Trpa1) Activity by Cdk5

Home , Carvacrol, Cinnamaldehyde, Diallyl disulfide, Icilin, Resiniferatoxin, Thyme

MODULATION OF TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL, SUBFAMILY A, MEMBER 1 (TRPA1) ACTIVITY BY CDK5

A dissertation submitted

to Kent State University in partial

fulfillment of the requirements for the

degree of Doctor of Philosophy

Michael A. Sulak

December 2011

Dissertation written by

Michael A. Sulak

B.S., Cleveland State University, 2002

Ph.D., Kent State University, 2011

Approved by

______, Chair, Doctoral Dissertation Committee Dr. Derek S. Damron

______, Member, Doctoral Dissertation Committee Dr. Robert V. Dorman

______, Member, Doctoral Dissertation Committee Dr. Ernest J. Freeman

______, Member, Doctoral Dissertation Committee Dr. Ian N. Bratz

______, Graduate Faculty Representative Dr. Bansidhar Datta

Accepted by

______, Director, School of Biomedical Sciences Dr. Robert V. Dorman

______, Dean, College of Arts and Sciences Dr. John R. D. Stalvey

TABLE OF CONTENTS

LIST OF FIGURES ...... iv

LIST OF TABLES ...... vi

DEDICATION ...... vii

ACKNOWLEDGEMENTS ...... viii

CHAPTER 1: Introduction ...... 1

Hypothesis and Project Rationale ...... 24

Specific Aims ...... 25

CHAPTER 2: Materials and Methods ...... 26

Experimental Protocols ...... 35

CHAPTER 3: Results ...... 39

CHAPTER 4: Discussion ...... 57

References ...... 68

iii

LIST OF FIGURES

Figure 1. TRP superfamily tree...... 8

Figure 2. TRPA1 reactive cysteines...... 11

Figure 3. Various TRPA1 agonist structures...... 12

Figure 4. Inflammatory soup cartoon...... 14

Figure 5. TRPA1 domain cartoon...... 17

Figure 6. Cdk5/activator structure figure...... 18

Figure 7. Serine 448 sequence alignment...... 21

Figure 8. DRG Neuron photo...... 22

Figure 9. GFP/RFP transfection photo...... 31

2+ Figure 10. Cdk5 inhibition attenuates TRPA1 agonist-induced rise in [Ca ]i in

DRG neurons...... 41

2+ Figure 11. Control trace illustrating TRPA1 agonist-induced rise in [Ca ]i in the absence of inhibition...... 42

Figure 12. Cdk5 inhibition does not attenuate TRPA1 agonist-induced rise in

2+ [Ca ]i in transfected HEK 293 cells, which lack Cdk5 activity...... 43

Figure 13. Summarized data illustrating TRPA1 response attenuation

by Cdk5 inhibition...... 44

Figure 14. Trace illustrating reversible TRPA1 inhibition by roscovitine...... 46

Figure 15. Reversible inhibition summarized data...... 47

Figure 16. Roscovitine dose-dependently inhibits TRPA1 responses...... 49

Figure 17. Phosphoserine western blot...... 51

Figure 18. Phosphoserine western blot summarized data...... 52

Figure 19. Serine 448 kinase assay...... 54

Figure 20. TRPA1 peptide kinase assay inhibitor effects...... 56

LIST OF TABLES

Table 1. Nocisensors (and other thermosensitive TRPs) ...... 5

Table 2. Partial list of TRPA1 agonists...... 13

DEDICATION

For Mom

vii

ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Derek Damron, for providing the research environment and tools necessary to complete this dissertation, and for allowing me the freedom to pursue a project of my own genesis. I thank my committee members, Dr. Robert Dorman, Dr. Ernest Freeman and Dr. Ian Bratz, along with Graduate Faculty Representative, Dr. Bansidhar Datta, for playing their essential roles in the dissertation process. I thank my labmates, Bethany

Prudner, Ryo Yuge and Hongyu Zhang for friendship and support over the years.

A special thanks goes out to Erin Kellams, for preparing many of the DRGs necessary to complete this project.

viii

CHAPTER 1

Introduction

In order to survive and thrive in a hostile environment, organisms require the capacity to detect and respond appropriately when potentially-harmful, tissue- damaging agents are encountered. The task of detecting such noxious mechanical, thermal and chemical stimuli is performed by a specialized class of sensory neurons of the peripheral nervous system (PNS) known as nociceptors

(Sherrington, 1906; Burgess & Perl, 1967). When activated by interaction with a potentially damaging agent (such as a hot coffee cup), nociceptors respond by producing an action potential – sending an electrical signal to inform the central nervous system (CNS) that all is not well. The unpleasant sensation which results, pain, provides the perceiver with an extremely potent and compelling behavioral cue for avoidance of interactions detrimental to their health and well- being.

Pain‟s importance as a guardian of bodily integrity is perhaps best illustrated by those rare cases where it‟s completely absent – in individuals suffering from neural disorders known as hereditary sensory and autonomic neuropathies

(HSANs), which render them unable to sense pain (Axelrod & Gold-von Simson,

2007). An example of one such neuropathy is congenital insensitivity to pain with anhydrosis (CIPA, a.k.a., HSAN IV), a disorder characterized by patients‟ complete lack of small-diameter sensory neurons. As this type of neuron is

1 2 associated with both nociception and innervation of the sweat glands, CIPA‟s hallmark symptoms – the inability to either sense pain or produce sweat (often leading to potentially fatal hyperpyrexia), are easily explained, physiologically.

Individuals afflicted with this rare and incurable condition frequently suffer burns, broken bones, self-mutilation and other serious injuries (leading to greatly shortened lifespans), as the „pain alarm‟ warning/informing of tissue damage is permanently off-line (Nagasako, Oaklander, & Dworkin, 2003; Axelrod & Gold- von Simson, 2007). While such disorders underline the value of pain and the perils of painlessness, at the other extreme can be found patients suffering from chronic pain syndromes – in these ailments, too much pain, rather than too little is the problem, as pain persists beyond its beneficial role as an acute warning device, and instead becomes a terrible burden in its own right.

This type of chronic pain, in marked contrast to the rare, „pain-free‟ sensory neuropathies, is far from uncommon, but instead afflicts tens of millions of Americans each year and is a leading cause of disability. Also in contrast to hereditary neuropathies, which involve neuronal loss or failure to develop and are therefore incurable, clinical pain syndromes are amenable to therapeutic intervention, offering help and hope to the afflicted, and placing a tangible goal in the sights of biomedical and pharmaceutical researchers seeking to improve the status quo. Relief from unnecessary suffering is the therapeutic goal in treatment of pain, but extant pharmaceuticals (NSAIDs and opioids), for all their positive attributes, still have significant shortcomings, leaving a considerable burden of suffering non-palliated. A promising strategy to help fill this „palliation gap‟ is to

3 scour known pain-related pathways in the hope of identifying novel molecular targets for rationally-designed therapeutics (Patapoutian, Tate, & Woolf, 2009).

To this end, advancing our understanding of the mechanisms of nociception at the subcellular, molecular level of detail is required.

Just as man-made thermometers and pressure gauges measure temperature and mechanical force, and reactive dyes are capable of detecting oxidizing agents and pH change, there must exist analogous elements within nociceptive neurons possessing the capacity to interact with each nociceptive stimulus and subsequently produce reliable, functional responses – enabling these elements to act as stimulus detectors, or nocisensors. What are these nocisensors – the functional molecular components within nociceptors that detect mechanical force, hot/cold, reactive chemicals, etc., acting as the biological equivalents of man-made measuring devices? A specific answer to that question was notably lacking until the late 1990‟s, at which time members of David Julius‟s lab at the University of California cloned TRPV1, a.k.a., the capsaicin receptor – the protein that binds specifically and with high affinity to the active ingredient

(capsaicin) found in “hot” chili peppers (Caterina, et al., 1997). This protein, a member of the Transient Receptor Potential (TRP) superfamily of non-specific cation channels, was found to be a critical detector of noxious thermal stimuli ( >

43° C) in addition to binding capsaicin – all at once answering the age-old question as to why chili peppers feel „hot‟ – they potently activate the very same nocisensor as does heat itself!

TRPV1 was the first nocisensor to be cloned, and remains the best studied and characterized, but the ensuing years have seen many more receptors – most of them members of the TRP superfamily – added to the nocisensor menagerie (Table 1.).

Table 1. Nocisensors (and other thermosensitive TRPs)

Receptor name Agonists

TRPV1 Noxious heat, > 43°C Capsaicin, Resiniferatoxin, Protons, Cannabinoids

TRPV2 High temperature > 53° C

TRPV3 Innocuous warmth > ~ 30-33°C

TRPV4 Non-noxious heat, mechanical shear force

TRPA1 Cinnamaldehyde, AITC, many other reactive electrophiles, propofol, icilin, prostaglandins, noxious cold?,

TRPM3 Noxious heat

TRPM8 Menthol, cold < 17 ° C

ASICs Protons

P2X3 ATP

BK receptor Bradykinin (BK)

The TRP superfamily

Transient receptor potential (TRP) cation channels are 6-pass transmembrane (TM) proteins found in a wide variety of cell types, in animals ranging from fruitflies and roundworms, to man. First characterized as a component of Drosophila photoreceptors (wherein a mutant fly produced a transient response, rather than a sustained response, to steady light exposure)

(Cosens & Manning, 1969), TRPs are classified into 7 subfamilies: TRPC

(Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPA (Ankyrin), TRPP

(Polycystin), TRPML (MucoLipin) and TRPN (NOMP-C, no mechanoreceptor potential C) (Owsianik, D'hoedt, Voets, & Nilius, 2006). A family tree illustrating the phylogeny of the TRP superfamily is presented in Figure 1.

TRP channels are believed to generally organize as homotetramers, with at least some heterotetrameric exceptions (Garcia-Sanz, et al., 2004;

,Hoenderop, et al., 2003a). The pore domain is thought to reside between TM helices 5 and 6, by analogy with prokaryotic K+ ion channels, the most similar proteins for which crystal structures are presently available. Differences in these regions account for the variable ion selectivity between channel subtypes

(Owsianik, Talavera, Voets, & Nilius, 2006). The cytoplasmic N- and C-termini vary widely between the different TRP subtypes, and play an essential role in specificity of channel function. The ankyrin repeat protein motif, a domain generally implicated in protein-protein interactions, is present in the N-termini of the majority of TRP channels, with the number of repeats ranging from three in

TRPV1, to 14-18 in TRPA1 (Subject to interpretation), to ~ 29 in TRPNs. While

7 many specific functional elements, such as Pleckstrin Homology (PH) domains,

CaM binding sites and at least one EF hand domain (in TRPA1), have been noted in TRP cytoplasmic tails based on sequence analyses, the mechanisms by which many of these domains specifically contribute to the overall function of each receptor often remain to be determined, given the absence of detailed crystal structures and knowledge of binding partners on which to base mechanistic models. Functional roles for many domains and residues within

TRP receptors have been established, however – even if the mechanistic details are lacking at the present time.

An example where a clear role for TRP domains has been demonstrated, involves the C-terminal tails of heat-sensing TRPV1 and cold-sensing TRPM8

(Brauchi, et al., 2006). In this chimeric study, the C-terminal tails of these thermo-sensitive TRPs were swapped-out, which led to a reversal of the channels‟ temperature sensitivities – the TRPV1 channel chimera with TRPM8‟s

C-terminus was cold sensitive, while the TRPM8 channel chimera with TRPV1‟s

C-terminus was heat-sensitive. These results left little doubt about the importance of the C-terminus as a determinant of thermo -TRP temperature specificity.

Figure 1. The TRP superfamily tree. Evolutionary relationships of TRP channels based on sequence analysis. Unless noted, human channels were used for analysis. TRPC2 is a pseudogene in humans, so the mouse channel sequence was substituted. The scale represents the evolutionary distance expressed in the number of substitutions per amino acid. Dr, Danio rerio; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans. Taken from Owsianik, D'hoedt, Voets, & Nilius, 2006.

TRP channels and pain

Not surprisingly for such a large group, TRP channels function in a myriad of physiological contexts aside from their originally described role in Drosophila photoreception (Cosens & Manning, 1969), and are involved in processes as diverse as: gustatation (TRPM5) (Perez, et al., 2002; Zhang, et al., 2003), Mg2+ homeostasis (TRPM6, TRPM7) (Nadler, et al., 2001; Schlingmann, et al., 2002;

Voets, et al., 2004; Walder, et al., 2002), and Ca2+ re-absorption in the kidneys and intestines (TRPV5 and TRPV6) (Nijenhuis, et al., 2003; Vennekens, et al,

2000; Vennekens, et al., 2001a; Vennekens, et al., 2001b; Hoenderop, et al.,

2002a; Hoenderop, et al., 2002b; Hoenderop, et al., 2003b; den Dekker, et al.,

2003), to name but a few.

Most relevant in the present context, however, is the emergence of TRP receptors as key „molecular machines‟ for sensation of diverse stimuli, including: noxious and innocuous cold and heat (Caterina, et al., 1997; McKemy,

Neuhausser, & Julius, 2002; Karashima, et al., 2009; Guler, et al., 2002), pungent chemicals such as capsaicin (Caterina, et al., 1997), thymol (Lee, et al.,

2008) and menthol (McKemy, et al., 2002), mechanical force (Kwan, Glazer,

Corey, Rice, & Stucky, 2009), and tissue acidification in an inflammatory milieu

(Bautista, et al., 2009; Dai, et al., 2007; Taylor-Clark, et al., 2008). The fact that many of the stimuli detected by TRPs are associated with pain has marked these receptors as potential targets for novel analgesics (Szallasi, Cortright, Blum, &

Eid, 2007; Jordt, McKemy, & Julius, 2003). As TRPs act at the most upstream point of the pain pathway – directly interacting with the stimulus, inhibitors of

10 these channels could potentially nullify a pain signal before it‟s ever generated, unlike extant analgesics (NSAIDs, opioids), which act further downstream, by modifying existing pain signals. The promise that a better understanding of receptor function will allow for modification of noxious signals at the source has helped make TRP receptor biology an extremely active area of research – but the field is still at an early stage, with much yet to be discovered.

Of the seven TRP subfamilies, receptors for pain and temperature sensation are known to be drawn from only three: TRPV, TRPA and TRPM. Of these channels, TRPA1, first described in 1999 in a non-nociceptive context

(Jaquemar, et al.,1999), is the receptor under investigation in this dissertation, and will now be discussed in more detail.

TRPA1

Transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is a polymodal ion channel expressed in a subset of small-diameter, capsaicin- sensitive, nociceptive neurons of the trigeminal and dorsal root ganglia (DRG), the activation of which produces pain and neurogenic inflammation (Bautista, et al., 2009). Like other TRP family members, TRPA1 is a 6-pass TM protein which organizes tetramerically to form a non-specific cation channel. The only member of the TRPA subfamily found in mammals, TRPA1 has emerged as a key nociceptive mediator, responsive to multiple stimuli, including pungent molecules

(mustard oil (aka, AITC), cinnamaldehyde) (Lee, et al., 2008; Bautista, et al.,

2009; Jordt, et al., 2004; Bautista, et al., 2005; Xu, Delling, Jun, & Clapham,

2006) and noxious cold temperature (Karashima, et al., 2009; Story, et al., 2003).

Acting as the body‟s „canary in a coal mine‟ for detection of chemical damage,

TRPA1 can be activated by a large number of „irritating‟ compounds – the list of

TRPA1 agonists has been growing at a rapid rate, with nicotine (Talavera, et al.,

2009), ozone (Taylor-Clark, et al., 2010) and the intravenous anesthetic, propofol

(Matta, et al., 2007), included among recent entries (see Table 2).

Unlike most ion channels, TRPA1

interacts covalently with many of its

agonists – a logical mode of binding

for a nocisensor tasked with

detecting reactive chemical species.

Cysteine nucleophiles (and at least

Figure 2. Human TRPA1’s channel gating one lysine residue) in the cysteine residues are highlighted in red. Taken from Hinman, et al., 2006. channel‟s N-terminal cytoplasmic domain are the sites of covalent interaction between the channel and its reactive electrophile agonists (Macpherson et al., 2007; Hinman Chuang, Bautista, &

Julius, 2006) (see Figure 2). The precise mechanism by which these covalent interactions gate the channel remains unknown, as there are presently no detailed structural data (X-ray, NMR or Cryo-EM) on which to base a working model.

While many of the channel‟s best- known ligands (cinnamaldehyde, mustard oil, etc.) are reactive molecules which act covalently, there are also quite a few non- reactive TRPA1 agonists (e.g., icilin, propofol, and menthol), which presumably gate the channel via non-covalent means

(see Figure 3.). As with the covalent agonists, the manner in which non-covalent ligand binding is linked to channel gating is unknown, and furthermore, the ligand Figure 3. Various TRPA1 agonist interaction sites of non-covalent agonists structures. Taken from Caterina, 2007. are also unknown (unlike the covalent agonists, for which the reactive TRPA1 moieties responsible for channel activation have been mapped). The fact thateexample non-covalent examples molecules examples such as propofol exhibit pore block at high concentrations, which is reversed upon washout (Matta, et al., 2007), suggests the possibility that at least one (perhaps low-affinity) binding site might exist in the region of the channel pore itself.

Other than by nature of channel interaction (covalent or non-covalent), TRPA1 agonists can also be categorized as extrinsic or endogenous. The extrinsic

Table 2. Partial list of TRPA1 activators

AITC (Mustard oil) UV light (acting via ROS)

Cinnamaldehyde Methylhydroxybenzoate

Allicin (from Garlic) Nifedipine

Acrolein Nitric Oxide

Propofol Noxious Cold temperature

Thymol Intracellular Ca2+

Ozone Zinc

Prostaglandins Formalin

4-Hydroxynonenal Hydrogen Peroxide

Menthol Hypochlorite

Icilin Tear gases

Sesquiterpenes Methyl isocyanate

Carvacrol (Oregano) Diallyl Disulfide (from Onions)

Eugenol (Cloves) Intracellular Alkalization

Hydroxysanshools Nitrooleic Acid

Nicotine Etomidate

14 agonists include both natural, plant-derived irritants (e.g., mustard oil), and pungent synthetic molecules (e.g., icilin, propofol) – in both cases the channel is acting as a nocisensor/alarm system for environmental chemicals capable of damaging tissue.

TRPA1 and Pain

Endogenous TRPA1 agonists include molecules such as 4- hydoxynonenal and the electrophilic prostaglandin, 15dPGJ2

(Trevisani, et al., 2007;

(Taylor-Clark, et al.,

2008); Cruz-Orengo, et al., 2008). Both of these molecules are Figure 4. Inflammatory soup. At the site of tissue found in the injury, peripheral terminals of sensory neurons are bathed in a potent concoction of inflammatory molecules „inflammatory soup‟ which contribute to inflammatory pain and hyperalgesia. Elements of this ‘soup,’ including certain prostaglandins, associated with tissue activate TRPA1. Taken from Julius & Basbaum, 2001. injury, and by activating

TRPA1, they contribute to the tissue sensitivity and neurogenic pain associated with inflammation.

The „inflammatory soup‟ is a potent concoction of pro-inflammatory signaling molecules released by cells in damaged tissues, and includes prostanoids, kinins, purines, growth factors, protons, amines, chemokines and proteases

(Figure 4). Peripheral sensitization, a state of altered physiology associated with inflammation in which sensory neurons display increased responsiveness to stimuli (manifested as hyperalgesia – heightened pain sensation) and a reduced threshold of activation (causing allodynia – pain in response to normally innocuous stimuli), has been linked to TRPA1 activation by components of the inflammatory soup. In one example, TRPA1 has been found to be essential for thermal hyperalgesia induced by bradykinin (BK) (Bautista, et al., 2009), a nonapeptide with potent pro-inflammatory properties (in this case, TRPA1 acts downstream of the BK receptor). Additionally, TRPA1 has also been implicated in airway inflammation and hyperreactivity in asthma (Caceres, et al., 2009) and in pain sensitivity in diabetes (Wei, et al., 2009). These recent findings indicating that TRPA1 is a key mediator of inflammation-related pain responses, heighten the clinical significance of its activation by endogenous ligands, and mark TRPA1 as a potential target for novel analgesic agents (Patapoutian, et al., 2009).

While recognition of TRPA1‟s significance has notably increased in recent years, most studies have focused on identifying TRPA1 ligands, with receptor regulation receiving less attention. Thus, at the present time, relatively little is known about how TRPA1 is regulated – a knowledge vacuum which is likely to be filled in the coming years, as more investigators set their sights on uncovering

16 this aspect of the channel‟s function. A cartoon illustrating the membrane topology of TRPA1 is presented in Figure 5.

Figure 5. TRPA1 topology cartoon. TRPA1, the only member of the TRPA family found in vertebrates, is a 6-pass TM protein 1100 + amino acids in length, with an extended N-terminal cytoplasmic region containing 14-18 ankyrin repeats. Taken from Caterina, 2007.

Cdk5 and Pain

Cyclin-dependent kinase 5 (Cdk5) could be termed a „misfit‟ member of the Cdk family, as it plays no known role in cell cycle regulation (Dhariwala &

Rajadhyaksha, 2008) and is not cyclin-dependent. While not regulated by

cyclins, Cdk5 activity is dependent

upon binding to one of its two

regulatory proteins, p35 or p39,

activators which bear no sequence

homology, but considerable 3-D

structural homology with cyclins

(Figure 6.) (Tarricone, et al.,

2001). A proline-directed serine- Figure 6. Cdk5/activator ribbon threonine kinase with a consensus structure. From Tarricone, et al., 2001. K S/T P X K/R/H phosphorylation

kjbjkbjbjkkbjbjbjbjkbjbjkbjbjkbjkbk sequence,

Cdk5 is principally active in post-mitotic neurons, due to the localized, transcriptionally and translationally regulated expression of its activators, p35/p39, in those cells (Tsai, Delalle, Caviness, Chae, & Harlow, 1994). Cdk5 plays many vital roles in the nervous system, and is essential for differentiation and migration of neurons during development (Cicero & Herrup, 2005; Hirasawa, et al., 2004). Cdk5 has also been implicated in regulation of synaptic transmission (Kim & Ryan, 2010), neuronal plasticity and learning (Fischer,

Sananbenesi, Schrick, Spiess, & Radulovic, 1997), memory (Fischer,

Sananbenisi, Pang, Lu, & Tsai, 2005), addiction (Takahashi, et al., 2005), apoptosis (Wang, Liu, Fu, Wang, & Lu, 2003), and pain signaling (Pareek, et al.,

2006; Pareek, et al., 2007; Pareek & Kulkarni, 2006) The involvement of the kinase in pain signaling is what‟s of greatest relevance in the present context.

A recent investigation demonstrated that p35 knockout mice and Cdk5 conditional (in neurons only) knockout mice are less sensitive to noxious thermal stimuli than wild-type mice, while mice overexpressing p35 are hyperalgesic.

Also in this study, Cdk5 activity was found to be increased in response to inflammatory insult (Pareek, et al., 2006). In two other, separate investigations,

Cdk5 inhibition was found to a.) attenuate formalin-induced pain responses in rats (Wang, et al., 2005), and b.) reduce capsaicin-induced Ca2+ influx in DRG neurons (Pareek, et al., 2007). The latter study pointed to direct Cdk5 phosphorylation of TRPV1 residue threonine 407, as a probable mechanism for its influence on channel activity. These findings, along with others indicating a role for Cdk5 in opioid tolerance (Xie, et al., 2009), have placed this unusual kinase squarely on the pain signaling map, as a potential „executive‟ or „master‟ regulator of multiple pain signaling pathways.

Regulation of TRPA1 by phosphorylation

While Cdk5 has been implicated in modulation of pain signaling, as yet no one has demonstrated a TRPA1/Cdk5 link. In fact, direct phosphorylation of

TRPA1 by any kinase has yet to be demonstrated – a remarkable fact, given the ubiquity of the phosphorylative mechanism in regulation of proteins in general

(Cohen, 2002), the size of the protein (1100+ amino acids), and the notable

20 phosphorylative regulation documented for other TRPs {Mandadi, et al., 2006;

Jung, et al., 2004; Hu, et al., 2002; Fan, et al., 2009; Yao, et al. 2005; Al-Ansary, et al., 2010). A survey of the TRPA1 literature reveals a great number of papers concerned with ligand activation of the channel – the number of pungent molecules known to exert their physiological effects either solely or partially through TRPA1 activation is growing all the time. Amidst this profusion of new data on ligand-mediated activation, however, one notes a lack of reports concerning covalent post-translational regulation of the TRPA1 receptor. Post- translational regulation of TRPA1 is an under-studied area, despite its potential importance for understanding this receptor‟s biology and roles in nociceptive signaling, and the promise such studies may offer toward the design of rational, novel modalities for the treatment of acute and chronic pain.

Scan for potential TRPA1 phosphorylation sites

Scanning the TRPA1 sequence for potential phosphorylation sites (Scansite and NetPhosK programs) shows that among several sites matching the Cdk5 consensus sequence, serine-448 (human numbering) returns the best score, a score which is nearly identical to that returned for threonine-407 of TRPV1, the residue directly phosphorylated by Cdk5 (Pareek, et al., 2007). Furthermore, this consensus sequence is conserved in at least 8 mammalian species – as one might expect for a motif which plays an important role in receptor regulation

(Figure 8).

Cdk5 Consensus site conservation

• TRPA1 sequence alignment

nCRQGVPVSVNNLLGFNVSIHSKSKDKKSPLHFAASYGRINTCQRLLQDMSDTRLLNEGD Horse n CRQGGPGSVNNLLGFNVSIHSKSKDKKSPLHFAASYGRINTCQRLLQDISDTRLLNEGD Human nn CRQGGPGSVNNLLGFNVSIHSKSKDKKSPLHFAASYGRINTCQRLLQDISDTRLLNEGD Chimp nn CRQGVPVSVNNLLNFNVSIHSKSKDK KSPLHFAASYGRINTCQRLLQDISDTRLLNEGD Cow nnCRQGVPVSVNNLLDFNVSIHSKNKDKKSPLHFAASYGRINTCQRLLQDMSDTRLLNEGD Pig nnCRQGVPVSVNNLLGFNVSIHSKSKDK KSPLHFAASYGRINTCQRLLQDISDTRLLNEGD Mouse nnCRQGAPVSVNNLLRFNVSVHSKSKDKKSPLHFAASYGRINTCQRLLQDISDTRLLNEGD Rat nnCRHGIPVSVNNLLDFNVSLRSKSKDK KSPLHFAASYGRINTCQRLLQDMSDTALLNEGD Dog

Figure 7. Multi-species sequence alignment of residues surrounding Serine 448 (Human numbering) of TRPA1.

Model Systems

A principal ex-vivo model used to study nociceptor function is the explanted, cultured sensory neuron of the dorsal root ganglion (DRG, illustrated in Figure

7.). These cells are generally excised from rat or mouse. When properly prepared, DRG neurons replicate many of the agonist responses and much of the regulatory milieu present in vivo

(Kress & Reeh,

1996; Cesare &

McNaughton,

1996) – and furthermore, Figure 8. DRG neurons grown in culture are express the regarded as an excellent model system for sensory nocisensors neuron function, as they replicate many of the typical of their physiological responses seen in-vivo. subtype on the plump, round cell body, itself – making them convenient for physiological study via the techniques of calcium imaging and patch clamping – no need to attempt imaging and patching of diminutive neurites. While cultured

23 primary DRG neurons may be seen to provide one of the best and most complete ex-vivo model systems for the molecular study of nociceptor behavior, heterologous cell lines are also often employed for the study of nocisensors.

When transfected with nocisensor complementary DNA (cDNA), heterologous cell lines, such as HEK 293 cells and CHO cells, provide an additional, highly-reductive model system for the study of nociciceptive mediators. Often, receptors expressed in these heterologous cells lines are found to exhibit characteristic response profiles very similar to those seen in primary DRG neurons (Savidge, Ranasinghe, & Rang, 2001) – offering a reassuringly reductive confirmation of experimental results, with a ready-made control – untransfected or empty-vector transfected cells.

It should be kept in mind, however, that transfected, heterologous cell lines are notably artificial systems, which fail to recapitulate all of the elements present in primary nociceptive cells. Thus, while „positive‟ results in these cell lines – where nocisensor behaviors match those seen in primary cell lines, seem safely subject to straightforward interpretation, „negative‟ results – where nocisensor behaviors differ between heterologous and primary cell lines, and can therefore be described as context-dependent, may be difficult to interpret. The best that can often be said in such cases is that one to many factors present/ absent in the primary vs. the heterologous cell line account(s) for the difference in results, and without knowing precisely what all of those factors are (and modifying the system accordingly), the more reductive, heterologous cell system is not an adequate model for the behavior in question.

Hypothesis and Project Rationale

The overall goal of this investigation is to identify the role of cyclin- dependent kinase 5 (Cdk5) in the modulation of transient receptor potential cation channel, subfamily A, member 1 (TRPA1) activity in sensory neurons.

The studies in Aim1 test the hypothesis that Cdk5 activity modulates TRPA1

2+ 2+ agonist-induced increases in intracellular free Ca concentration ([Ca ]i) in sensory neurons. Such modulation could provide a molecular basis for Cdk5‟s ability to alter some forms of pain sensation. Aim 2 seeks insight into the mechanism of Cdk5 influence on TRPA1 activity – and considering the fact that

Cdk5 is a kinase, the possibility that its modulation of TRPA1 activity involves receptor phosphorylation will be investigated. The following experimental findings led to the construction of the hypothesis: 1) Inhibition of Cdk5 activity was found to reduce formalin-induced pain in rats (Wang, et al., 2005); 2) TRPA1 is the principal mediator of formalin-induced pain (McNamara, et al., 2007;

Macpherson, et al., 2007; Kerstein, Camino, Moran, & Stucky, 2009). Based on the results of these recent studies, the possibility that Cdk5 might influence

TRPA1 activity emerges as a logical hypothesis. With both TRPV1 signaling

(Pareek, et al., 2007) and opioid tolerance (Pareek & Kulkarni, 2006; Xie, et al.,

2009) already demonstrated as being influenced by Cdk5 activity, the extension of Cdk5 regulation to TRPA1 presents no conceptual stretch, but would only strengthen the emerging view of Cdk5 as a potential „master regulator‟ of pain sensation pathways, and a promising target for novel analgesic drugs

Specific Aims:

1. To determine the role that Cdk5 plays in modulation of TRPA1 activity.

We will test the hypothesis that inhibition of Cdk5 activity leads to decreased

TRPA1 response to agonists. We will investigate whether:

A. Inhibition of Cdk5 activity attenuates TRPA1 agonist-induced increases in

2+ [Ca ]i in DRG neurons. B. Inhibition of Cdk5 activity fails to attenuate TRPA1 agonist-induced

2+ increases in [Ca ]i in HEK 293 cells, which lack significant Cdk5 activity.

2+ C. The attenuation of TRPA1-agonist-induced increases in [Ca ]i in DRG

neurons is readily reversible.

D. Inhibition of Cdk5 activity attenuates TRPA1 agonist-induced increases in

2+ [Ca ]i in DRG neurons in a manner dependent upon the dose of the inhibitor

2. To determine whether Cdk5 modulates TRPA1 activity via a mechanism

involving direct or indirect phosphorylation of the receptor. We will test

the hypothesis that active Cdk5 results in increased phosphorylation of the

TRPA1 receptor. We will:

A. Investigate whether the presence of active Cdk5 increases TRPA1

phosphorylation when expressed in a heterologous system.

B. Determine whether Cdk5 is able to phosphorylate specific TRPA1 peptides

via a direct molecular interaction.

We will utilize a multi-pronged experimental approach encompassing cell physiology, biochemistry and molecular biology to investigate these hypotheses.

These studies will contribute to our fundamental understanding of the TRPA1 receptor, an important nociceptive mediator.

CHAPTER 2

Materials and Methods

DRG Cell Isolation and Culture

DRG neurons from wild-type (30-40g) mice were used in this study. The ganglia were dissected from the lumbar (L1-L6) segments of the spinal cord and incubated with collagenase (Type IV, 0.15%) at 37°C for 50 min and dissociated by gentle trituration. Neurons were plated in 6-well dishes onto cover glasses pre-coated with poly-D-lysine and laminin (both from Sigma-Aldrich, St. Louis.

MO.), and cultured in a humidified atmosphere at 37°C and 5% CO2 in Ham‟s F-

12K medium supplemented with 10% fetal bovine serum, 100 ng/ml nerve growth factor and antibiotics. Proliferation of fibroblasts and Schwann cells was prevented through inclusion of cytosine arabinoside (5-10 µM) in the medium.

Cells begin to develop neurites within 24 hours and studies are performed within 24-48 hours of isolation.

Intracellular Ca2+ Measurements

DRG neurons were incubated (37ºC, 5% CO2) for 45 min with fura-2 acetoxy methylester (fura-2/AM; 2 µM,TEF labs, Austin, TX.) in Hepes-buffered saline solution (HBSS) containing the following: 118 mM NaCl, 4.8 mM KCl, 1.2 mM

MgCl2, 1.25 mM CaCl2, 11.0 mM dextrose, 5 mM pyruvate and 25 mM HEPES

26 27

(pH 7.35). Cover slips containing the fura-2-loaded DRG neurons were placed in a chamber (Warner Instruments, Hamden, CT.) mounted on the stage of an

Olympus IX-81 inverted fluorescence microscope (Olympus America, Center

Valley, PA.). The cells were superfused continuously with HBSS at a flow rate of

2 ml/min. Exposure of the DRG neurons to the interventions was performed by switching from control buffer to buffer containing the intervention for periods generally ranging from 20 to 60 seconds (noted in the legend), followed by a

2+ switch back to HBSS for washout. [Ca ]i measurements were performed on individual DRG neurons using a fluorescence imaging system (Easy Ratio Pro,

Photon Technology International, Lawrenceville, NJ.) equipped with a multi wavelength spectrofluorometer (DeltaRAM X) and a QuantEM 512SC electron multiplying CCD camera (Photometrics, Tuscon, AZ.). Images and photometric data were acquired by alternating excitation wavelengths between 340 and 380 nm (20 Hz) and monitoring an emission wavelength of 510 nm. Due to the fact that calibration procedures rely on a number of assumptions, the ratio of the light intensities at the two wavelengths was used to measure qualitative changes in

2+ [Ca ]i. Just before data acquisition, background fluorescence was measured and automatically subtracted from the subsequent experimental measurement using software from Photon Technology International (Easy Ratio Pro).

Culture of Immortalized Cell lines

HEK 293 cells were obtained from the American Type Culture Collection

(ATCC, Manassas, VA.), and were cultured in Dulbecco‟s Modified Eagle‟s

Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and

28 antibiotics (penicillin (100 U/ml), streptomycin (100 g/ml)) in a humidified atmosphere at 37° Celsius and 5% CO2.

Plasmid Transfection

Plasmids were transfected via lipofection (Lipofectamine 2000, Invitrogen,

Carlsbad, CA.) according to the manufacturer‟s recommendations. Transfection efficiency was estimated by co-transfecting an RFP-expressing vector (pIRES2,

DsRed2, Clontech, Mountain View, CA.) along with the vector containing the gene under study, or by using plasmids coding for GFP-chimeric versions of one or more genes of interest. Myc-tagged TRPA1 cloned into pcDNA5-FRT was the kind gift of Dr. Armen Akopian (University of Texas Health Science Center at San

Antonio). GFP-tagged Cdk5 and p25 were obtained from the Addgene plasmid bank (Cambridge, MA.), through the courteous donation of Li-Huei Tsai (Picower

Institute, Cambridge, MA.).

Peptides

Wild type and S448A mutant peptides corresponding to the sequence surrounding residue serine 448 of Human TRPA1 were obtained from Biomatik

(Wilmington, DE.), for in-vitro kinase assays. The 13-mer peptide sequences used were KSKDKKSPLHFAA (wild-type) and KSKDKKAPLHFAA (S-A mutant).

Chemicals

Unless noted otherwise, all chemicals and reagents were obtained from

Sigma-Aldrich. Propofol was obtained from the Cleveland Clinic pharmacy.

Statistical Analysis: Experiments were replicated a minimum of three times.

Statistical evaluation was done with Sigmaplot software (Systat Software, Inc.,

San Jose, CA.). Significant differences between experimental groups were assessed by one-way ANOVA (more than two groups) or unpaired Student‟s t-test (two groups). ANOVA was followed by Student‟s t-tests with a Bonferroni post-hoc correction for multiple comparisons, where significance was set at

p < 0.05.

Cell culture and transfection

HEK 293 cells were plated out onto 10 cm dishes and grown to 90 % confluency prior to transfection. Cells were co-transfected with plasmids coding for myc-tagged mouse TRPA1 along with either an RFP-coding empty vector

(control dishes) or with GFP-tagged Cdk5 and p25 plasmids (experimental dishes). Transfection was performed via lipofection with Lipofectamine 2000

(Invitrogen, Carlsbad, California), according to the manufacturer‟s instructions.

Twenty-four hours after lipofection, cells were briefly observed and photographed through an Olympus IX-81 epifluorescence microscope (Olympus America,

Center Valley, PA.), using FITC (for GFP) and Cy3 (for RFP) filters, to determine transfection efficiency (see figure 9).

Cell Lysis

Thirty to sixty minutes after being photographed, cell dishes were removed from the incubator and placed on ice. Growth medium was aspirated from plates and cells were briefly rinsed in ice-cold PBS. Following PBS aspiration, 500 L of

30 cell lysis buffer was added to each 10 cm plate, and cells were removed with a cell scraper (Costar, Corning Life Sciences, Lowell, MA.) and pipetted into 1.5 mL eppendorf tubes. Cell lysis was completed by rotating tubes for 45 minutes at 4° Celsius, and was followed by centrifugation at 7,000x g for 20 minutes (also at 4° Celsius). Supernatants were assayed for protein content via a modified

Lowry assay (Biorad DC, Biorad, Hercules, CA.).

Magnification = 100x Scale bars = 200 M

Figure 9. Representative fluorescence micrographs of green fluorescent protein (GFP, Panel A) and red fluorescent protein (RFP, panel B) -expressing HEK 293 cells, illustrating transfection efficiency of experimental (GFP) and control (RFP) plasmids used for phosphoserine western blotting experiments (Experiment 2a).

Preparation of tissue lysate (Kinase assay)

Rat Brain tissue was polytron-homogenized in a lysis buffer consisting of 150 mM NaCl, 50 mM Tris HCl (pH 7.4), 5 mM EDTA, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, Halt Protease inhibitor cocktail (1:100, Pierce/Thermo), and

PhosSTOP phosphatase inhibitor cocktail (1:9, Roche Applied Science,

Indianapolis, IN.) Homogenized lysates were then mixed on a lab rotator for 45 minutes and centrifuged at 10,000x g for 20 minutes (both at 4° Celsius). The resulting supernatant comprised the rat brain lysate, which then underwent a modified Lowry assay (Biorad DC) for determination of protein concentration.

Immunoprecipitation

HEK 293 cell lysates were diluted to the same concentration in lysis buffer such that equal quantities of protein (500-1500 g) and equal volumes (500-600

L) were loaded into experimental and control immunoprecipitation tubes.

Samples were pre-cleared for 60 minutes in 40 L of pre-washed protein A/G

PLUS agarose bead slurry (Santa Cruz Biotechnology, Santa Cruz, CA.), then incubated overnight with 7 g of anti-myc tag IgG (sc-40, Santa Cruz

Biotechnology) at 4° Celsius on a rotator. The following day, samples were added to 70 L of pre-washed protein A/G PLUS agarose bead slurry and rotated for two hours at 4° Celsius. Sample tubes were centrifuged briefly and „after IP‟ supernatants were removed. Beads were then washed four times in lysis buffer and one time in PBS. After the final wash, residual buffer was aspirated from the beads with a fine-gauge needle, 70 L of SDS-PAGE sample buffer was added

33 to each tube, and samples were boiled for 10 minutes in preparation for electrophoretic separation.

For kinase assays, Rat brain lysates were diluted to 1 g/ L in the aforementioned lysis buffer, and 500 L samples were pre-cleared by incubation with 50 L of Protein A/G PLUS agarose bead slurry (Santa Cruz Biotechnology) for 1 hour at 4° Celsius with rotation. Pre-cleared lysates were combined with 5

g of anti-Cdk5 IgG, (sc-173, Santa Cruz Biotechnology) and immunoprecipitated overnight at 4° Celsius on a rotator. The next day, samples were added to 25 L of Protein A/G PLUS agarose bead slurry (Santa Cruz Biotechnology), and incubated for an additional 3 hours at 4° Celsius. Samples were briefly centrifuged, „after IP‟ supernatants were removed, and beads were washed twice in lysis buffer and twice in kinase buffer (20 M Tris HCl (pH 7.4), 10 mM MgCl2, 1 mM EDTA, and PhosSTOP (1:9) phosphatase inhibitor, then resuspended in 30

L of H2O along with 10 L of kinase assay mixture (100 mM Tris HCl (pH 7.4),

50 mM MgCl2, 5 mM EDTA, and PhosSTOP phosphatase inhibitor cocktail (1:1))

This rat brain lysate-derived, immunoprecipitated, Cdk5 served as the enzyme source for kinase assays.

Electrophoresis and Western Blotting

35-40 L of bead supernatant per well were loaded into 4-15% gradient polyacylamide gels (Biorad), and run for 1 hour at 140 volts. Proteins were electrophoretically transferred to nitrocellulose membranes (All from Biorad) for 1 hour at 85 volts.

Membranes were blocked in 7% milk in TBST containing 50 mM NaF

(TBSTF), washed 3-4x in TBSTF, then incubated overnight in anti-phosphoserine

IgG (Sigma-Aldrich, St. Louis, Missouri), diluted 1:1000 in TBSTF containing 5% bovine serum albumin (BSA). Membranes were washed 3-4x in TBSTF, then incubated for 1 hour with horseradish peroxidase-conjugated, goat anti-mouse secondary antibody (1:10,000, sc-2005, Santa Cruz Biotechnology), followed by

4 additional washes in TBSTF. Immunoreactive bands were visualized via enhanced chemiluminescence (Supersignal West Pico or ECL substrate kits,

Pierce/Thermo, Rockford, IL.) and exposed to X-ray film. Band density for each lane was normalized to total TRPA1 after stripping membranes and reprobing with anti-myc tag IgG (sc-40, Santa Cruz Biotechnology). Signal intensity was quantified via densitometry using the ImageJ program (NIH, Bethesda, MD.).

In vitro peptide kinase assay reactions

TRPA1 peptides (wild-type and S-A mutant) (each at 0.2 M, Biomatik,

Wilmington, DE.) and 5 Ci of [ -32P] ATP (MP Biomedicals, Solon, OH.) were added to the resuspended, immunoprecipitated Cdk5 beads (Total volume of each reaction = 50 L), and incubated for 40 to 70 minutes at 30° Celsius. All reaction conditions were performed in triplicate. Reactions were quenched by the addition of 70 L of 10% trichloroacetic acid (TCA). Samples were subsequently centrifuged for 5 minutes at 10,000x g, and 20 L aliquots of supernatant were spotted onto P81 phosphocellulose squares in triplicate and air-dried in a fume hood. Squares were then individually washed in 6-well plates

35 with 75 mM Phosphoric acid (5 washes, 15-20 minutes each wash). A final 15 minute wash in acetone was followed by air-drying. Dried squares were placed in scintillation vials containing biosafe scintillation fluid, (ScintiVerse™ BD

Cocktail, Fisher Scientific, Pittsburgh, PA.) and quantified in a Beckman LS-6500 scintillation counter (Beckman-Coulter, Brea, CA.). Results were analyzed via one-way ANOVA followed by Student‟s t- tests with a Bonferroni post-hoc correction for multiple comparisons using Sigmaplot software (Systat Software,

Inc.).

For inhibitor kinase assays, the same procedure was followed as above, but

PD-98059 or Roscovitine (both from LC Laboratories, Woburn, Massachusetts), at a final concentration of 15 M, was added to immunoprecipitates prior to the addition of peptides and [ -32P] ATP.

Experimental Protocols

Protocol 1a: Hypothesis: Inhibition of Cdk5 activity will attenuate TRPA1-

2+ agonist-induced rise in [Ca ]i in DRG neurons.

Fura-2 loaded mouse DRG neurons were initially exposed to the known

TRPA1 agonists, Allyl isothiocyanate (AITC) and propofol, to establish baseline responsiveness. After return to baseline, incubation with HBSS containing either roscovitine or vehicle (dimethylsulfoxide (DMSO)) began, and continued for 30-

40 minutes. Challenge with TRPA1 agonists was then repeated in the same

36 manner used prior to inhibitor/control treatment. Inhibition protocols concluded with exposure to 50 mM KCl, to ensure the overall health/viability of cells.

Protocol 1b: Hypothesis: Cdk5 inhibition will not attenuate TRPA1-agonist-

2+ induced rise in [Ca ]i in transfected HEK 293 cells, which lack Cdk5 activity

HEK 293 cells were transiently-transfected with TRPA1 cDNA 24-48 hours prior to experiments. Fura-2 loaded HEK cells were initially exposed to known

TRPA1 agonists to establish baseline responsiveness. After return to baseline, incubation with HBSS containing either roscovitine or vehicle (DMSO) began, and continued for 30-40 minutes. Challenge with TRPA1 agonists was then repeated in the same manner used prior to inhibitor/control treatment.

Protocol 1c: Hypothesis: Cdk5 inhibition attenuates TRPA1-agonist-

2+ induced rise in [Ca ]i in DRG neurons by a readily reversible mechanism.

Fura-2 loaded mouse DRG neurons were initially exposed to the known

TRPA1 agonists, propofol and cinnamaldehyde, to establish baseline responsiveness. After return to baseline, incubation with HBSS containing roscovitine began, and continued for 20 minutes. Challenge with TRPA1 agonists was then repeated in the same manner used prior to inhibitor/control treatment. A 20 minute washout in HBSS followed, preceding a final challenge with TRPA1 agonist.

Protocol 1d: Hypothesis: Cdk5 inhibition attenuates TRPA1-agonist-

2+ induced rise in [Ca ]i in DRG neurons in a dose-dependent manner.

The same protocol was followed as was used in Protocol 1c, with the variable being the dose of inhibitor used (roscovitine, 10-100 M).

Protocol 2a: Hypothesis: The presence of active Cdk5 increases TRPA1 phosphorylation, when both are expressed in a heterologous system.

10 cm dishes of HEK 293 cells were grown to ~90% confluency, and were then either triple-transfected with myc-TRPA1/Cdk5/p25 DNAs (experimental cells), or double-transfected with myc-TRPA1/RFP-empty vector cDNAs (control cells). Twenty-four to thirty hours post-transfection, cells were lysed, subsequent lysates were assayed for total protein content, and normalized volumes/protein amounts were immunoprecipitated with anti-myc IgG. Post-immunoprecipitation samples underwent western blotting with anti-phosphoserine mouse primary antibody and HRP-conjugated anti-mouse IgG secondary antibody.

Phosphoserine (PS) immunoreactive bands were visualized via enhanced chemiluminescence, and bands at the molecular weight of myc-TRPA1 were quantified by densitometry. Membranes were then stripped and re-probed with anti-myc IgG, to normalize the PS signals to total TRPA1 content.

Protocol 2b: Hypothesis: Cdk5 can phosphorylate TRPA1 residue serine

448, in-vitro.

Cdk5 immunoprecipitated from rat brain lysate was combined with TRPA1 peptides and [ -32P] ATP, and incubated for 40-70 minutes at 30° Celsius.

Reactions were subsequently quenched with TCA, and sample tubes were centrifuged. Supernatant aliquots were then spotted onto P81 nitrocellulose squares, extensively washed, and radioactively quantified by liquid scintillation counting. Data were analyzed via one (more than two groups) or unpaired

Student‟s t test (two groups). ANOVA was followed by Student‟s t tests with a

Bonferroni post-hoc correction for multiple comparisons, where significance was set at p < 0.05.

Protocol 2c: Hypothesis: Cdk5 inhibition, but not MAPK inhibition, will block in-vitro phosphorylation of TRPA1 residue serine 448. The same basic protocol was followed as was used in Protocol 2b, except that either the Cdk5 inhibitor, roscovitine (15 M), or the MAPK inhibitor, PD-98059 (15 M), were added to some reaction tubes, prior to the addition of peptides and [ -32P] ATP.

CHAPTER 3

Results

Experiments 1a and 1b: Inhibition of Cdk5 activity attenuates TRPA1

2+ agonist-induced increases in [Ca ]i in DRG neurons, but not in TRPA1- transfected HEK 293 cells, which lack Cdk5 activity

Fura-2 loaded mouse DRG neurons or TRPA1-transfected HEK293 cells were first exposed to TRPA1 agonists (propofol and/or AITC, both at 100 M), to establish baseline response levels, and then underwent a 20 minute superfusion with either HBSS Ca2+ plus vehicle (DMSO), or with HBSS Ca2+ containing the

Cdk5 inhibitor, roscovitine (25-100 M). A 2nd exposure to TRPA1 agonists followed, testing TRPA1 responses in cells exposed to Cdk5 inhibition vs. controls. Typical traces are shown for control and inhibitor exposure experiments in mouse DRG neurons and for inhibitor exposure experiments in TRPA1- transfected HEK 293 cells. Summarized data are given as the ratio of peak heights post-exposure to vehicle or inhibitor compared to pre-exposure peak heights ± the standard error of the mean (SEM). Mean post:pre-exposure peak height ratios were 0.97 in control DRG neurons, 0.157 in inhibitor-exposed DRG neurons and 1.01 in inhibitor-exposed HEK 293 cells. Inhibitor-exposed DRG ratios differ significantly from both control DRG ratios and inhibitor-exposed HEK

39 40

293 cell ratios (p ≤ 0.01). Control DRG neuron ratios and inhibitor-exposed HEK cell ratios were not significantly different.

)

i allyl allyl

]

at at the

2+ M),

[Ca

(

bar) with the Cdk5

red

incubation incubation (

intracellular intracellular free calcium concentration or or potassium chloride (KCl) for 30 seconds

neurons, neurons, subsequent to

)

induced rise in

DRG

- M) for 60 seconds

M). DRG neurons were exposed to propofol (Prop, 100

agonist -

Representative calcium imaging trace illustrating attenuation of Transient Receptor

Figure Figure 10. mouse dorsal root ganglion ( inhibitor, roscovitine (50 isothiocyanate (AITC, 100 arrows. timeby points marked Potential A1 (TRPA1)

in in

)

i ]

[Ca (

for 60 seconds for at

)

Mouse dorsal root ganglion (DRG) neurons

intracellular intracellular free calcium concentration allyl isothiocyanate100 (AITC,

M)or

induced induced rise in -

agonist -

Representative control calcium imaging trace illustrating Transient Receptor

igure 11

F DRG DRG neurons in the absence of Cdk5 inhibition. exposed were to propofol (Prop, 100 arrows marked theby points time Potential A1 (TRPA1)

tine

roscovi

(which lack Cdk5

Transient Receptor TransientReceptor

for 60 seconds at the time

intracellular intracellular free calcium

) ) M

illustrating

Cells Cells were exposed to propofol

transfected HEK 293 cells

induced induced rise in -

agonist

in TRPA1

)

) i

]

allyl isothiocyanate (AITC, 100

TRPA1

( [Ca

(

M) M)

Representative calcium imaging trace Representative imaging calcium

both both before and after incubation with the Cdk5 inhibitor,

, , , , incubation time indicated by red bar).

igure12

100

F Potential A1 concentration activity) ( (Prop, 100 bypointsarrows marked

*= p < 0.01

Figure 13. Summarized data for the calcium imaging experiments represented in figures 10-12, illustrating the attenuation of Transient Receptor Potential A1 (TRPA1) -agonist-induced rise in intracellular 2+ free calcium concentration ([Ca ]i) by roscovitine (50 M) in mouse dorsal root ganglion (DRG) neurons, and the lack of such attenuation in TRPA1- transfected HEK 293 cells. Summarized data are given as the ratio of mean peak height post-exposure to inhibitor or vehicle compared to mean pre-exposure peak height. Mean post:pre-exposure peak height ratios were 0.97 in control DRG neurons (Control DRG, illustrated in figure 11), 0.157 in inhibitor-exposed DRG neurons (Inhibitor DRG, illustrated in figure 10) and 1.01 in inhibitor-exposed HEK 293 cells (Inhibitor HEK 293, illustrated in figure 12). Inhibitor- exposed DRG neuron ratios differ significantly from both control vehicle exposed DRG neuron ratios and inhibitor-exposed HEK 293 cell ratios. Control DRG neuron and inhibitor-exposed HEK 293 cell ratios did not differ significantly. Error bars = SEM. n = 6. NS = Not significant. = p < 0.01

Experiment 1c: Inhibition of Cdk5 activity attenuates TRPA1 agonist-

2+ induced increases in [Ca ]i in DRG neurons in a reversible manner

Fura-2 loaded mouse DRG neurons were first exposed to TRPA1 agonist, to establish baseline response levels, and then underwent a 20 minute superfusion with HBSS Ca2+ containing the Cdk5 inhibitor, roscovitine (10-100 M). A 2nd exposure to TRPA1 agonist followed, testing TRPA1 responses subsequent to

Cdk5 inhibition. A 20 minute inhibitor washout in HBSS Ca2+ came next, followed by a final exposure to TRPA1 agonist, testing the reversibility of the

TRPA1 response attenuation. A representative trace illustrating reversible inhibition is shown. Summarized data are given as the ratios of peak heights post-exposure vs. pre-exposure to inhibitor compared with peak heights post- washout vs. pre-exposure to inhibitor. Mean peak heights in inhibitor exposed cells were 16.5% of pre-exposure levels, while mean post-washout peak heights were at 99% of pre-exposure levels. * = p < 0.05.

) )

I ]

[Ca (

M, incubation time indicated

(50

for 60 seconds at the time points

reversible attenuation of Transient

) )

the the 0

(10 roscovitine

intracellular free calcium concentration

induced induced rise in -

agonist -

Cells were exposed to propofol

(TRPA1)

During During washout (time indicated by green bar), DRG neurons were superfused with buffer

Representative calcium imaging tracedemonstrating

. . . dorsal root ganglion (DRG) neurons induced by

igure 14

F in mouse by red bar) alone (no inhibitor present). by marked arrows Receptor Potential A1

Figure 15. Summarized data for the calcium imaging experiments represented in figure 14, demonstrating the reversibility of the attenuation in Transient Receptor Potential A1 (TRPA1) -agonist- 2+ induced rise in intracellular free calcium concentration ([Ca ]i) in mouse dorsal root ganglion (DRG) neurons induced by the Cdk5 inhibitor, roscovitine (50 M). Data represent mean ratios of peak heights post-exposure vs. pre-exposure to inhibitor (labeled Cdk5 inhibitor exposed) compared with peak heights post-washout vs. pre- exposure to inhibitor (labeled Post-washout).

Error bars = SEM n = 5. = p < 0.05

Experiment 1d: Inhibition of Cdk5 activity attenuates TRPA1 agonist-

2+ induced increases in [Ca ]i in DRG neurons in a dose-dependent manner

Cdk5 inhibition. A 20 minute inhibitor washout in HBSS Ca2+ came next, followed by a final exposure to TRPA1 agonist, demonstrating the reversible nature of the TRPA1 response attenuation. The same protocol was followed to establish the dose-dependent nature of the inhibition as was followed in experiment 1c, with the variable being the concentration of inhibitor present during the superfusion step. Summarized data are given as the ratios of peak heights post-exposure vs. pre-exposure to inhibitor. * = p < 0.05.

*= P < 0.05

P ≤ 0.001

Figure 16. Summarized data from calcium imaging experiments demonstrating the dose-dependent nature of attenuation in Transient Receptor Potential A1 (TRPA1) -agonist-induced rise in 2+ intracellular free calcium concentration ([Ca ]i) in mouse dorsal root ganglion (DRG) neurons induced by the Cdk5 inhibitor, roscovitine

(10-100 M, as labeled). Data represent mean ratios of peak heights post-exposure vs. pre-exposure to inhibitor. Error bars = SEM. n ≥ 4. = p < 0.05

Experiment 2a: Cdk5 phosphorylates TRPA1 in transfected HEK293 cells, as demonstrated by phosphoserine western blot

HEK 293 cells were either triply-transfected with myc-tagged TRPA1/Cdk5-

GFP/p25 (experimental), or were doubly-transfected with myc-tagged TRPA1/

RFP empty vector (control). Myc-tagged mouse TRPA1 was immunoprecipitated from transfected HEK 293 cells with anti-myc IgG, and subjected to western blot with anti-phosphoserine IgG. TRPA1 phosphoserine immunoreactivity was quantified and normalized to total TRPA1 after stripping and reprobing with anti- myc IgG, to correct for differences in transfection efficiency and loading. A certain amount of TRPA1 phosphoserine immunoreactivity was observed in the control, non-Cdk5/p25-transfected state. In Cdk5/p25-transfected cells, mean normalized band density increased by 3.3 fold =+/- 0.49 compared to control, p <

0.05. The blots shown are typical of three similar results.

RFP vector control Cdk5/p25 experimental

Figure 17. Phosphoserine western blots: The upper blot shows* =P ≤ 0.05 phosphoserine immunoreactivity in control RFP vector vs. experimental Cdk5/p25Sryeryeryehgerhedrhgedbhgftjngfhjgjkhgkbvkmbv,mbvmbmbnm co-transfected HEK 293 cells expressing myc- tagged Transient Receptor Potential A1 (TRPA1). Myc-tagged TRPA1 was immunoprecipitated (IP) from transfected cell lysates with anti- myc IgG. Membranes were subsequently subjected to western blot (WB) with anti-phosphoserine antibodies (upper blot). The lower blot shows myc-TRPA1 immunoreactivity in the same lanes after stripping and reprobing. The blots shown are typical of three similar results.

*= P < 0.05

P ≤ 0.001

Figure 18. Summarized data for western blotting experiments illustrated in figure 17, depicting mean phosphoserine band densities normalized to total myc-tagged Transient Receptor Potential A1 (myc-TRPA1) immunoreactivities in myc-TRPA1- transfected HEK 293 cells co-transfected with either RFP vector (Control) or Cdk5/p25 (Cdk5/p25 transfected). Error bar = SEM.

n = 3. = p < 0.05

Experiment 2b: Cdk5 phosphorylates TRPA1 peptide in vitro, but fails to phosphorylate Ser448Ala mutant peptide

Cdk5 immunoprecipitated from rat brain was combined with TRPA1 wild-type and control peptides (each at 0.2 M) and 5 Ci of -32P-ATP, then incubated at

30° Celsius for 70 minutes. After TCA precipitation and centrifugation, 20 L supernatant aliquots were spotted onto P81 phosphocellulose squares and air dried, then extensively washed to remove unbound signal prior to radioactive quantification via liquid scintillation counting. Each reaction condition was performed in triplicate, and each replicate reaction was spotted onto phosphocellulose squares in triplicate (A total of nine squares per reaction condition). The replicates from individual reactions were averaged prior to statistical analysis. Data were analyzed via Student‟s t-test , using Sigmaplot software (Systat Software, Inc., San Jose, CA). Mean disintegrations per minute

(DPMs) were 15.4-fold higher in wild type peptide vs. S448A mutant peptide, with p < 0.001.

**= P ≤ 0.001

Figure 19. TRPA1 peptide in-vitro kinase assay. Cdk5 was immunoprecipitated from rat brain lysate and used as the kinase source for kinase assays. Bars represent mean disintegrations per minute (DPM) 32P per 1.66 picomoles of peptide spotted onto P81 phosphocellulose squares for S448A mutant (S-A mutant peptide) and wild-type (Serine 448 peptide) peptides after undergoing an in- vitro kinase assay. Data are given as mean disintegrations per minute. (DPM) ± SEM. n = 3. P ≤ 0.001 Student’s t-test

Experiment 2c: In-vitro phosphorylation of wild-type TRPA1 peptide is abrogated by Cdk5 inhibition, but not by MAPK inhibition

Cdk5 immunoprecipitated from rat brain lysate was combined with TRPA1 wild-type peptide (0.2 M), kinase inhibitor (Roscovitine or PD-98059, 15 M) or vehicle (DMSO, 1:3333), and 5 M of 32P-ATP, then incubated at 30° Celsius for 40 minutes. After TCA precipitation and centrifugation, 17 L supernatant aliquots were spotted onto P81 phosphocellulose squares and air dried, then extensively washed to remove unbound signal prior to radioactive quantification via liquid scintillation counting. Each reaction condition was performed in triplicate, and each replicate reaction was spotted onto phosphocellulose squares in triplicate (A total of nine squares per reaction condition). The replicates from individual reactions were averaged prior to statistical analysis. Data were analyzed via one-way ANOVA followed by Student‟s t-tests with Bonferroni‟s correction for multiple comparisons, using Sigmaplot software (Systat Software,

Inc.). Radioactive counts were 17.3-fold higher in vehicle vs. roscovitine-treated peptides, with p < 0.001, while PD-98059-treated peptides did not differ significantly from those treated with vehicle.

**= P ≤ 0.001

. P ≤ 0.001

**= P ≤ 0.001

Figure 20. TRPA1 peptide in-vitro kinase assay with Inhibitors. Cdk5 was immunoprecipitated from rat brain lysate and used as the kinase source for kinase assays. Bars represent mean disintegrations per minute (DPM) 32P per 1.13 picomoles of peptide spotted onto P81 phosphocellulose squares for wild-type peptides after undergoing an in-vitro kinase assay in the presence of Cdk5 inhibitor (Roscovitine, 15 M), MAPK inhibitor (PD-98059, 15 M), or dimethylsulfoxide vehicle alone (Control untreated). Control peptides and MAPK inhibitor exposed peptides DPMs did not differ significantly, while Cdk5 inhibition significantly reduced DPMs. Data are given as mean disintegrations per minute. (DPM) ± SEM. n = 3. NS = Not significant. P ≤ 0.001. One-way ANOVA followed by Student’s t-tests with Bonferroni’s correction for multiple comparisons.

CHAPTER 4

Discussion

TRPA1 is a large protein more than 1100 amino acids in length, clearly offering abundant scope for rich and varied regulation of channel function. Yet despite widespread recognition of the receptor‟s importance, to a great degree,

TRPA1 remains a vast and largely unexplored protein realm. The studies detailed in this dissertation were untaken in an effort to provide additional insight into TRPA1 function and regulation, and thereby contribute to our limited pool of knowledge about this important nociceptive mediator.

The results of this investigation provide physiological evidence for Cdk5 regulation of TRPA1 activity in DRG neurons, and are consistent with a regulatory mechanism involving direct phosphorylation of the receptor by Cdk5.

Notably, no previous studies have implicated Cdk5 as a modulator of TRPA1 activity, nor have previous studies provided evidence for phosphorylation of

TRPA1 residues by any kinase.

Recent findings by other investigators provided the intellectual impetus for the initiation of the present investigation. An in-vivo study reported that inhibition of Cdk5 activity reduced formalin-induced pain in rats (Wang, et al., 2005), while other separate investigations have recently determined that TRPA1 is the principal mediator of formalin-induced pain (McNamara, et al., 2007;

Macpherson, et al., 2007; Kerstein, et al., 2009). Putting two and two together,

57 58 the possibility that Cdk5 might act as a regulator of TRPA1 signaling emerges.

Considering these earlier reports, the present study tested the hypothesis that

Cdk5 inhibition would attenuate TRPA1 responses in DRG neurons. Due to the fact that Cdk5 is a kinase, this investigation also tested the hypothesis that Cdk5 activity leads to phosphorylation of TRPA1. The main findings of the present

2+ study are that Cdk5 inhibition attenuates rise in [Ca ]i in response to TRPA1 agonists in DRG neurons, that Cdk5 activity increases TRPA1 phosphoserine immunoreactivity in transfected HEK 293 cells, and that Cdk5 directly phosphorylates a substrate corresponding to TRPA1 residue Serine 448 in-vitro.

Effect and characteristics of Cdk5 inhibition on TRPA1 agonist-induced

2+ increases in [Ca ]i in DRG neurons, and in TRPA1-transfected HEK 293 cells.

As a previous in-vivo study indicated that Cdk5 inhibition attenuates the formalin-induced flinch response in rats (Wang, et al., 2005), and other, subsequent investigations have demonstrated the primacy of TRPA1 as a mediator of formalin-induced pain (McNamara, et al., 2007; Macpherson, et al.,

2007; Kerstein, et al., 2009), experiments were designed to test the hypothesis that Cdk5 modulates the activity of TRPA1 in DRG neurons. Fura-2 calcium imaging, an extremely-sensitive technique for the analysis of ion flux through calcium permeable channels, was the experimental method of choice. DRG neurons were first treated with the TRPA1 agonists, AITC and propofol, then incubated with the Cdk5 inhibitor, roscovitine, and subsequently re-treated with

59 the TRPA1 agonists. A final depolarizing exposure to KCl was performed, to ensure that inhibitor-exposed cells were still healthy and viable at the end of the protocol, and not suffering from a generalized neurotoxic effect.

2+ The results indicate a significant attenuation of rise in [Ca ]i in response to

TRPA1 agonist after incubation with the Cdk5 inhibitor. Control DRG neurons

2+ not exposed to Cdk5 inhibitor did not exhibit a reduction in [Ca ]i signal at the time of re-treatment with TRPA1 agonists. Together, the results from experiment

1a indicate that Cdk5 inhibition is the variable responsible for the observed attenuation of TRPA1 response to agonist in DRG neurons – a result which matches the pre-experimental hypothesis.

As an additional control, inhibitor experiments were repeated using TRPA1- transfected HEK 293 cells, rather than DRG neurons, as the model system. The

Cdk5 antagonist is expected to be without effect in this cell line, due to a lack of significant Cdk5 activity (Nair, Simonetti, Fabbretti, & Nistri, 2010; Jämsä, et al.,

2+ 2009). The results show that Cdk5 inhibition has no influence on rise in [Ca ]i in response to TRPA1 agonists in transfected HEK 293 cells, even at the highest dose used (100 M roscovitine), providing evidence for the target-specific action

2+ of the Cdk5 inhibitor, and confirming that attenuation of rise in [Ca ]i is not due to direct inhibitor interaction with TRPA1 itself.

The differing behavior of TRPA1 channels expressed in cultured DRG neurons vs. transfected cell lines is of notable interest in light of the findings of this study – when TRPA1 is natively expressed in DRG neurons, response to receptor agonist is attenuated by Cdk5 inhibition, but when transfected into HEK

293 cells, TRPA1 responses are unaffected by Cdk5 inhibition. As HEK 293 cells have been demonstrated to lack significant Cdk5 activity (Tsai, et al., 1994;

Tang & Wang, 1996; Nair, et al., 2010; Jämsä, et al., 2009; Li, Zhang, Gu, &

Amin, 2000), it naturally follows that application of Cdk5 inhibitor to these cells should have no effect on calcium flux through transfected TRPA1 channels – unless the Cdk5 inhibitor were acting on a target other than Cdk5. Thus, the

TRPA1-transfected HEK cells inhibition experiment represents an important control against the possibility of non-specific inhibitor effects. The interpretation of these results from this „control experiment perspective‟ appears clear, straightforward and unambiguous.

An additional aspect of these results concerns the context-dependence of the requirement for Cdk5 activity for TRPA1 function. When transfected into the

„artificial,‟ non-nociceptor context of HEK 293 cells, the presumably more „naked‟

TRPA1 receptor (lacking in regulatory factors and binding partners present in nociceptive neurons) responds robustly to agonist, in a manner independent from

Cdk5 activity. When endogenously expressed in the „native‟ nociceptor environment of DRG neurons (with the full coterie of regulatory and binding partners present), however, TRPA1 response to agonist acquires a dependence upon Cdk5 activity. These results would seem to imply that a tonic inhibition of

TRPA1 activity is present in DRG neurons (but not in HEK 293 cells), and that this tonic inhibition is relieved by Cdk5 activity. Determining the identity of the regulatory factors involved in this nociceptor-specific tonic inhibition of TRPA1 function stands as a goal for future studies.

That a more layered and intricate regulatory machinery would have evolved to control and modulate TRPA1 nocisensor function within the highly- specialized environment of the nociceptive sensory neuron appears eminently logical. The placement of Cdk5 within such a regulatory framework is also far from surprising – as a factor principally active in post-mitotic neurons and which has previously been demonstrated to play key roles in multiple aspects of neuronal function – including pain signaling (Pareek et al., 2006; Pareek et al.,

2007; Pareek & Kulkarni, 2006; Xie et al., 2009), Cdk5 seems a most apt factor to play a modulatory role in TRPA1-related nociception.

Experiment 1c was designed to test the idea that Cdk5 inhibition attenuates

2+ rise in [Ca ]i in response to TRPA1 agonist in DRG neurons by a fully reversible mechanism. Kinase phosphorylation of substrates is generally a short-term modification unless actively maintained, due to the ubiquitous activity of protein phosphatases. For this reason, incubation with kinase inhibitor leads to a rapid de-phosphorylation of kinase substrates – and just as readily, washout of inhibitor, by returning the kinase to an active state, should lead to rapid re- phosphorylation of substrates. Therefore, TRPA1 modulation by Cdk5 activity, whether by direct or indirect interaction, would be expected to be a reversible on/off process. The results of experiment 1c demonstrate that TRPA1 responses exhibit the anticipated reversibility under the influence of Cdk5 inhibition/washout, congruent with channel modulation by kinase action. Additionally, Cdk5 inhibition with roscovitine exhibited a dose-dependent effect on TRPA1 responses, as

62 would be expected for a biological response to enzyme inhibition (Experiment

1d).

Effect of Cdk5 activity on TRPA1 phosphorylation

In light of the altered TRPA1 physiological responses observed with Cdk5 inhibition in DRG neurons, attempting to ascertain the mechanism(s) by which

Cdk5 influences TRPA1 activation is a logical next step. As kinases are enzymes which add phosphoryl groups to their substrates, investigating whether

Cdk5 activity is associated with alterations in the phosphorylation status of

TRPA1 appears to be a good place to start a search for mechanism.

Phosphorylation of channel residues as a means of regulation has been previously reported in multiple other members of the TRP superfamily, including

TRPV1, TRPV4 and TRPV6 (Mandadi, et al., 2006; Jung, et al., 2004; Hu, et al.,

2002; Lee, et al., 2008; Macpherson, et al., 2007), but not in TRPA1. To investigate the effect of Cdk5 activity on global serine phosphorylation of the ion channel, phosphoserine immunoreactivity was probed via western blot of TRPA1 immunoprecipitated from TRPA1/Cdk5/p25 co-transfected (experimental) and

TRPA1/RFP empty vector co-transfected (control) HEK 293 cells. Data indicate that the presence of active Cdk5 increases total phosphoserine immunoreactivity of TRPA1. This result is consistent with kinase modulation of receptor activity by a phosphorylative mechanism, but sheds no light on which residues might be phosphorylated, or on whether Cdk5 is capable of directly phosphorylating the receptor (as opposed to working through a downstream kinase).

In the interest of locating potential Cdk5 phosphorylation sites on TRPA1, bioinformatics searches were performed using the NetPhosK and Scansite programs. The TRPA1 residue which returned the highest score as a potential

Cdk5 consensus site was serine 448 (human numbering). To investigate whether S448 could act as a Cdk5 substrate, an in-vitro Cdk5 kinase assay was performed using wild-type and S448A mutant peptide substrates corresponding to the region encompassing Serine 448 of TRPA1. The results indicate that

Cdk5 robustly phosphorylates the wild-type peptide, while radioactive counts for the S448A mutant were close to background levels – clearly demonstrating that

S448 can act as a Cdk5 substrate in-vitro. These results were further bolstered by performing additional Cdk5 kinase assays in the presence of either the MAPK inhibitor, PD-98059, or the Cdk5 inhibitor, roscovitine. The Cdk5 specificity of the observed phosphorylation was reliably confirmed in these assays, as Cdk5 inhibition significantly reduced radioactive counts – taking them down to levels comparable to background, while MAPK inhibition did not significantly alter counts, relative to uninhibited control.

The mechanism(s) by which Cdk5 affects TRPA1 ion channel activity is an important question to be answered. Phosphorylation, by itself, is often adequate as a means of modifying protein function – that is, the addition of the negatively- charged phosphate group alone is sufficient to activate/deactivate, sensitize/desensitize, etc. In the case of modulation of TRPA1 by Cdk5, such a

„phosphoryl only‟ mechanism would appear unlikely, as the channel is functional when transfected into cells which lack Cdk5 activity, but is dysfunctional in DRG

64 neurons, in the absence of Cdk5 activity. Thus, Cdk5 influence on TRPA1 channel function (and of TRPV1 function (Pareek, et al., 2007), whether exerted solely via direct phosphorylation or not, appears to require the presence of additional regulatory factors.

The simplest model consistent with the data would be one in which Cdk5 phosphorylation of TRPA1 prevents binding of an allosteric inhibitory factor (a factor present in DRG neurons, but not in HEK cells) to the receptor. Such an inhibitory factor might act by sequestering de-phosphorylated TRPA1 away from the plasma membrane, or might help place the ion channel into an inactive conformation or complex. Notably, either of these mechanisms have the potential to be easily reversible – which matches the results observed in experiment 1c of the present investigation.

This sort of indirect model differs from that seen in instances such as

PKC regulation of TRPV1 (Mandadi, et al., 2006), where Serine 800 phosphorylation alone is sufficient to alter receptor function (sensitizes) in both heterologous cell lines and in DRG neurons. In cases of this PKC /TRPV1 type, where mode of regulation is less context-dependent, and can therefore be readily elucidated in reductive systems such as heterologous cell lines, study and characterization are simplified. In more context-dependent cases, as is observed in Cdk5 regulation of TRPA1 (in the present investigation) and TRPV1 (Pareek, et al., 2007), study and characterization are somewhat less straightforward, as the optimally-reductive system is hard to define/create.

Our understanding of nociceptor regulation, and perhaps most other biological subfields, inevitably tends to skew towards those facets which are most readily studied – how could it be any other way? (as is illustrated by the extreme case where a subject can‟t be studied at all, and therefore we‟re in no position to say anything about the matter, from an empirical, scientific perspective). As amenability to investigation and biological significance are unlikely to be perfectly correlated, however, the low-hanging fruit analogy comes into play – if you‟re truly hungry (for knowledge), it‟s best not to overlook the slightly less accessible, somewhat higher-placed fruit, though a step ladder may be required for access – at the end of the day, the lower and the slightly-higher placed fruit may both prove equally filling (enlightening).

It might be useful to think along these lines when considering cell signaling research, as there very likely exist numerous undescribed regulatory pathways/networks. The unraveling of at least some of these presently-unknown regulatory modes might hold the potential to contribute significantly, both to our fundamental biological knowledge and to the development of novel therapeutic agents. And though hypothetical until characterized, such modes of regulation, while presently-cryptic, may well be equally as real (or perhaps, in the case of

TRP channels, painfully as real) as presently-described pathways – but merely more problematic to study.

Conclusions and future directions

As improved understanding of TRPA1 regulation and functional interactions is an essential step toward the development of rationally-designed therapeutics targeting the receptor, the novel findings described in this study have potential utilitarian implications, in addition to enhancing our knowledge of basic nociceptor biology. Further insight into the mechanistic interactions underlying Cdk5 regulation of TRPA1 (and TRPV1) can only help in this regard.

Yet to be determined is whether Cdk5 activity affects intracellular trafficking of TRPA1 in DRG neurons – with inhibition sequestering the receptor away from the plasma membrane. The role of TRPA1-TRPV1 interactions

(which have been described elsewhere (Salas, Hargreaves, & Akopian, 2009;

Staruschenko, Jeske, & Akopian, 2010; Akopian, Ruparel, Jeske, & Hargreaves,

2007)) in Cdk5 regulation of nociception has also not been investigated. Notably,

TRPA1 is expressed in a subset of capsaicin-sensitive (i.e., TRPV1-expressing) sensory neurons – so if TRPA1 is present, then TRPV1 is there also (Jordt, et al.,

2004; Story, et al., 2003). The possibility that TRPA1/TRPV1 cross-talk may play a role in Cdk5 modulation of nociception therefore exists, and is a notion worthy of examination – especially considering the fact that both receptors have now been separately found to be influenced by Cdk5 activity (Pareek, et al., 2007;

Sulak, 2011). Detailed proteomic analyses of TRPA1 binding proteins in DRG neurons – from both wild-type and Cdk5 knockout mice, also hold the potential to go a long way towards unraveling the nature of nociceptor-specific modulation of

TRPA1 activation by Cdk5. Such studies could indicate which factors associate

67 with TRPA1 in a Cdk5-dependent manner. Calcium imaging experiments using

DRG neurons from these same Cdk5 knockout mice would also be of obvious interest. The transfection of TRPA1 cDNA into DRG neurons from TRPA1 knockout mice would create another potentially informative experimental system.

If Cdk5 modulation of TRPA1 activity in this system is found to mirror that seen in wild-type DRGs, the possibility for physiological analysis of an entire series of mutant and chimeric TRPA1 variants would be opened up– allowing additional light to be shed on the residues and domains of TRPA1 necessary for Cdk5 influence. In short, our understanding of nocisensory biology at the molecular level is still in a nascent state – with so much still to learn, there should be no shortage of research projects for quite a long time into the future.

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