co-application of 15 µM capsaicin with increasing concentrations of lidocaine was flanked by two control applications of 15 µM capsaicin. (A)/(B) Representative agonist-evoked current traces before and after the serial co-application of (A) capsaicin or (B) pH 5.5 buffered solution with lidocaine at a holding potential of −60 mV. Calibration bars: horizontal = 1 min; vertical = 4 µA. (C) Concentration-response data comparing the inhibition of pH 5.5 proton-evoked inward currents by lidocaine, to capsaicin-evoked current inhibition at pH 7.4. Data points at each concentration represent mean ± SEM of remaining current, normalized to the mean peak current. (D) Electrostatic potential maps representing the approximate ratios of neutral vs. charged lidocaine molecules at pH 7.4. (E) Bar graph comparing 300 µM lidocaine inhibition observed with capsaicin (37.2 ± 6 %) vs. pH 5.5 (14.8 ± 3 %). (D) Calculated log IC50 values between capsaicin (17.2 ± 2 µM) and proton activation (23.1 ± 9 µM).

DISCUSSION

The experiments summarized in this chapter characterized the effects of extracellular proton-evoked TRPV1 channel current inhibition by amino-amide LAs. It has long been assumed that acidosis plays an important role in the development of inflammatory and hypoxia-induced pain (Steen et al., 1995). It is not uncommon for localized pH to drop below 6 during inflammation, ischemia, bone cancer metastasis, and even following intense anaerobic activity (Nagae et al., 2006; Wemmie et al., 2006). The pH sensitivity of TRPV1 is unique from other acid sensing channels, such as ASIC receptors, in that they are directly gated by extracellular pH < 6 (Jordt et al., 2000;

Tominaga et al., 1998). The present results show, using a multi-agonist approach, that

QX-314 is able to potently inhibit proton-evoked TRPV1 currents in a similar manner as capsaicin-mediated inhibition [Figure 18C]. The permanently charged quaternary QX-

314 molecule, unperturbed by changes in pH, demonstrated more potent TRPV1 inhibition under acidic conditions [Figure18D]. One possibility for this change may be the coupling mechanism underlying proton activation exposes the binding site for QX-

314, leading to easier access by this molecule. Site-directed mutagenesis performed on proton-sensitive residues has demonstrated that proton-induced TRPV1 activation is mediated through a pathway functionally distinct from vanilloid activation (Gavva et al.,

2005; Jordt et al., 2000). Residues Y511, S512, and R491, located in the intracellular loop between S2 and S3, have been shown to participate in capsaicin binding (Jordt and

Julius, 2002). On the other hand, proton-induced TRPV1 activation has been isolated to extracellular binding-sites; V538 in the linker between S3 and S4, T633 in the pore helix and E648A adjacent to the selectivity filter (Jordt et al., 2000; Ryu et al., 2007). Another

83 possibility for the decrease in potency observed between acid- and capsaicin-evoked

TRPV1 QX-314 inhibition is a decrease in QX-314 permeation through the proton- activated TRPV1 pore. Prolonged capsaicin application has been shown to produce time- dependent increases in large cation permeation (Chung et al., 2008). Extracellular co- application of capsaicin with the membrane impermeable fluorescent dye, YO-PRO1, time-dependently increases the extracellular uptake rate of the dye through the TRPV1 channel, presumably via vanilloid induced pore dilation (Chung et al., 2008). This has been supported by the observed extracellular influx of other large organic cations, such as

NMDG, FM1-43 (dye), and aminoglycoside antibiotics through the capsaicin-activated

TRPV1 pore (Chung et al., 2008; Meyers et al., 2003; Myrdal and Steyger, 2005).

To more directly evaluate the intrinsic properties of LAs on multimodal TRPV1 inhibition, parent compound of QX-314 was tested in this chapter, lidocaine. As an amphipathic tertiary amine base with a pKa of ~7.9, lidocaine is strongly regulated by the manipulation of external pH (Butterworth and Strichartz, 1990). At physiological conditions (pH 7.4), lidocaine exists in a dynamic equilibrium between neutral and ionized molecular conformations [Figure 19D]. It is generally accepted that the ionized form of LAs is the primary mediator of Nav channel inhibition leading to nerve block

(Butterworth and Strichartz, 1990). In the present experiments, micromolar lidocaine inhibited capsaicin-activated TRPV1 inward currents at pH 7.4, albeit incompletely, with similar potency as QX-314 [Figure 19A & C]. Corresponding to the fraction of neutral

LA molecules at pH 7.4, approximately ~30% of the peak current remained following administration of the highest concentration of lidocaine (300 µM).

Co-application of lidocaine with pH 5.5 had a two-fold effect. Firstly, it helped to identify whether amphipathic LAs are able to inhibit TRPV1 inward cationic currents under conditions of severe extracellular acidification in Xenopus laevis oocytes. Secondly, by shifting the equilibrium of this mildly basic tertiary amino-amide LA to the primarily ionized fraction, it hinted towards the molecular determinants of TRPV1-mediated extracellular inhibition. In the presence of proton-evoked inward cationic currents, the results show that the potency of lidocaine block was relatively unaltered. The main distinction between capsaicin- and proton-evoked TRPV1 inhibition was a significant increase in the efficacy of lidocaine to reduce TRPV1 inward cationic currents [Figure

19E]. These findings hint towards the direct role of protonated LAs in multimodal

TRPV1 channel inhibition.

Despite the broad conformational changes that lead to gating of TRPV1, proton- and vanilloid-induced activation are separated at multiple levels following agonist binding. Primarily acidic (glutamic acid) residues in the extracellular S5-S6 loops mediate proton-induced activation, while vanilloid activation occurs mainly through aromatic and nucleophilic intracellular binding sites between the S2-S3 linker (Gavva et al., 2004; Jordt et al., 2000). The interface between the S3 and S4 domains of the TRPV1 channel is thought to mediate the general coupling mechanism for proton- and capsaicin- induced pore opening (Ryu et al., 2007). Although all TRPV1 antagonists block capsaicin activation, not all of these compounds block proton activation (Gavva et al., 2005).

TRPV1 antagonists that have been found to block all modes of activation show pronounced efficacy in reducing inflammatory pain in multi-species models (Gavva et al.,

2005). For example, in marked contrast to QX-314 and lidocaine inhibition, a >10 fold

85 decrease in potency was observed for inhibition of capsaicin-evoked currents by the TRP pore blocker, ruthenium red, at pH 6.4 compared with physiological pH 7.4 (Garcia-

Martinez et al., 2000). Nonetheless, additional mechanistic insights into the pore properties of TRPV1 are needed in order to identify the structural determinants of LA inhibition. Due to the similarities between TRPV1 and the Shaker K+ channel, extracellular residues located between the S5 and S6 segments, near the selectivity filter threshold, may be directly involved in LA-TRPV1 coupling. Under these circumstances, a putative binding site for LAs may be found in aromatic amino-acids lining the extracellular pore of TRPV1 (Ahern, 2006; Lopez-Barneo et al., 1993).

Chapter 4: Extracellular quaternary ammonium blockade of transient receptor potential vanilloid subtype 1 channels expressed in Xenopus laevis oocytes

INTRODUCTION

As summarized in chapter 2, lidocaine and QX-314 exerted biphasic effects on

TRPV1 channels expressed in Xenopus laevis oocytes, inhibiting capsaicin-evoked

TRPV1 currents at lower (micromolar) concentrations and activating TRPV1 channels at higher (millimolar) concentrations. Whereas the mechanisms are unknown, studies into the permeation and gating pathway of TRPV1 have found - similar to results obtained with Kv channels - that permanently charged quaternary ammonium compounds are able to potently block TRPV1 intracellularly, thus delimiting similarities amongst these seemingly disparate family members (Jara-Oseguera et al., 2008; Oseguera et al., 2007).

However, it remains unclear if TRPV1 is also susceptible to extracellular inhibition by quaternary ammonium compounds. LA molecules, which as previously alluded to have both amphipathic and amphiphilic properties and maintain a dynamic equilibrium between charged and neutral forms, generally contain a tritratable amine group linked to a hydrophobic aromatic moiety via an amine or ester linkage. It has been shown previously in Nav channels that these different chemical moieties have distinct pharmacological effects (Butterworth and Strichartz, 1990; Liu et al., 2003).

On the basis of these findings, the goal of experiments in this chapter was to determine if the observed inhibition of TRPV1 channels represents a general characteristic of LAs, and, if so, to define the structure-activity relationships and molecular determinants of this blockade. The results provide evidence that TRPV1, expressed in Xenopus laevis oocytes, is potently inhibited by extracellular quaternary

87 ammonium compounds. Furthermore, the findings demonstrate that it is the charged (or chargeable) amine of LAs that is critical for the inhibition of TRPV1. Finally, in an attempt to delineate a possible binding site for quaternary ammonium compounds, it is shown that point mutations near the putative TRPV1 pore region reduce these compounds’ inhibitory efficacy and affinity for these channels.

RESULTS

Lidocaine inhibition of TRPV1 channels in Xenopus laevis oocytes. To define the molecular determinants of LA blockade observed in chapter 2, rat TRPV1 channels were expressed in Xenopus laevis oocytes. An approximate EC50 concentration of capsaicin

(15 M) was co-applied with increasing concentrations of lidocaine, flanked by two control applications of capsaicin alone. As shown in Fig. 1A, addition of 100 nM lidocaine produced a ~20% inhibition in inward current. Increasing the lidocaine concentration to 10 M produced a ~70% inhibition of capsaicin-elicited currents [Figure

20B]. The resulting IC50 for lidocaine inhibition of TRPV1 was 82 ± 65 nM, with a Hill coefficient of 0.7 ± 0.5. To characterize the voltage-dependence of lidocaine inhibition, blockade was subsequently investigated at a more depolarized holding potential. The Erev for the system, determined from current-voltage plots, was −20 mV (data not shown). To test the voltage-dependence of lidocaine inhibition in oocytes, blockade was examined at

−25mV, the closest membrane potential where inward currents could still be observed.

Compared with the effects at −60 mV, we observed a significant decrease in the efficacy of lidocaine current inhibition when oocytes were held at −25 mV, consistent with a charged pore blocking mechanism [Figure 20C & D]. Complete inhibition of TRPV1 activation was not achieved, even at the highest concentrations of blockers that were employed in this study; and a residual inward current remained even at high (100 M) lidocaine concentrations. Our data thus clearly show that extracellular lidocaine produces concentration-dependent and reversible inhibition of capsaicin-induced inward currents in

Xenopus laevis oocytes under our new experimental conditions [cf. General Materials and

Methods, Electrophysiology].

Figure 20. Lidocaine inhibits capsaicin-evoked TRPV1 currents in Xenopus laevis oocytes in a concentration-dependent and reversible manner. The co-application of 15 μM capsaicin with different concentrations of lidocaine was flanked by two control applications of 15 µM capsaicin to control for (de)sensitization. Only one drug application was performed per oocyte and 2 min washout intervals were used between all applications. Capsaicin-evoked currents were not observed in uninjected oocytes (data not shown). (A)/(B) Representative capsaicin-evoked current traces before and after the co-application of capsaicin with 100 nM (A) or 10 M (B) lidocaine at a holding potential of −60 mV. Calibration bars: horizontal = 1 min; vertical = 0.1 μA. (C) Concentration-response data for the inhibition of capsaicin-evoked inward currents by lidocaine at −60 mV (black, n = 4–5) and −25 mV (grey, n = 4). Data points at each concentration represent mean ± SEM of remaining current, normalized to the mean peak current. Data was fit with the Hill equation (here and in all following fits: Hill four- parameter, non-linear regression). (D) Bar graph comparing the inhibition of 100 µM lidocaine observed at a holding potential of −60 mV (black, 24 ± 4 %) vs. −25 mV (grey, 50 ± 8 %) represented as % current remaining. Efficacy of inhibition at −60 mV vs. −25 mV displays significant voltage-dependence (P = 0.02). The inset depicts the chemical structure of lidocaine.

The neutral LA benzocaine does not strongly inhibit TRPV1 channels. To test the role of cationic charge in the inhibition of TRPV1 by lidocaine and QX-314, we investigated the blocking abilities of the neutral LA, benzocaine as less than 1% of benzocaine molecules are charged under our experimental conditions (pH 7.4). As shown in Figure

20A, administration of 10 μM benzocaine produced no effects on TRPV1 currents, and even concentrations of up to 100 μM resulted in only a modest inhibition of around 25%

[Figure 21B]. Plotting the effect of a wide range of benzocaine concentrations on TRPV1 currents demonstrates that benzocaine does not significantly inhibit capsaicin-evoked

TRPV1 currents in the range of concentrations for lidocaine or QX-314 that would produce near complete inhibition. For example, a comparison of the effects produced by the lowest (1 nM) and highest (100 μM) benzocaine concentrations studied shows no difference p-value = 0.13). Moreover, application of very high benzocaine concentrations

(> 1 mM) did not inhibit TRPV1 channels further but a small degree of channel potentiation was observed when co-applied with capsaicin (data not shown).

Figure 21. Benzocaine does not strongly inhibit capsaicin-evoked TRPV1 currents in Xenopus laevis oocytes. (A)/(B) Representative capsaicin-evoked current traces before and after the co-application of capsaicin with 10 M (A) or 100 M (B) benzocaine. Calibration bars: horizontal = 1 min; vertical = 0.1 μA. (C) Data points at each concentration represent mean ± SEM of remaining current, normalized to the mean peak current (n = 4–5). Note that data means are connected by straight lines. The inset depicts the chemical structure of benzocaine.

Quaternary ammonium compounds potently inhibit capsaicin-induced TRPV1 currents in oocytes. We previously showed that the quaternary lidocaine derivative, QX-314, inhibits capsaicin activated TRPV1 channels (Rivera-Acevedo et al., 2011). Benzocaine, on the other hand, which features a lipophilic aromatic moiety that is shared with both lidocaine and QX-314 but lacks a nitrogen that can be charged under physiological

92 conditions, did not inhibit TRPV1 currents (see above). We next sought to determine if it is the charged ammonium moiety of lidocaine and QX-314 which mediates inhibition of

TRPV1. Tetraethylammonium (TEA) represents an ideal candidate to test this hypothesis as it mimics the charged amine tail group of QX-314 but does not contain the amino- amide linker or the lipophilic head. When co-applied with capsaicin, we found that TEA potently inhibits TRPV1, even at nanomolar concentrations as co-application of 100 nM

TEA with capsaicin reduced peak current amplitudes by more than 50% and TEA concentrations of 10 M almost completely inhibited capsaicin-elicited TRPV1 currents

[Figure 22A, upper and middle panel]. The IC50 for TEA inhibition of TRPV1 was 41 ±

13 nM, with a Hill coefficient of 0.9 ± 0.1 [Figure 22A, lower panel]. We next tested the smallest of the quaternary ammonium salts, tetramethylammonium (TMA). As shown in

Figure 22B, TMA also robustly inhibited capsaicin-induced TRPV1 currents in oocytes, although with lower efficacy and potency than TEA: at 100 nM and 10 M, TMA produced approximately 40% and 70% inhibition of TRPV1 currents, respectively

[Figure 22B, upper and middle panel]. The corresponding IC50 for TMA inhibition of

TRPV1 was 104 ± 60 nM, with a Hill coefficient of 0.5 ± 0.1 [Figure 22B, lower panel].

TRPV1 inhibition by quaternary ammonium compounds is voltage-dependent. Similar to lidocaine, we characterized the voltage-dependence of TEA and TMA inhibition by investigating blockade at a holding potential of −25 mV. Compared with the effects at

−60 mV, we observed a marked decrease in both TEA and TMA current inhibition

[Figure 22C & D]. Administration of 100 nM TEA and TMA produced little to no reduction in currents elicited by capsaicin, while at 100 M approximately 30% current

93 remained present with TEA, and approximately 60% with TMA [Figure 22C & D].

Compared with the effects at −60 mV, both QA’s produced an inhibition-response curve that was shifted to the right and had a decreased slope at −25 mV. The IC50 for TEA increased to 1.5 ± 0.8 μM; with a Hill coefficient of 0.4 ± 0.1 [Figure 22A]. For TMA, the IC50 increased to >1 mM [Figure 22B]. Attempts to more thoroughly characterize this voltage-dependence where not feasible given the contaminating effects of the inherent weak voltage-dependence of gating and strong desensitization at depolarized potentials.

Nonetheless, the data indicate that TRPV1 inhibition by quaternary ammoniums is more effective at hyperpolarized potentials, consistent with the notion that TEA and TMA enter the electric field during blockade, possibly by occupying a binding site in the selectivity filter.

Figure 22. The quaternary ammonium compounds, tetraethylammonium and tetramethylammonium, potently inhibit capsaicin-evoked TRPV1 currents.

Figure 22. The quaternary ammonium compounds, tetraethylammonium and tetramethylammonium, potently inhibit capsaicin-evoked TRPV1 currents. (A)/(B) Representative capsaicin-evoked current traces before and after the co-application of capsaicin with 100 nM (upper panels) or 10 M (middle panels) tetraethylammonium (TEA; A) or tetramethylammonium (TMA; B). Calibration bars: horizontal = 1 min; vertical = 0.1 μA. Lower panels show concentration-response data for the inhibition of capsaicin-evoked inward currents by TEA (A) or TMA (B) at −60 mV (black, TEA n = 10–20, TMA n = 5–11) and −25 mV (grey, TEA n = 4, TMA n = 4) . Data points at each concentration represent mean ± SEM of remaining current, normalized to the mean peak current. (C) Bar graph comparing 100 µM TEA inhibition observed at a holding potential of −60 mV (black, 17 ± 3 %) vs. −25 mV (grey, 29 ± 4 %). (D) Values of 100 µM TMA observed at a holding potential of −60 mV (black, 30 ± 4 %) vs. −25 mV (grey, 55 ± 3 %). Both TEA and TMA display significant voltage-dependence of block (P = 0.03 and 0.002, respectively). Insets depict the chemical structures of TEA and TMA.

Mutations near the selectivity filter weaken TEA inhibition. As TRPV1 shares sequence homology with Kv channels from the Shaker family at the putative selectivity filter [Figure 24A] (Moiseenkova-Bell et al., 2008; Owsianik et al., 2006), we mutated amino acids close to the putative selectivity filter of TRPV1 to investigate their possible role in TEA inhibition. The F649A and E648A mutations resulted in channels with detectable expression, normal gating and sensitivity to capsaicin [Figure 23], however the

L647A mutation was non-functional. In terms of blockade, we found that compared to the robust inhibition by 10 M TEA observed with WT TRPV1 [Figure 21A], the mutant channels F649A [Figure 24B] and E648A [Figure 24C] displayed a significant reduction in inhibition levels (P ≤ 0.001, data not shown). These effects were consistent over a wide range of TEA concentrations [Figure 24E & F], suggesting that both E648 and F649 could be involved in TEA inhibition of TRPV1. TEA block was also demonstrated to be voltage-dependent for the TRPV1 mutants, although a significant decrease between −60 mV vs. −25 mV was only detected in the F649A mutant [Figure 24D].

Figure 23. Capsaicin activation of TRPV1 channel mutants in Xenopus laevis oocytes. Concentration-response curves for capsaicin-evoked F649A and E648A TRPV1 currents in oocytes. Increasing capsaicin concentrations were applied to each cell, until saturation was observed. TRPV1 F648A channel (EC50 = 14 ± 0.2 µM; n = 12) and E648A (EC50 = 41 ± 20 µM; n = 6). The data were fitted using the Hill equation; error bars denote SEM. Cells were held at −60 mV.

Figure 24. Mutation of residues near the selectivity filter ameliorates TRPV1 inhibition by tetraethylammonium. (A) Sequence comparison between rTRPV1 and the Shaker voltage-gated K+ channel. Site-directed mutagenesis was carried out on amino acid residues indicated by black arrows. (B) & (C) Representative capsaicin-evoked current traces before and after the co-application of capsaicin with 10 M tetraethylammonium (TEA) for F649A (B) and E648A (C; details, see body text). Calibration bars: horizontal = 1 min; vertical = 0.1 μA).

Figure 24. Mutation of residues near the selectivity filter ameliorates TRPV1 inhibition by tetraethylammonium. (D) Bar graph comparing 100 µM TEA inhibition observed for the TRPV1 mutants at a holding potential of −60 mV (black, F649A 35 ± 3 %; E648A 51 ± 11 %) vs. −25 mV (grey, F649A 69 ± 5 %; E648A 78 ± 8 %). In the presence of TEA the F649A mutant exhibits significant voltage dependent relief of inhibition (P = 0.001), but not the E648A mutant. (E) Concentration-response data for the inhibition of capsaicin-evoked inward currents by TEA for F649A at −60 mV (black, n = 4–6) and −25 mV (grey, n = 4–5) and (F) E648A at −60 mV (black, n = 4–7) and −25 mV (grey, n = 4) black dashed regression represents WT TEA inhibition. A significant decrease in inhibition was observed between WT vs. both F649A and E648A mutants at −60 mV (P ≤ 0.001, data not shown). Data points at each concentration represent mean ± SEM of remaining current, normalized to the mean peak current.

DISCUSSION

The results in this chapter have demonstrated that TRPV1 channels, expressed in

Xenopus laevis oocytes, are potently inhibited by extracellular application of LAs and quaternary ammonium compounds. These findings are in good agreement with an earlier observation that capsaicin-evoked TRPV1 inward currents can be inhibited by extracellular lidocaine in mammalian HEK293t cells, although with significantly lower potency (Leffler et al., 2008). Together with the findings from chapter 2 that the extracellular administration of a quaternary lidocaine derivative, QX-314, inhibits

TRPV1-mediated currents in Xenopus laevis oocytes, these results suggest that it is a general trait of titratable LAs to inhibit TRPV1-mediated inward currents. The experiments in this chapter sought to test which specific structural aspects of LA molecules mediate extracellular TRPV1 inhibition in Xenopus laevis oocytes.

Specifically, the studies assessed the individual contributions of the ubiquitous lipophilic aromatic head and linker regions found in all LA compounds and that of the often charged (or chargeable) amine tail groups. A first hint towards a possible contribution of the latter came from the observation that even at the highest lidocaine concentration employed (100 μM), approximately 30% of the current remained [Figure 20C]. Indeed, the pharmacological efficacy of lidocaine, a weak amphipathic base with a pKa of 7.9, and for that matter LAs in general, is linked to pH (Butterworth and Strichartz, 1990).

The protonatable amine group of lidocaine, attached to a lipophilic head by an amide linker, exists in a dynamic equilibrium between charged and uncharged states and it is generally accepted that it is the charged form of LAs that is pharmacologically active, e.g., responsible for blocking Nav channels (Butterworth and Strichartz, 1990). It is thus

100 interesting to note that the current level remaining (around 30%) at high lidocaine concentrations is similar to the approximate 30% proportion of uncharged lidocaine molecules present at pH 7.4. If the chargeable amine of lidocaine mediates the potent inhibition of TRPV1, then LAs with a similar overall structure, but lacking a charged or chargeable amine should not produce strong inhibition of TRPV1, an experimental possibility supported by the clear lack of benzocaine-mediated inhibition of TRPV1 activation [Figure 21]. To more directly assess the role of charge in TRPV1 inhibition, experiments here characterized extracellular block by the quaternary permanently charged amines, TEA and TMA, which have been instrumental in the structure-function study of K+ channel pore regions. More recently, these compounds have been successfully employed to help define the structure and basic biophysical properties of the intracellular TRPV1 channel pore (Jara-Oseguera et al., 2008; Oseguera et al., 2007). In the current study, similar to both lidocaine and QX-314, extracellular application of TEA produced a reduction in capsaicin-evoked inward currents, albeit with substantially higher affinity [Figure 22A]. Of note, even a saturating (1 µM) concentration of TEA did not result in complete TRPV1 inhibition, as we generally observed 10–15% of residual current. This effect could potentially be attributed to TEA behaving like a partially permeant ion, as observed for other voltage-dependent inhibitors (Huang, 2001; Huang et al., 2000). Considering the size of TEA (~8 Å) and the estimated TRPV1 pore diameter of 7 Å (Owsianik et al., 2006), this seems entirely possible. Consistent with this hypothesis, the smaller TMA (~6 Å) (Briend, 1993) displayed significantly lower efficacy than TEA [Figure 22B]: The TMA-mediated inhibition also saturated near 1 μM but more than 35% of the current remained. Moreover, it has previously been shown that

101 large organic cations can permeate TRPV1 channels, albeit relatively slowly (Chung et al., 2008). Indeed, this mechanism has been suggested to take place in explanted DRG cultures exposed to prolonged applications of QX-314 in the presence of capsaicin

(Binshtok et al., 2007). Collectively, the data available to date indicate that QAs may act as permeant blockers of TRP channels. Consistent with this possibility, TEA and TMA displayed strong voltage dependent relief of inhibition at −25 mV compared to −60 mV, similar to the extracellular open channel block of voltage-gated K+ channels and intracellular pore block in TRPV1 channels by other QAs (Jara-Oseguera et al., 2008).

On the basis of the predicted structural homology between TRPV1 and Shaker- like K+ channels (Latorre et al., 2009), investigations were then carried out here into the potential molecular binding sites in the area surrounding the selectivity filter. It has been established that TEA produces extracellular block through the actions of a specific residue (T449) near the Shaker voltage-gated K+ channel pore (Heginbotham and

MacKinnon, 1992; MacKinnon and Yellen, 1990). Phenylalanine or tyrosine substitution at this site supports TEA block through cation-π interactions (noncovalent bonds between positive monopoles and electron-rich quadropoles) with the negative electrostatic surface potential at the face of the aromatic ring (mediated through the π-electron cloud) (Ahern,

2006; Heginbotham and MacKinnon, 1992). Given this precedent, it was tested if F649, a phenylalanine side chain that aligns with the residue adjacent to T449 in Shaker, plays a similar role in TEA inhibition of TRPV1. Mutation of this residue to a nonaromatic alanine dramatically altered TEA inhibition, but did not abolish it completely (wild-type:

14% vs. F649A: 35% of current remaining at 300 µM TEA). However, due to the TRPV1 channels apparent intolerance at the F649 site of the in vivo nonsense suppression method,

102 required for the expression of fluorinated phenylalanine residues, it was not possible to directly test for a possible cation-π interaction between F649 and TEA (data not shown), which has been demonstrated to occur between lidocaine and a conserved phenylalanine residue in Nav channels (Ahern et al., 2008; Pless et al., 2011). Next, attention was turned to E648, the residue that aligns directly with T449 in Shaker, and has also been shown to be critical for proton-evoked TRP channel activation without affecting heat or capsaicin sensitivity (Jordt et al., 2000). Mutation of E648 to alanine ameliorated TEA inhibition, suggesting that this negative charge interacts electrostatically with the positively charged amine found in TEA, TMA, and LAs. Importantly, both E648A and

F649A channels displayed activation properties and reversal potentials similar to WT channels (data not shown), suggesting that these substitutions were tolerated and did not result in general dysfunction of the selectivity filter region. Taken together, these results indicate that both the aromatic F649 and acidic E648 contribute to inhibition of TRPV1 by compounds with charged (or chargeable) amines.

The choice of the Xenopus oocyte expression system was made because it is highly amenable for high-throughput pharmacologic characterization of drug-receptor activity, and for many years has been successfully used to investigate the molecular properties of a diverse range of ion channels. It is important to note that although general

TRPV1 channel activity is conserved throughout various expression systems, differences in the constitution of membrane lipids, intracellular ions, second messenger molecules and experimental conditions do appear to affect the efficacy of agonist and antagonist activity of TRP channels (Chung et al., 2008; Lukacs et al., 2007; Novakova-Tousova et al., 2007). The polymodal nature of TRPV1 channels makes them particularly sensitive to

103 regulation by a wide range of endogenous lipids such as PIP2, PKC, PKA (cAMP- dependent protein kinase), PLC, DAG, arachidonic acid metabolites, and PUFAs (poly unsaturated fatty acids) (Bhave et al., 2002; Matta et al., 2007; Pingle et al., 2007;

Prescott and Julius, 2003; Woo et al., 2008), and changes in the presence and concentrations of these molecules have been shown to alter TRP channels activity

(Lukacs et al., 2007). Thus, although the specific drug-receptor affinity may change depending on the expression system (Ahern et al., 2008; Lev et al., 2012), the observation of QA blockade likely is universal, regardless of the cell system used. This is further supported by the findings that LAs and QAs can block TRPV1 in mammalian cells (Jara-

Oseguera et al., 2008; Leffler et al., 2008; Oseguera et al., 2007).

In summary, the results in the present chapter indicate that quaternary amine moieties underlie the potent LA-mediated TRPV1 inhibition. Mutations of residues near the TRPV1 pore significantly alter the potency of QA-mediated inhibition, providing a starting point to further elucidate the extracellular binding site for these molecules.

Ultimately, the present results introduce a basic molecular model for the observed, but so far unexplained, phenomenon of LA-mediated TRPV1 inhibition in vitro and serve as a basis for understanding the complex LA-mediated effects on TRPV1 channels.

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Chapter 5: Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes

INTRODUCTION

Ionotropic membrane proteins are assumed to possess static structural features that regulate channel behavior. The general consensus has been that mechanisms underlying ionic selectivity, ligand binding, and channel gating are controlled by rigid changes in molecular conformation. However, in some ligand-gated ion channels this rigid mechanistic view may not be applicable (Chung et al., 2008; Fujiwara and Kubo,

2004; Legendre et al., 2002; Taleb and Betz, 1994). First characterized in glycine receptors, Taleb and Betz observed pharmacological changes to glycine activation in these channels at high expression levels (Taleb and Betz, 1994). Notably, they have been shown to exhibit dynamic agonist and antagonist affinity, dependent upon channel expression in Xenopus laevis oocytes and heterologous mammalian expression systems

(Legendre et al., 2002; Taleb and Betz, 1994). In addition, changes in glycine receptor gating kinetics were documented as channel desensitization increased with receptor expression in vitro (Legendre et al., 2002). Similarly, the ATP-gated P2X2 receptor demonstrates both time- and expression-dependent cationic selectivity during agonist exposure (Fujiwara and Kubo, 2004). More recently, studies testing the ionic selectivity of TRP isoforms expressed in sensory neurons TRPV5, TRPA1, and TRPV1 indicate that these channels undergo time- and agonist-dependent pore dilation indicating the presence of dynamic structural features (Chen et al., 2009; Chung et al., 2008; Yeh et al., 2005).

These unique observations have only been detected in channels predominantly expressed

105 throughout the CNS and sensory ganglia. The mechanisms underlying the biophysical and pharmacological changes due to expression levels in vitro have not been identified.

It was discovered in the course of the work for this thesis that in Xenopus laevis oocytes the TRPV1 channel demonstrates expression-dependent inhibition by the quaternary lidocaine derivative, QX-314. The TRPV1 channel has been shown to permit the influx of large molecular cations due to a relatively large pore diameter of at least >

6.8 Å (Baez-Nieto et al., 2011). In particular, the ionic selectivity for monovalent, divalent, and large cationic molecules has been shown to change with time, agonist type, and drug concentration to approximately 12.3 Å (Baez-Nieto et al., 2011; Chung et al.,

2008). Previous evidence in heterologous expression systems indicates that changes in

TRPV1 channel conduction are strongly correlated to functional channel density determined as the magnitude of peak outward currents without alteration in single channel conductance (Chung et al., 2008). Like the mechanisms that underlie pore- dilation in TRP channels however, the relationship between TRPV1 receptor density and

LA binding efficacy still remains unclear. Here, the two-electrode voltage clamp technique was used with optimized ligand-binding assays to characterize how the functional pharmacology of TRPV1 channels changes with increasing receptor expression levels in the Xenopus laevis oocyte system.

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RESULTS

QX-314 exhibits expression-dependent block of TRPV1 channels. In order to determine whether the degree of current inhibition by QX-314 is dependent on TRPV1 channel expression levels, oocytes injected with TRPV1 mRNA were incubated for varying lengths of time during a 36 h window. Expression levels were calculated for each oocyte by measuring baseline peak inward currents following application of an ~EC50 capsaicin concentration (15 µM) at the beginning of each experiment. Once baseline expression was established we performed a single co-application of capsaicin with (100 pM, 1 nM,

100 nM, 1 µM, 10 µM, or 100 µM) QX-314. A minimum (0.1 µA) and maximum (15

µA) current cutoff was set for oocyte expression in all the experiments. Variable non- selective inward current inhibition in TRPV1 channels was observed for QX-314 (1 µM and 10 µM) between low [Figure 25A & C, top] and high [Figure 25A & C, bottom] levels of TRPV1 expression. Linear regressions performed on the data demonstrated a strong positive correlation between the % current remaining for QX-314 inhibition and baseline TRPV1 activation [Figure 25B & C]. The coefficients of correlation (R-value) for 1 and 10 µM QX-314 were 0.8 and 0.7, respectively. A decrease in current inhibition via a reciprocal increase in TRPV1 expression occurred in all QX-314 concentrations tested (data not shown), suggesting this to be a general effect of QX-314 in oocytes. The least amount of inhibitory variability was detected between 0.1–3 µA baseline peak current amplitude.

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Figure 25. QX-314 inhibition dependent on TRPV1 expression levels in Xenopus oocytes. Co-application of 15 μM capsaicin with QX-314 was flanked by two applications of 15 µM capsaicin to control for (de)sensitization. Only one 10 s drug application was performed per oocyte with 2 min washout intervals between all applications. (A/C) Representative capsaicin-evoked current traces observed before and after the co-application of (A) 1 µM and (C) 10 µM QX-314 in oocytes expressing low (top) or high (bottom) levels of TRPV1. Calibration bars: (top) horizontal = 1 min, vertical 0.3 µA; (bottom) horizontal = 1 min, vertical 3 µA. (B/D) A strong positive correlation is seen between capsaicin-evoked TRPV1 inhibition following co-application of (B) 1 µM and (D) 10 µM QX-314, normalized to baseline TRPV1 peak current amplitudes (µA). Recordings were performed on oocytes demonstrating > 0.1 µA baseline inward currents (1 µM n = 32; 10 µM n = 38). A holding potential of −60 mV was employed during all experiments.

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TEA does not exhibit expression-dependent inhibition of TRPV1 channels.

Experiments performed under the same conditions as QX-314 demonstrated that TEA does not display expression-dependent inhibition of TRPV1 channels in Xenopus laevis oocytes. Oocytes were once again injected with TRPV1 mRNA and incubated for varying lengths of time during a 36 h period. Baseline TRPV1 expression was normalized to inward cationic currents elicited by 15 µM capsaicin at the beginning of each experiment, followed by single co-application of TEA (100 pM–100 µM). Marked changes in the amount of current inhibition produced by TEA (1 µM and 10 µM) between low [Figure 26A & C, top] and high [Figure 26A & C, bottom] TRPV1 expression levels in oocytes were not readily apparent. The results showed little to no correlation between the % current remaining for TEA inhibition and baseline TRPV1 activation [Figure 26B & C]. The coefficient of correlation (R) for 1 and 10 µM QX-314 was 0.1 and 0.2, respectively. Findings were similar for all concentrations of TEA employed (data not shown).

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Figure 26. TEA inhibition of TRPV1 channels does not demonstrate expression- dependence in Xenopus laevis oocytes. (A/C) Representative capsaicin-evoked current traces observed before and after the co-application of (A) 1 µM and (C) 10 µM TEA in oocytes expressing low (top) or high (bottom) levels of TRPV1. Calibration bars: (top) horizontal = 1 min, vertical 0.3 µA; (bottom) horizontal = 1 min, vertical 3 µA. (B/D) No significant correlation was detected between TRPV1 baseline expression levels for capsaicin-evoked activation and TEA inhibition following co-application of (B) 1 µM and (D) 10 µM TEA. Recordings were performed in oocytes demonstrating > 0.1 µA baseline inward currents (1 µA, n = 46; 10 µA, n = 51).

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Both QX-314 and TEA block of TRPV1demonstrate voltage dependence. The body of experimental findings obtained gave rise to the prediction that TEA and QX-314 produce

TRPV1 inhibition through open channel pore block. Open channel pore block by charged molecules often displays voltage dependence as the blocking molecules bind at a site that interacts with the electric field. If the binding site for a cationic blocker lies within the membrane electric field, the affinity for the binding site will be affected by the electrostatic potential from an applied voltage. The affinity for a positively charged blocker situated on the extracellular side of the field will be reduced by increasing positive intracellular voltages. In order to test this hypothesis, voltage-dependent experiments were performed to calculate the fractional distance traversed by these compounds through the channels’ electric field during open channel block. Fractional block (Fb) was determined comparing the normalized activated currents (1 - IQA/Icontrol) against the test potential (mV) [Figure 27]. The fraction of the electric field through which the drug passes (δ) was calculated using the Woodhull equation for an impermeable blocker:

Where z (valence of the molecule), F (Faraday constant: 96,485.3 C/mol), R (gas constant 8.3 J/mol K), and T (temperature in Kelvin), Kd (binding constant at 0 mV), [D]

(drug concentration), and V (membrane voltage) (Bett, 2002; Wang et al., 2003). These results suggest that QX-314 and TEA senses at least 39% of the transmembrane electric field of TRPV1 channels from an extracellular site [Figure 27A & B]. TEA demonstrates similar blocking properties by crossing approximately 38% of the transmembrane electric

111 field [Figure 27B] corresponding with a value higher than the reported δ of TEA block in the Shaker K+ channel (Ahern, 2006; Heginbotham and MacKinnon, 1992).

Figure 27. QX-314 and TEA mediate inhibition of TRPV1 channels through open channel pore block. In order to control for variations in expression-dependent inhibition, we only tested oocytes exhibiting < 3 µA of baseline current activation. Voltage ramp experiments were performed from −40 to +80 mV using capsaicin control (15 µM), then on the same cell co-applied with QX-314 or TEA (100 nM), and used to calculate δ, representing the fraction of the transmembrane field traversed by these cationic molecules. (A) For QX-314, δ was 0.39 ± 0.02 (n = 4) (B) For TEA, δ was 0.38 ± 0.03 (n = 5). Data points represent mean ± SEM.

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DISCUSSION

The work summarized in this chapter strongly suggests that potency of TRPV1 inhibition by the experimental quaternary LA, QX-314, is dependent on TRPV1 expression levels in Xenopus laevis oocytes. We characterized the inhibitory effects of

QX-314 on vanilloid-activated TRPV1 channels expressing 100 nA–15 µA peak inward currents. In these experiments, oocytes were injected with increasing volumes of TRPV1 mRNA (10–50 nl of TRPV1 mRNA [500 ng/µl]) and incubated for progressively longer time periods (12–36 h) post injection. The effect was a dramatic enhancement of inward currents with larger mRNA volumes and longer incubation times indicating an increase in

TRPV1 membrane expression. Oocytes displaying higher TRPV1 expression levels were less sensitive to QX-314 inhibition, displaying both a weaker potency and efficacy, when co-applied with capsaicin [Figure 25].

The idea for expression-dependent pharmacology may seem surprising, but relevant observations from other ion channels exist. Previous studies have shown that the biophysical and pharmacological properties of membrane proteins can change at high density. For example, the gating and conductance properties of nicotinic acetylcholine receptors and L-type Ca2+ channels become altered at high expression levels in lipid bilayers (Hymel et al., 1988; Schindler et al., 1984). It may be a property of membrane proteins to interact allosterically at high densities (Taleb and Betz, 1994). In the case of

TRPV1 channels, changes in receptor density could affect antagonist binding in multiple ways. First, rising channel expression could influence the balance of downstream secondary modulators of TRPV1 channel activity, such as PIP2 binding and PKA- or

PKC-mediated phosphorylation (Bhave et al., 2003; Bhave et al., 2002; Brauchi et al.,

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2007). PIP2 coupling has been linked to increased TRPV1 channel desensitization

(Prescott and Julius, 2003), while phosphorylation by PKC and PKA has been associated with channel sensitization during agonist stimulation (Bhave et al., 2003; Bhave et al.,

2002). Dysregulation or depletion of all three second messenger molecules alters agonist and antagonist behavior in heterologous expression systems. However, capsaicin exposure during the present experiments was kept to a minimum in order to prevent these non-specific effects, and capsaicin controls do not display changes in the kinetics of

TRPV1 gating regardless of channel expression. A second possibility may be that TRPV1 channels are not evenly expressed throughout the oocyte membrane, but cluster at specific locations with high receptor density, where intermolecular interactions could occur (Fujiwara and Kubo, 2004; Meier et al., 2000). The analysis of the single channel properties of P2X2 receptors in excised Xenopus oocyte membrane patches containing one or more channels indicates that open channel lifetimes are significantly longer for patches with multiple receptors (Ding and Sachs, 2002). The data suggests that increased receptor expression promotes multiple channel contacts increasing positive cooperativity between each neighboring receptor, leading to a reciprocal reduction in pore closing kinetics (Ding and Sachs, 2002). This implies that the properties of a single ligand-gated channel may change depending on whether they are found clustered or homogenously distributed throughout the membrane (Ding and Sachs, 2002).

Unlike QX-314, the quaternary ammonium molecule, TEA, did not demonstrate expression-dependent changes in TRPV1 inhibition [Figure 26]. Various possibilities may underlie this effect. Firstly, the pore properties of TRPV1 have been shown to undergo dynamic time- and concentration-dependent widening in the presence of

114 vanilloids (Chung et al., 2008). Perhaps, similar to P2X2 receptors, the pore properties also change depending on the density of open channels. Although we did not observe significant expression dependent changes in the agonist (capsaicin) sensitivity with

TRPV1, the conformational arrangements that ion channels undergo when they transition between conducting and non-conducting states can strongly influence channel affinity and efficacy, as drug binding sites can be occluded during conformational and environmental changes (Wang et al., 2003). Secondly, the inherent properties of the QX-

314 molecule must factor into the management of TRPV1 expression-dependent inhibition. The large hydrophobic aromatic head region of QX-314 may interact with structures in the TRPV1 channel to produce these effects unlike the quaternary ammonium molecule, TEA, lacking both the amide linker and hydrophobic aromatic head. There exists no conclusive evidence to exclude or prove these possibilities.

Another goal of the present studies was to delineate whether QX-314- and TEA- mediated TRPV1 inhibition occurs through similar mechanisms. These blockers have a net positive charge, suggesting that they must enter the transmembrane electric field to reach the binding site. The fraction of the field crossed by the drug before reaching the binding site is called the effective electrical distance (δ). It does not necessarily correspond to the physical portion of the binding-site location in the membrane, as the drop in potential across the membrane is not uniform. The relative depths of various binding sites can be compared by calculating the apparent electrical distance as a fraction of the total transmembrane voltage (Bett, 2002). The results indicate that both QX-314 and TEA are open channel blockers that must cross a significant portion of the extracellular transmembrane electric field (~39% and ~38%, respectively) in order to

115 inhibit TRPV1 currents. Furthermore, the similarity in electric distance traversed between

QX-314 and TEA supports the idea that they share a similar binding site. Due to the inherently weak voltage-dependent nature of TRPV1 activation (V1/2 ~70 mV) and the presence of natively expressed Ca2+-activated Cl- channels in Xenopus oocytes, a precise voltage dependence of TRPV1 channel block was difficult to isolate (Matta and Ahern,

2007). For this reason, only the voltage-dependence of QX-314 and TEA inhibition on oocytes stably expressing low levels of TRPV1 channels between 0.1–3 µA baseline current amplitudes could be defined. Taken together, these results provide evidence that the properties of the TRPV1 channel are not static but change dynamically depending on channel density in vitro.

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Chapter 6: General discussion

In the present thesis, pharmacological characterization of mammalian TRPV1 channels using the two-electrode voltage clamp technique has enabled the dissection of the diverse range of concentration-dependent effects produced by the prototype amino- amide LA, lidocaine, and its experimental quaternary derivative, QX-314, in vitro.

Previous studies had suggested that facilitated entry of quaternary LAs through the activated TRPV1 pore can produce nociceptive-specific block in vivo (Binshtok et al.,

2007). Although the present body of work was not able to disprove this result in vitro, it was not able to reproduce it either. This thesis has instead identified a novel and reproducible molecular mechanism that underlies the interactions between clinically available LAs and the nociceptive ion channel TRPV1.

Based on the research conducted for this thesis, it is shown that amino-amide LAs produce biphasic concentration-dependent activity on TRPV1 channels. It is confirmed that clinical-relevant concentrations of LAs activate TRPV1 channels in vitro, supporting clinical observations for LA-induced side effects in vivo (Leffler et al., 2008; Schwarz et al., 2010). Moreover, the present work discovered that nanomolar concentrations below the activation threshold for TRPV1 potently inhibit multi-agonist-induced TRPV1 activation. This novel observation introduces conventional LA compounds (at sub- activating concentrations) as potentially safe molecular probes for investigating TRPV1 pharmacology in vivo and in vitro. In addition to the characterization of LA-mediated

TRPV1 channel regulation, the results also provide a unifying molecular theory to describe these effects using structure-activity relation assays and site-directed mutagenesis. The major findings in this thesis introduce a new molecular target for pain

117 control that can be dynamically regulated using established and well characterized LA compounds.

TRPV1 CHANNEL ACTIVATION BY LOCAL ANESTHETICS

This work shows that QX-314, and for that matter LAs in general, are concentration-dependent TRPV1 channel agonists. In conjunction with recent in vivo and in vitro observations, it is shown here that LA compounds activate TRPV1 at clinically relevant millimolar concentrations (Leffler et al., 2008; Rivera-Acevedo et al., 2011;

Schwarz et al., 2010). Whereas the precise interaction for LA agonism has not been resolved molecularly, the following considerations endeavor to reconcile the observations using a phenomological and structural interpretation.

Direct insight into local anesthetic-mediated toxicity in vitro

Conventional LA administration represents a generally safe alternative to general anesthesia, associated with a low occurrence of severe adverse events. Although rare, toxic reactions to LAs are a feared potential complication. LA toxicity can be categorized into two main classes: Local and systemic toxicity. Local toxicity can be further divided into neurotoxicity, transient neurological symptoms, and myotoxicity, while systemic toxicity includes CNS and cardiovascular system (CVS) toxicity (Johnson, 2000; Parry,

2011). LAs injected below the dural layer into the cerebral spinal fluid are linked to both transient neurological symptoms and persistent lumbosacral neuropathy (Perez-Castro et al., 2009; Yamashita et al., 2003). It is not uncommon in patients receiving high (5%) lidocaine concentrations for spinal anesthesia to experience transient neurological

118 symptoms of variable severity that resolve within 1–2 weeks (Perez-Castro et al., 2009;

Pollock, 2003). As a result of sustained high millimolar LA intrathecal infusions, a more rare and serious complication known as cauda equina syndrome may develop (Pollock,

2003). The cauda equina or “horse-tail” is a nerve bundle comprising the nerve roots from the L1–5 and S1–5 segments (Gardner et al., 2011). LA toxicity affecting this region is often irreversible and characterized by anal sphincter incontinence, sexual dysfunction, paresthesias, and leg muscle weakness (Johnson, 2000). Irreversible LA- induced toxicities have been reported following intrathecal 5% lidocaine, 1% tetracaine, and even 0.5% bupivacaine administration (Yamashita et al., 2003). The current model for LA-induced neurotoxicity suggests various overlapping mechanisms independent from Nav channel block, including depletion of ATP, mitochondrial injury, and prolonged elevation of cytosolic Ca2+ (Hogan, 2008; Werdehausen et al., 2009). In fact, using both in vivo and in vitro models, LAs have been shown to produce neuronal death largely by increasing intracellular Ca2+ levels initiating necrotic and apoptotic downstream cellular effects through an unknown mechanism (Perez-Castro et al., 2009; Werdehausen et al.,

2009).

Activation of TRPV1 channels by millimolar QX-314 and lidocaine in vitro provides a conceivable and plausible molecular explanation for persistent neurological injury observed during high-dose intrathecal LAs administration in vivo. This work supports previous observations from Leffler and colleagues providing a basic molecular model for LA-mediated neurogenic-inflammation and cell death (Leffler et al., 2008).

The model proposes that direct exposure to high millimolar LA concentrations initiates a sudden rise in cytosolic Ca2+ levels in neurons through the activation of TRPV1 channels.

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Persistent and prolonged activation of these nociceptors will not only lead to cell death in some neuronal populations due to Ca2+ overload, but also recruit the release of second messenger proteins such as substance P and CGRP from peripheral nerve endings exacerbating surrounding spinal tissue inflammation. This effect has been replicated in cultured capsaicin-sensitive DRG neurons which show a significant intracellular increase in Ca2+ mediated by lidocaine TRPV1 activation compared to TRPV1-/- mice (Leffler et al., 2008). It has been well established that pungent TRPV1 agonists directly kill adult sensory neurons through TRPV1 activation in vitro (Jeftinija et al., 1992; Szallasi and

Blumberg, 1999). Similar results have been verified in cultured capsaicin-sensitive DRG populations exposed to TRPV1 agonists, capsaicin and resiniferatoxin (Jin et al., 2005).

For example, a single intrathecal injection of the ultra-potent TRPV1 agonist resiniferatoxin in vivo, produces significant neural dysfunction in TRPV1-expressing neurons, leading to permanent analgesia in dogs suffering terminal canine osteosarcoma

(Brown et al., 2005). In humans, the intracutaneous injection of capsaicin at high concentrations induces neural degeneration of epidermal nerve fibers (Simone et al.,

1998). However, with LAs, an intracellular increase in Ca2+ and CGRP was still detected in TRPV1-/- mice, suggesting that additional mechanisms underlie LA-mediated neuronal toxicity. The results in this thesis demonstrate that the closest TRPV1 homologue,

TRPV4 was not activated by QX-314 (or lidocaine; data not shown), indicating that another molecular target must mediate this effect. Acrolein activation of the lidocaine sensitive TRPV1-/- DRG population suggests that TRPA1 channels reconcile the TRPV1- independent rise of intracellular Ca2+.

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As a fellow TRP family member and polymodal nociceptor, TRPA1 channel expression occurs mainly in sensory neurons (Story et al., 2003). This channel is abundantly expressed in trigeminal, nodose and dorsal root ganglionic neurons (Anand et al., 2008; Yang et al., 2008). In both rodent and human DRG neuronal populations,

TRPA1 is colocalized with TRPV1 and CGRP (Brierley et al., 2009; Cattaruzza et al.,

2010). In fact, it has been demonstrated that most TRPA1-expressing neurons contain

TRPV1, but not the other way around (Malin et al., 2011). TRPA1, like TRPV1, is activated by a wide range of chemical ligands – particularly, an extensive list of endogenous and exogenous irritant compounds (Holzer, 2011). Many of these chemicals need to be sensed by the body and avoided because of their potential for tissue damage

(Kang et al., 2010). The properties of TRPA1 allow it to sense spicy compounds present in mustard, horseradish, wasabi (allyl isothiocyanate) (Jordt et al., 2004; Story et al.,

2003), black pepper (Okumura et al., 2010), garlic, onion (allicin) (Bautista et al., 2005;

Macpherson et al., 2005), cinnamon (cinnamaldehyde) (Bandell et al., 2004), ginger

(gingerol) (Bandell et al., 2004), oregano (carvacrol) (Xu et al., 2006), wintergreen

(methyl salicylate) (Bandell et al., 2004), and clove (eugenol) (Bandell et al., 2004). It can also detect toxic environmental stimuli such as tear gas (morphanthridine analogs)

(Bessac et al., 2009), nicotine (Talavera et al., 2009), formaldehyde (Macpherson et al.,

2007), acrolein (Bautista et al., 2006), 4-hydroxy-2-nonenal (Macpherson et al., 2007), and acetaldehyde (Bang et al., 2007). The current body of evidence suggests that TRPA1 activation (together with TRPV1) forms a significant component for LA-induced neurogenic toxicity and inflammation (Leffler et al., 2011). Consistent with this idea, intrathecal TRPA1 and TRPV1 activation have been shown to release both glutamate and

121

CGRP from central nerve terminals regulating spinal nociceptive signaling and cell homeostasis (Caterina et al., 2000; Leffler et al., 2008; Leffler et al., 2011). Thus, the data establishes a direct role for TRPV1, and to a lesser extent TRPA1 in LA-mediated neurotoxicity in vivo.

Structure-activity relation of local anesthetic TRPV1 agonism

As previously mentioned, TRPV1 channels are exquisitely sensitive to chemical gating, particularly to naturally occurring vanilloids, such as capsaicinoids and capsinoids found in plants of the genus Capsicum (chili and bell peppers), resiniferatoxin found in

Euphorbia resinifera, as well as a wide array of similarly structured synthetic compounds

(Vriens et al., 2009). The present results demonstrate that LAs are direct activators of

TRPV1 channels in a similar manner as vanilloids, albeit with significantly weaker potency. Previous in vitro studies alluded to the possibility that LAs interact with the putative vanilloid-binding sites found in TM3 (Leffler et al., 2008). Three seminal papers published by the Sandoz Institute for Medical Research in London have outlined an approach to the systemic investigation of the structure-activity profile of capsaicin-like molecules (Walpole et al., 1993a; Walpole et al., 1993b; Walpole et al., 1993c). They subdivided the capsaicin molecule into three regions, labeled A, B, and C, modifying one region while maintaining the others constant. Compounds were evaluated using in vitro screening assays measuring 45Ca2+ influx into the polymodal subset of small and medium neonatal rat DRG neurons. If activity was detected, the compound’s activity was further analyzed using guinea pig ileum contraction assays to quantify potency. Lastly, promising compounds would be tested using in vivo mice models (tail flick latency to

122 noxious thermal stimuli) to calculate therapeutic index. Their findings have established the comprehensive SAR blueprint for the synthesis of TRPV1 agonists as novel synthetic analgesics. Due to some structural similarities between capsaicin and LAs, the following sections will dissect the activity of these molecular functional groups using the A, B, and

C-region designations established by Walpole and colleagues [Figure 28].

Figure 28. Structural composition of capsaicin and local anesthetic compounds used in this study.

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The aromatic “A-region”

Like capsaicin, all clinically employed LAs possess an aromatic head group. In fact, all natural and synthetic vanilloids possess a substituted benzyl function. Only endogenous hybrid TRPV1/cannabinoid agonists, such as anandamide and 12-HPETE, do not possess this region, although they demonstrate robust poly-unsaturation similar to aromaticity (Messeguer et al., 2006). Methylene substitutions in LAs at positions 2, 5, and 6 on the aromatic ring lead to a significant reduction in efficacy for TRPV1 activation compared with capsaicin, but does not abolish agonist activity (Walpole et al.,

1993c). In LAs, the variation of the 4-hydroxyl on the aromatic ring present in vanilloids decreases activity, but significant residual activation can still be retained with nitro- substituents, helping to explain the apparent increase in efficacy observed with amino- ester LAs procaine and tetracaine in vitro (Komai and McDowell, 2005; Leffler et al.,

2008). However, the designation of a minimal structure for a potent (< 1 µM) agonist is a

4-hydroxyl (4-OH) substituent with an adjacent 3-hydroxyl (3-OH) or 3-methoxy (3-

OCH3). In a further attempt to evaluate the function of the 4-hydroxy substituent, it has been shown that under certain circumstances, there exists bioisosterism between a phenolic hydroxyl group and a pyrrole or pyrimidine nitrogen atom. This would suggest that one of the most potent and toxic amino-amide LAs, dibucaine (cinchocaine), used mostly in veterinary anesthesia, would also be a more potent TRPV1 agonist compared with lidocaine. The presence of the aromatic moiety found in capsaicinoids and LAs is presumed essential for the capacity to form hydrogen bonds and π-π electron interactions with tyrosine residues in the putative vanilloid binding pocket critical for TRPV1 agonist activity (Messeguer et al., 2006; Walpole et al., 1993c).

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The amide bond “B-region”

The aromatic A-region of vanilloids is attached via an amide (capsaicinoids) or ester (capsinoids) linker similar to lidocaine and benzoic acid LA derivatives. SAR studies demonstrate that the amide and altered reverse amide structures in vanilloids are equipotent. The exception lies with lower homologue amides (similar in structure to lidocaine derivatives) – NHCO- -CONH– where the former amide configuration is significantly more active (Walpole et al., 1993a). Generally, a one carbon atom “bridge” is necessary between the A-region and dipolar B-region for potent (< 1 µM) TRPV1 activation. Removal of the dipolar functions of the B-region leads to a significant loss in potency. The carbonyl portion of the linker contains polar groups necessary for the formation of hydrogen bonds with the putative vanilloid binding sites initiating sensory neuron excitation (Messeguer et al., 2006; Morita et al., 2006). For TRPV1 activity, compounds must possess some component of a hydrogen-bond-donor-acceptor pair, which must ideally assume a trans coplanar configuration that is seen with LAs (Walpole et al., 1993a). The higher the number of H-bonds made in this region, the higher the observed affinity for channel activation. For compounds with an EC50 < 1 µM, two H- bond donors are necessary, whereas less potent compounds such as LAs form only one

H-bond interaction. Reduction, removal, or steric constraints placed on the nitrogen atom significantly reduce activity. The presence of the flexible dipolar B-region found in LAs is key for TRPV1 agonist activity (Walpole et al., 1993a).

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The hydrophilic “C-region”

Unlike the two regions described previously, the SAR of the C-region is the most dissimilar to the structure of LAs. Overall size and hydrophobicity in the C-region are the most important predictors for agonist potency, both of which are lacking in all LAs including benzocaine. Potency increases with increasing chain length up to a plateau of

8–12 carbon atoms. This property is highlighted by the lack of activity shown by compounds with short side chains, long polar side chains, or with polar functional groups

(such as amines) attached to the end of hydrophobic chains as is the case with most LAs

(Walpole et al., 1993b). It has been shown, however, that diverse substitutions in the C- region can be accommodated into the vanilloid binding sites, with the mode of B-region attachment being the critical factor. Steric bulk at the end of a molecule, as is the case with QX-314, does not appear to affect TRPV1 agonism, as other permanently charged vanilloids still display extracellular activity (Li et al., 2011; Walpole et al., 1993b).

Local anesthetic structure-activity relationship overview

The functional molecular groups found in clinically employed amino-amide LAs share some similarity to the capsaicin homologues. In the case of lidocaine and QX-314, they both possess an m-substituted aromatic head group essential to form π-π stacking associated with vanilloid binding of critical tyrosine residues in the proposed vanilloid binding site (Lee et al., 2011). In LAs, the aromatic group directly attached to a carbonyl function (amino-esters) or a 2,6-dimethyl phenyl group attached to a carbonyl function through an –NH– group (amino-amide) make these regions highly lipophilic. The aromatic group of LAs shares similar properties to both the aromatic A-region and highly

126 lipophilic C-region found in vanilloids. A- and C-regions are thought to stabilize the molecule inside the TRPV1 hydrophobic binding pocket (Vriens et al., 2009). Through resonance in the amide, one could expect that in lidocaine, for example, the electrons from the nitrogen can be resonance-delocalized onto the carbonyl oxygen to form a zwitterion [Figure 29]. In the case of QX-314, conventional wisdom would suggest that converting lidocaine to its quaternary ammonium analog does not prevent hydrogen bond formation to the amide component of the molecule. SAR studies with LAs have established that similar to vanilloids, the zwitterionic state formed by the amide or ester linkers directly mediate compound-receptor binding through the formation of H-bonds with the nucleophilic serine, and basic arginine and lysine residues that line the putative vanilloid binding pocket (Chou et al., 2004; Williams et al., 2002). Although it seems that compounds with 3-alkoxy-4-substituted benzyl rings have the highest TRPV1 channel activity, novel vanilloid-like agonists exist that lack this vanillyl function and instead possess unsaturated 1,4-dialdehydes or triphenyl phenols, such as warbuganal, polygodial, isovelleral, and scutigeral (Szallasi and Blumberg, 1999). The TRPV1 channel, first identified in 1990 as a specific vanilloid receptor, has been consistently demonstrated to be activated by compounds lacking the vanillyl moiety, while still presenting vanilloid- like activity (Szallasi and Blumberg, 1990).

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Figure 29. Functional interactions between amino-amide local anesthetic molecules and TRPV1 residues associated with vanilloid binding. Possible molecular interactions mediated by amino-amide local anesthetics at millimolar concentrations with TRPV1 residues in and around the putative vanilloid binding sites (Chou et al., 2004; Jordt and Julius, 2002).

Like vanilloids though, LAs show slow TRPV1 gating when exposed extracellularly (Vriens et al., 2009). This is thought to occur in part because of delayed access to the binding site. In 2002, Jordt and Julius discovered that the TRPV1 residues

Y511 and S512, located in the intracellular loop between S2 and S3, participate in capsaicin binding (Jordt and Julius, 2002). More refined structural analysis has revealed

128 that the binding pocket for vanilloid-like agonists may be found more extracellularly along the S3-S4 interface (Chou et al., 2004; Johnson et al., 2006). Extensive functional mapping of residues along the S3 and S4 α-helix have identified a methionine at position

547 near the extracellular region of rTRPV1 involved in RTX binding (Chou et al., 2004).

The hypothetical TRPV1 model based on the KvAP crystal structure predicts additional contributions for vanilloid-like binding from the Tyr511, Leu515, Phe543, Asn551,

Met547, and Tyr555 along the S3–S4 (Chou et al., 2004). Furthermore, “cross-talk” seems to occur between the binding of more extracellular residues, such as Met547 and

Tyr511 present in the cytoplasmic face of TRPV1 (Johnson et al., 2006). This would suggest that QX-314 and lidocaine would not need to traverse the entire TRPV1 electric field in order to elicit the conformational change necessary for pore opening.

Amphipathic amino-amide and amino-ester LAs have been suggested to interact with the vanilloid binding region in TM3 to produce pore opening (Leffler et al., 2008). It is noteworthy then that similar effects are observed here with QX-314. This permanently charged molecule activates TRPV1 channels expressed in both amphibian and mammalian heterologous expression systems at similar concentrations to conventional

LAs. Various plausible mechanisms may mediate this effect. First, due to the permanent positive charge found in QX-314, it may be interacting with extracellular residues involved in proton-induced excitation that line the TRPV1 extracellular pore region in a similar fashion as polyamine activation (Ahern et al., 2006). This possibility appears unlikely, as the effects for polyamine activation and sensitization occur at concentrations where robust QX-314 inhibition is seen here. Additionally, the extracellular application of millimolar concentrations of TEA (data not shown) and the permanently charged

129 generic organic cation, NMDG, failed to elicit TRPV1 activation in vitro. Extracellular small monovalent molecule activation of TRPV1 seems unlikely, but it does not preclude the documented divalent potentiation and activation of TRPV1 by endogenous regulatory amines (Ahern et al., 2006). Secondly, although the intracellular administration of quaternary capsaicin analogues has been shown to be significantly more potent and effective, extracellular application still demonstrates agonist activity (Jung et al., 1999; Li et al., 2011). Capsazepine inhibition of QX-314- and lidocaine-induced activation further validates a vanilloid-dependent activation pathway, as this competitive TRPV1 antagonist has been demonstrated to be ineffective at blocking extracellular proton-induced activation in rat TRPV1 channels (McIntyre et al., 2001). Similarly to capsaicin, capsazepine has been described to bind with residues in the S3, S4, and S2–S3 intracellular loop, directly competing with vanilloids for this binding site (Gavva et al.,

2005). This would suggest that capsazepine binding to TRPV1 interferes on-side with agonist binding, and also by blocking the communication between the activation sensor and the gate. The coupling mechanisms between the structures that integrate chemical ligand binding and the channel gate still remain unknown. Employing a more physicochemical interpretation, phosphate and Cl- counter-ions are known to regularly form ion- pairs with amines to produce a significant enhancement of diffusion across membranes even in aqueous solutions (Sharma, 2005). Furthermore, experiments performed on liposomal cell membrane models show that the permanently charged QX-314 molecule can indeed cross lipid bilayers through phospholipid facilitation (Tsuchiya and Mizogami,

2008). Similar to the penetration of small cationic peptides, quaternary LAs form hydrophobic ion pairs with anionic membrane lipids, such as acidic phosphatadylserine

130 and cardiolipin, facilitating intracellular diffusion (Esbjorner et al., 2007; Tsuchiya and

Mizogami, 2008). It seems that although delayed, QX-314 may indeed cross the lipid bilayer to interact with portions of the putative vanilloid binding pocket in a similar fashion as capsaicin (Schwarz et al., 2010).

MOLECULAR BASIS FOR TRPV1 CHANNEL INHIBITION BY LOCAL ANESTHETICS

The primary question of this thesis was answered with the establishment that LAs activate TRPV1 at millimolar concentrations used during clinical nerve block. Focus then was shifted to explore the possible pharmacological interactions of these molecules at the sub-activating range. A thorough in vitro pharmacological analysis followed using three representative LA compounds: The quaternary QX-314, the amphipathic lidocaine, and the neutral benzocaine molecule. SAR activity was further dissected with the quaternary ammoniums TEA and TMA. The results from these experiments have revealed that LAs at sub-activating concentrations are potent inhibitors of TRPV1 channel activity, mediated through onium open channel pore block.

It has been known for some time that LAs interact with ion channel targets other

than Nav channels, primarily Kv and Cav channels found in DRG and dorsal horn neurons

(Komai and McDowell, 2001; Olschewski et al., 1998; Sugiyama and Muteki, 1994;

Xiong and Strichartz, 1998). The main distinction between the blocking action of LAs on

Kv and Cav channels, compared with TRPV1 receptors, are the significantly lower affinities of the former compared with Nav channel blockade. Furthermore, extracellular

QX-314 has been shown to be ineffective at blocking K+ currents, suggesting a distinct molecular mechanism between LA interactions on the Kv channels and the TRPV1

131 receptor leading to block (Komai and McDowell, 2001). These observations provide a novel, potentially therapeutic use for LAs at concentrations significantly below what is necessary for Nav channel blockade in the presence of noxious chemical agonists. This is an intriguing pharmacological profile as it exemplifies the adage immortalized by

Philippus von Hohenheim (aka Paracelsus) that “allein die Dosis macht, daß ein Ding kein Gift ist” (“The dose makes the poison”). This section of the study highlights the important aspects of investigating the pharmacological spectrum of drugs over a broad range of concentrations.

Local anesthetics are multimodal TRPV1 channel inhibitors

TRPV1 activation directly modulates peripheral pain and inflammatory processes in the human body. It is not surprising that diverse target-based approaches have been implemented in order to inhibit TRPV1 channels (Holzer, 2008). This has not been easily achieved, as TRPV1 can be gated by a plethora of noxious stimuli as previously noted

(Tominaga et al., 1998). These gating mechanisms are not simply selective on-off switches mediated by distinct stimuli; instead, it appears that all TRPV1 regulators modify the intrinsic activity of the channel in different ways. The activation of TRPV1 by agonists when applied alone often produces only partial stimulation. A maximal response is achieved through a synergistic interaction of two or more stimuli which work together to open the channel at a lower activation threshold consistent with an allosteric binding model (Brauchi et al., 2007; Tominaga et al., 1998). Of particular interest in the development of therapeutic TRPV1-targeting analgesic agents are compounds that are able to inhibit multimodal channel activity (Gavva et al., 2005).

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In support of this idea by the body of work in this thesis, amphoteric and permanently charged LAs now belong to the expanding list of multimodal TRPV1 channel antagonists. Thorough concentration-dependent characterization of LA compounds has revealed that these molecules potently inhibit both capsaicin- and proton- induced TRPV1 activation with nanomolar affinity in vitro. In fact, concentration- response data acquired from QX-314 and lidocaine inhibition during stimulation with pH

5.5 containing solutions demonstrated more potent and effective TRPV1 antagonism compared with capsaicin. Unlike LAs, the standard TRP non-competitive antagonist, ruthenium red, exhibits a ~10-fold decrease in efficacy when applied at pH 6.4 (Garcia-

Martinez et al., 2000). If the inhibitory effects of QX-314 and lidocaine are conserved in other TRPV1 expression systems, these results would advocate the use of low dose LAs for non-specific TRPV1 antagonism during experimental controls.

Currently available competitive and non-competitive TRPV1 channel antagonists can be divided into two major groups. Group A antagonists are able to block both vanilloid and proton activation, while group B antagonists block only vanilloid activation in a species-dependent manner (Gavva et al., 2005). The capacity to block both capsaicin and proton-induced TRPV1 activation strongly correlates with in vivo analgesia in animal pain models (Gavva et al., 2005; Honore et al., 2005; Lehto et al., 2008; Pomonis et al.,

2003). Nevertheless, during clinical trials, most competitive TRPV1 antagonists frequently display mild but significant hyperthermia compared to placebo (Gavva et al.,

2007). An avenue currently being explored to avoid these side effects is the design of non-competitive multimodal TRPV1 open-channel pore blockers which may “use- dependently” target active TRPV1 channels (Garcia-Martinez et al., 2006; Garcia-

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Martinez et al., 2002). QX-314 and lidocaine as both Nav and TRPV1 channel pore blockers could be redesigned for a similar indication. It is noteworthy that although intrathecal QX-314 has been demonstrated to be a central irritant (Schwarz et al., 2010), these effects were not observed during subcutaneous and peripheral administration (Lim et al., 2007; Ries et al., 2009). This raises the intriguing possibility that the toxicity profile of these molecules is highly dependent on the route of administration. In fact, it has been previously documented that administration of a 0.2% QX-314 solution near the sciatic nerve prior to an injection of capsaicin inhibits the noxious response elicited from this irritant vanilloid (Binshtok et al., 2007). As this effect occurs prior to the documented onset for motor and sensory block from Nav channel inhibition, one could reason that

TRPV1 channel inhibition is likely involved (Lim et al., 2007). Currently, one therapeutic avenue being explored to produce long-lasting analgesia with LAs involves the co- application of lidocaine with low-dose QX-314 (Binshtok et al., 2009; Roberson et al.,

2011). Although regarded as a novel and promising therapeutic opportunity for long- lasting (and potentially nociceptive-selective) analgesia, extensive preclinical studies will be required to establish safety. The main breakthrough from the present work lies in the discovery that LAs can indeed produce multimodal TRPV1 block in a concentration- dependent manner, with the possibility of mechanical and thermal peripheral analgesia in vivo.

Quaternary ammonium determines local anesthetics inhibition

In the model based on the results of this thesis, TRPV1 channel inhibition by LA molecules requires the presence of a protonatable amine tail. From the list of clinically

134 available LAs, only benzocaine lacks this group. As expected, no significant extracellular inhibitory effects produced by benzocaine occurred in the presence of capsaicin. On the other hand, this work shows for the first time that extracellular TEA acts as a high- affinity TRPV1 channel blocker in a voltage-dependent fashion (Rivera-Acevedo et al.,

2012). The weak voltage-dependence of antagonism suggests that LAs are TRPV1 open channel blockers. This was validated by the observations that at more depolarized potentials lidocaine, as well as TEA and TMA, displays a significant reduction in the potency and efficacy of TRPV1 channel inhibition. Consistent with previous results, the weak voltage dependence of block observed with QX-314 and TEA implies that the charged ammonium molecules cross a small fraction of the TRPV1 electric field to reach their binding site, with an estimated electrical distance (δ) of ~39% and 38% respectively

(Woodhull, 1973). This finding is consistent with the notion that extracellular QAs bind to a region in TRPV1 located slightly deeper into the electric field compared with similar open-channel blockers in Shaker and Kv1.2 (Ahern, 2006; Wang et al., 2003). These results agree with δ values acquired from the voltage-dependence of extracellular arginine-rich hexapeptide block in TRPV1. These compounds which are much larger than

QX-314 and TEA, have a δ of 0.2 suggesting they partially cross the TRPV1 electric field to reach a shallow binding site near the permeation pathway (Planells-Cases et al., 2000).

This indicates that similar to the effects produced by QAs in voltage-gated K+ channels, extracellular LAs inhibit the TRPV1 channel via electrostatic interactions with structures near the outer pore region (Heginbotham et al., 1999; Heginbotham and MacKinnon,

1992; MacKinnon and Yellen, 1990)

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So far, only a select few extracellular open channel blockers for TRPV1 have been identified. Most are large polycationic amines or trialkylglycines, such as ruthenium red, methoctramine, DD161515, and DD191515 (Garcia-Martinez et al., 2002; Maggi et al., 1988; Mellor et al., 2004). All non-competitive TRPV1 open channel pore blockers tested have been shown to produce significant analgesic activity when administered in vivo (Garcia-Martinez et al., 2002; Mellor et al., 2004; Ohkubo et al., 1993). These compounds demonstrate considerable efficacy at increasing thresholds for mechanical and thermal hyperalgesia in chronic and neuropathic pain models (Garcia-Martinez et al.,

2006; Garcia-Martinez et al., 2002; Mellor et al., 2004). Thus, it would be prudent to assume that a similar result may be observed with both lidocaine and QX-314 at lower concentrations than those required for Nav channel blockade, or the co-application of both for combinational therapy (Roberson et al., 2011).

Intriguingly, similar to what has been observed with intracellular QA administration, the present experiments found that both LAs and QAs produce incomplete current inhibition even at saturating concentrations (Jara-Oseguera et al.,

2008; Oseguera et al., 2007). Although this effect has been poorly characterized, a possible explanation proposes that channel blockers applied externally likely exist in a dynamic equilibrium between bound and unbound states. The rate at which this process occurs may be fast enough to allow the small molecular blocker to unbind and permeate through the pore helping to explain the observed relief-of-block (Huang et al., 2000;

Huang and Moczydlowski, 2001). Huang and colleagues described a similar result in Nav channels, where the intracellular application of QAs and polyamines demonstrated relief- of-block dependent on the interactions with charged residues lining the pore. Passage of a

136 blocking cation through the pore at a rate of less than 0.1% has the capacity to significantly alter the form of an I-V curve in relation to single ion permeation (Huang,

2001). An interesting observation from the intracellular administration of quaternary agents in Nav channels is that unlike QAs, the administration of QX-314 internally does not display relief-of-block, suggesting distinct structural properties for pore permeation in different ion channel types (Huang et al. 2000). The block produced by intracellular TEA has been shown to occur with very fast on-and-off rates in single channel experiments

(Salazar et al., 2009). It seems likely that the same would be true for extracellular block by TEA and LAs as well.

Properties of the extracellular TRPV1 pore illuminated by local anesthetics

A precise extracellular model for the TRPV1 pore has so far eluded structural biophysicists. At present, the TRPV1 channel lacks a high-resolution crystal structure to aid the identification of residues that may mediate gating and chemical interactions. The only full-length TRPV1 structural model achieved so far comprises a 19-Å resolution electron cryomicroscopy 3D structure (Moiseenkova-Bell et al., 2008). In this model,

+ TRPV1 exhibits 4-fold symmetry, fitting well with the crystal structure of the Kv1.2 K channel, but very little is known of the precise interactions mediated throughout specific regions of the channel (Long et al., 2005a). The intracellular application of QAs of linearly increasing alkyl chains have been instrumental in revealing that TRPV1 channels possess two constriction sites in the pore. One anchored by A681, located in the intracellular S6 region, and another by Y671, located deeper in the pore near the selectivity filter (Salazar et al., 2009). Apart from the basic channel architecture, very

137 little is known about the specific regions involved in extracellular channel gating, and only a few studies have been performed delimiting extracellular small molecular binding regions for TRPV1. Hence, the structure obtained from Shaker-like K+ channels has been an important reference not just to define the pore, but also to elucidate the coupling models for TRPV1 (Cuello et al., 2010; del Camino et al., 2000; Jiang et al., 2002; Long et al., 2005a). Consequently, similar to previous observations with polycationic amines, this thesis shows that extracellular ammonium block occurs through binding of aromatic and acidic residues that line the extracellular TRPV1 ionic permeation pathway (Garcia-

Martinez et al., 2000). Due to well-known interactions that occur between TEA and the extracellular residues that line the Shaker K+ channel pore, this thesis’ search for the

TRPV1 putative ammonium binding site focused on this region (MacKinnon and Yellen,

1990). Together with the studies previously performed by Garcia-Martinez and colleagues, it seems that Asp646, Glu648, and Phe649 directly bind to charged amine compounds of varying sizes through an electrostatic reaction impeding (at least partially) permeation of molecular monovalent cations in this region (Garcia-Martinez et al., 2000).

QX-314 and lidocaine are approximately 10-12 Å in diameter, while TEA is 8 Å (Salazar et al., 2009; Yamagishi et al., 2009). This indicates that under conditions were the extracellular pore does not dilate from prolonged agonist stimulation the penetration by molecules at least 8 Å in diameter are inhibited from traversing the pore (Chung et al.,

2008).

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Strengths and limitations of the study

The work performed for this thesis illuminated the behavior of LAs on TRPV1 channels, primarily using two-electrode voltage clamp (TEVC) electrophysiology on

Xenopus laevis oocytes in vitro. Electrophysiological assays in oocytes, similar to mammalian cells, allow for detailed observations of drug activity on a specific molecular target. These techniques are particularly useful for the functional structure-activity relationship studies needed to identify regions involved in ion channel regulation. Unlike mammalian cells, oocytes have the advantage of easily expressing a wide variety of channel isoforms and mutations (Goldin, 2006). Another advantage over mammalian cells is the quicker screening of experimental drug-receptor interactions using oocytes.

Moreover, this approach allows for precise measurements of TRPV1 activity alone, whereas native expression systems such as DRG or trigeminal neuronal cells express additional channels whose effects may occlude the desired target activity.

The major disadvantage of using the Xenopus oocyte system lies in the fact that it is a less physiologically relevant system compared to mammalian expression platforms and does not always replicate precisely the spectrum of possible responses observed in vivo (Goldin, 2006). Another disadvantage is the often increased EC50 and IC50 values observed in oocytes compared with mammalian cells. To be able to adequately extrapolate physiologically relevant data from the present observations, it would have been useful to test the effects of LA inhibition on DRG and HEK293 cells using patch- clamp electrophysiology. Performing TEVC on oocytes did not allow to accurately resolve the kinetics of LA-mediated biphasic effects with the level of resolution afforded by single-channel patch-clamp experiments.

139

Even though experiments demonstrated that TRPV4 channels are not activated by

QX-314, the difficulties at the onset in perfecting the ligand-binding assays did not allow a proper investigation whether QX-314 and lidocaine were also able to inhibit TRPV4 activation. This was mostly due to the very slow gating kinetics presented by the synthetic vanilloid, GSK101, one of the very few TRPV4 agonists available. The redesigned and optimized ligand binding assays employed during chapters 4 and 5 of this thesis could have resolved this issue. Furthermore, although it appears that valid conclusions could be extrapolated from the amelioration of inhibition produced by TEA on the TRPV1 pore mutants, the effects of QX-314 and lidocaine inhibition were not pursued directly using TRPV1 F649A and E648A channel mutants. It would have been valuable to measure the degree of inhibitory change compared with TEA. In order to dissect some of these and other possible ambiguities, a series of future experiments could be performed.

FUTURE DIRECTIONS FOR LOCAL ANESTHETIC TRPV1 RESEARCH

The activation of TRPV1 channels has been explained mainly using allosteric binding models (Baez-Nieto et al., 2011; Rivera-Acevedo et al., 2011). For the case of

TRPV1 identifying chemical agonist binding sites, as well as the integration of temperature and voltage sensors into the general gating responses are still important research objectives. Future work pertaining to the effects of LAs on TRPV1 should elaborate on the kinetics of block and activation as well as delineate some of the intimate details amongst the clinically available LAs in order to establish a proper SAR blueprint for the activity of these compounds on the TRPV1 channel isoform.

140

 The next step is to perform single channel experiments on TRPV1 with

extracellular QX-314, lidocaine, and TEA to explore the kinetics of block by

these compounds.

 In order to properly identify whether the amide or aromatic functional groups

mediate TRPV1 activation, it would be useful to test activation produced by

amino-amide and amino-ester LAs, particularly benzocaine, at millimolar

concentrations.

 Site-directed mutagenesis performed on the key residues involved in vanilloid-

and proton-induced TRPV1 regulation would help reveal the precise mechanisms

employed in LA-mediated channel gating.

 Conduct Ca2+ fluorescence imaging on physiologically relevant DRG neuronal

cells, but also the applicability of LA-mediated TRPV1 inhibition using enteric

Caco-2 and epithelial cell lines.

 Investigate the toxicity of the QX-314 bromide and QX-314 chloride salts.

Distinct toxicities were observed between each salt, the bromide salt being highly

toxic to Xenopus oocytes at millimolar concentrations in our study [cf. Chapter 2],

but has been the main compound used in many of the studies characterizing QX-

314.

 QX-314 is just one member in a vast catalogue of quaternary lidocaine derivatives.

It would be useful to qualify and quantify the possible effects produced by

compounds such as the N-methyl lidocaine derivative, QX-222, on TRPV1

inhibition under whole-cell patch clamp conditions in DRG neurons.

141

 Further explore whether QX-314 inhibits other TRP channels involved in

nociception such as TRPA1.

 It would also be useful to thoroughly characterize the effects produced by other

LAs on the Xenopus oocytes and mammalian cell lines under the present

conditions. Differential effects have been recorded between distinct LAs on

TRPV1 channels expressed in heterologous expression systems, but the effects

appear contradictory. Lidocaine and prilocaine significantly attenuated

intracellular Ca2+ entry from capsaicin induced currents in HEK293 cells at 1 mM

(Hirota et al., 2003). An opposite effect was observed with bupivacaine and

tetracaine in isolated DRG neurons, where bupivacaine produced insignificant

capsaicin current inhibition at 1 mM, but produced a secondary current increase

after application, while the amino-ester, tetracaine, produced significant

potentiation of capsaicin-induced activation (Komai and McDowell, 2005).

 Finally, from the extrapolation of the present body of data, it would be useful to

further investigate whether low-dose QX-314 is able to produce positive

therapeutic analgesia in neuropathic or inflammatory in vivo pain models using

different routes of administration.

142

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