Molecular Mechanisms and Regulation of Cold Sensing

Molecular mechanisms and regulation of cold sensing

A dissertation submitted to the

Division of Research and Advanced Studies Of the University of Cincinnati in partial fulfillment of the requirements for the degree of

DOCTORATE OF PHILOSOPHY (Ph.D.)

in the Neuroscience Graduate Program in the College of Medicine

Ignacio Sarria

B.A. St. Thomas University, Miami, Florida 2006

October 2011

Dissertation Committee:

Jianguo Gu, Ph.D., Advisor Steve Kleene, Ph.D., Committee Chair: Mark Baccei, Ph.D. Jun-Ming Zhang, M.D, Ph.D. David Richards, Ph.D.

Sarria, I

General Abstract

TRPM8 is the principal sensor of cold temperatures in mammalian primary sensory neurons. Cold temperatures 28~8°C and the cooling compound menthol activate TRPM8.

TRPM8 is expressed on nociceptive and non-nociceptive primary sensory neurons and mediates innocuous and painful cold sensations. Using calcium imaging, I examined menthol responses and role of protein kinases in two functionally distinct populations of cold-sensing DRGs that use TRPM8 receptors to convey innocuous (menthol-sensitive/capsaicin-insensitive, MS/CI) and noxious (menthol-sensitive/capsaicin-sensitive, MS/CS) cold sensation. PKC activation decreased menthol response in all neurons. MS/CI neurons had larger menthol responses with greater adaptation and adaptation was attenuated by blocking PKC and CaMKII. In contrast

MS/CS neurons had smaller menthol responses with less adaptation that was not affected by blocking PKC or CaMKII. In both MS/CI and MS/CS neurons, menthol responses were not affected by PKA activation or inhibition. Taken together, these results suggest that TRPM8- mediated responses are different between non-nociceptive-like and nociceptive-like neurons

(Chapter II).

Calcium influx causes a feedback regulation of TRPM8 currents that when analyzed under whole-cell voltage-clamp exhibit a Ca2+-dependent functional downregulation with two distinctive phases, a shorter, faster acute desensitization and a prolonged tachyphylaxis. Using acutely dissociated rat DRGs I examined TRPM8 whole-cell currents while pharmacologically manipulating several intracellular targets. TRPM8 acute desensitization is caused by calmodulin and requires phosphatidylinositol 4,5-bisphosphate (PIP2). Conversely, tachyphylaxis is mediated by hydrolysis of PIP2 and activation of PKC/phosphatase 1,2A. Consequently, I set out to determine the mechanisms underlying the mentioned findings by studying inside-out

Sarria, I recordings of TRPM8 channels stably expressed in HEK 293 cells. PIP2 switches TRPM8 channel gating to a high open probability state with short closed times and Ca2+-calmodulin reverses the effect of PIP2, switching channel gating to a low open probability state with long closed times. Thus, through gating modulation, Ca2+-calmodulin provides a mechanism to rapidly regulate TRPM8 functions in the somatosensory system (Chapter III).

It is not well understood how cooling temperatures have multiple sensory effects ranging from generating cooling or painful cold sensation to modifying sensory modalities like touch, itch and pain. With electrophysiology I studied how temperature modulates excitability in

DRGs. Cooling temperatures differentially modify the excitability of non-nocicepetive and nociceptive neurons. Cold aborts repeated action potential firing in non-nociceptive neurons by increasing the voltage-dependent inactivation of TTXs Na+ channels and reducing A-type K+ currents. Cooling temperatures also inhibit IA in nociceptive-like neurons, which possessed

TTXr Nav 1.8 channels, but these neurons largely retain or increase firing rate. Cold had less inhibition on TTXr Na+ channels, allowing nociceptive neurons to fire at painful cold temperatures. Like cold, IA blocker 4-AP reduced IA-K+ currents in TTXs and TTXr cells, but this led to higher spike frequency only in the latter. Finally, the molecular determinants for neuron excitability under cooling temperatures play a role in defining temperature threshold and ranges for which innocuous and noxious cold directly elicit impulses in nociceptive and non- nociceptive cold-sensing neurons respectively, providing a molecular mechanism for sensory distinction between innocuous and noxious cold stimuli (Chapter IV).

Sarria, I

Acknowledgments

I would like to thank the following people:

Advisor: I would like to deeply thank Dr.Gu for welcoming me in his lab and providing an encouraging scientific niche where I could thrive. You have been truly a very good mentor and I will certainly miss our almost daily discussions. Thank you for introducing me to the world of electrophysiology and for the valuable training and teaching you offered me throughout graduate school.

Committee members: Thank you Dr. Steve Kleene, Dr. Mark Baccei, Dr. Jun-Ming Zhang, and

Dr. David Richards, for your help and guidance.

Lab members: Myeounghoon Cha, and Jennifer Ling. Thank you for your help, friendship, and tenderness Jennifer. You made looking at DRGs exciting.

Friends: Balu, Freddy, and Kostas. Good friends make the best buffer from grad school’s hardships.

Girlfriend: Thank you Megan for all your support, patience and levity. Stacking the freezer with food has saved me this last month.

Parents: Thank you for being so wonderful throughout the years. Since I was a child, you have fostered me and my inquisitive nature with patience and affection. I remember you never discouraged me, even when I asked a hundred questions in our trips to San Jose. Thanks to you I became an older kid who still likes asking the what, why, and how of things; I just do it a lab now. You are simply the best parents a son could ever have and I dedicate this to you.

Sarria, I

Table of Contents

Page

GENERAL ABSTRACT………………………………………………………………...... iii

ACKNOWLEDGEMENTS…….………………….…...…………………………...... …vi

TABLE OF CONTENTS……………….……………………………………………...... …vii

LIST OFTABLES AND FIGURES……….………………………………...…………...... ix

CHAPTER 1: General Introduction.…………...……………………………………...... 12

CHAPTER 2: TRPM8-mediated responses and adaptation are different in nociceptive-like vs. nonnociceptive-like neurons: role of protein kinase ...... …...………………………………………………...... 24

Abstract…..………...... ……...... …...…..………………...25

Introduction………...... 26

Materials and Methods……...…...... …...... ……...... ……………………….....29

Results...... ………...... ………………………………………………...... 33

Discussion ..……...... ….…………………………………………………..…...... 39

Figures……………………...…...... …….…………...... ………………………...... 43

Notes to Chapter2…..………...... …………………………...…………...…...... 53

CHAPTER 3: TRPM8 acute desensitization is mediated by Calmodulin and requires PIP2: Distinction from Tachyphylaxis ...... 54

Abstract………………….……………………………………...... …55

Introduction…...... …………………………………………………………56

Materials and Methods....………………...... ………………………………...60

Results…………………………...... ………………………………………....64

Discussion.....……………………………………...... ………………………..72

Sarria, I

Figures………...... …………………………………………77

Notes to Chapter 3……...…...... ……………………………………...... ……..92

CHAPTER 4: Cold differentially modifies sensory neuron action potential firing properties: Contribution to sensory distinction between innocuous and noxious cold...... 93

Abstract…………...... ……………………………………………………..…94

Introduction……………...... …………...……………………………………96

Materials and Methods………...... …..……………………………………...100

Results……………………...... ……..……………………………………....105

Discussion...…………...... …………………………………………………..116

Tables and Figures…...... ………………………………………………...….121

Notes to Chapter 4...... ….…….………………………...………………….135

CHAPTER 5: Summary...... 136

Figures………...... …………………………………………143

REFERENCES...... 146

Sarria, I

List of tables and figures

Page

CHAPTER 1

CHAPTER 2

Figure 2.1. Responses to menthol, capsaicin and AIT in dorsal root ganglion

neurons of rats...... …...... …...... ……...………....…...... 43

Figure 2.2. Menthol responses in menthol-sensitive/capsaicin-insensitive and

menthol-sensitive/capsaicin-sensitive neurons following prolonged menthol application...... 45

Figure 2.3. Recovery after menthol-induced adaptation in

menthol-sensitive/capsaicin-insensitive and menthol-sensitive/capsaicin sensitive neurons..46

Figure 2.4. Responses to multiple brief applications of menthol...... …….…...... ……….47

Figure 2.5. Effect of PKC activator PDBu on menthol responses...... …..…....…..……….48

Figure 2.6. Lack of effect by PKA activators on menthol responses...... …...... …..……….50

Figure 2.7. Effect of protein kinase inhibitors

on menthol responses in MS/CI and MS/CS neurons...... 51

CHAPTER 3

Figure 3.1. Menthol-and cold-evoked currents in DRG neurons

bath-perfused at room temperature...... 77

Figure 3.2. Menthol-and cold-induced responses in DRG neurons bath-perfused at 32°C...79

Figure 3.3. PIP2 attenuates tachyphylaxis and confers TRPM8

functional state for acute desensitization...... 80

Figure 3.4. Both acute desensitization and tachyphylaxis

are Ca2+-dependent but show different Ca2+-sensitivity...... …….…....…..……….82

Sarria, I

Figure 3.5. Inhibition of calmodulin attenuates acute desensitization,

blocking PKC and protein phoshpatase 1,2A reduces tachyphylaxis...... ….84

Figure 3.6. Single channels characteristics of TRPM8...... …...... …..……….86

Figure 3.7. Modulation of TRPM8 channel gating by PIP2...... 87

Figure 3.8. Differential modulation of TRPM8 gating by PIP2 and calmodulin...... 88

Figure 3.9. Schematic diagram of Ca2+-dependent modulation of TRPM8 channels...... 90

CHAPTER 4

Table 4.1. Comparison of membrane parameters between TTXrh, TTXrl, and TTXs

DRGs at different temperatures...... 121

Table 4.2. Effects of 4-AP on membrane parameters of TTXr and TTXs cells...... 122

Table 4.3 Comparison of membrane parameters between cold sensing

TTXs and TTXr cells...... 123

Fig 4.1. Action potential firing patterns of TTXr and TTXs DRGs at different temperatures.124

Fig 4.2. Effect of temperature on action potential firing of TTXrh, TTXrl, and TTXs cells...125

Fig 4.3. Low temperatures differentially affect TTXs and Nav 1.8 TTXr VGSN...... 126

Fig 4.4. Identification of Kv potassium currents in DRGs...... 128

Fig 4.5. Kv currents in TTXr and TTXs DRGs...... 129

Fig 4.6. Low temperatures inhibit activation of IA Kv currents...... 130

Fig 4.7. Transient A-type Kv channel blocker 4-AP

enhances AP firing in TTXr, but not in TTXs cells...... 131

Fig 4.8. Effect of cold temperature on IA in TRPM8/cold sensing cells...... 132

Fig 4.9. Cold sensitive TTXr and TTXs DRGs fire action potentials

at different temperature ranges...... 133

Sarria, I

Fig 4.10. Membrane and cold-induced action potential firing properties

of TTXs/TRPM8 and TTXr/TRPM8 DRGs...... 134

CHAPTER 5

Fig 5.1 Whole-cell currents of wildtype and nonfunctional TRPM8

mutant without calmodulin binding site…….….…………………………………………….143

Fig 5.2 Single-channel currents of wildtype and nonfunctional TRPM8

mutant without calmodulin binding site…….….…………………………………………….144

Fig 5.3 NGF lowers excitability threshold in nociceptive-cold sensing DRGs…….….……145

Sarria, I

Chapter I:

General Introduction

Sarria, I

Temperature sensation and TRP channels

Primary afferent sensory neurons of the dorsal root and trigeminal ganglia allow mammals to perceive ambient temperature (Julius & Basbaum 2001; Patapoutian et al 2003).

The thermal information from peripheral tissues is transmitted by these cells to the spinal cord and brain, where the signals are integrated and interpreted, resulting in adequate motor responses.

Mammals have a sensory system that is capable of noticing and discerning thermal stimuli over a wide temperature spectrum, ranging from painful heat (>52°C), to noxious cold (<8°C), which points to the existence of various types of temperature sensors with discrete thermal sensitivities

(Patapoutian et al 2003). It is well documented that transient receptor potential (TRP) superfamily of cation channels are the principal temperature sensors in the sensory nerve endings of mammals (Clapham 2003; Voets & Nilius 2003). TRP channels are putative six- transmembrane (6TM) polypeptide subunits that assemble as tetramers to form cation-permeable pores. Generally, they are ubiquitously expressed and most have splice variants. Temperature- activated transient receptor potential channels (Thermo TRPs) are sensory ion channels gated by physical stimuli and up to date, there are six thermo-TRP channels (Patapoutian et al 2003) that have been described, and they cover almost the whole range of temperatures that mammals are able to detect.

Cold sensing and identification of TRPM8, the cold transducer

In 1951 Hensel and Zotterman recorded action potentials from cold receptors in lingual nerve and proposed that menthol specifically acts upon a cold receptor by shifting their thermal activation thresholds to warmer temperatures (Hensel & Zotterman 1951). This theory was

Sarria, I supported by studies in cultured sensory neurons where 10-20% of cells were sensitive to cold and menthol (Reid & Flonta 2001b; Viana et al 2002a). Moreover, these studies also showed that cold and menthol elicited non-selective cation currents in these neurons (threshold temperature near 28°C) and that the responses induced by menthol were temperature dependent

(Reid & Flonta 2001b). The desire to identify a cold activated ion channel led to the identification of the cool/menthol receptor Transient receptor potential cation channel subfamily

M member 8 (TRPM8) by two independent groups in 2002 (McKemy et al 2002; Peier et al

2002). Using menthol as a stimulus, Julius’s group used expression cloning to isolate TRPM8 from a rat trigeminal neuron cDNA library in heterologous expression systems (McKemy et al

2002). Patapoutian’s team isolated TRPM8 from dorsal root ganglia (DRG) and consequently heterologously expressed it as a cold- and menthol sensitive cation channel (Peier et al 2002).

Between 1950 and the recent identification of TRPM8 as the principal receptor of cold-mediated currents, other mechanisms for cold sensing have been suggested. Some have fallen out favor

(Na+/K+ -ATPase inhibition) (Pierau et al 1974), or have yet to produce thermosensory deficits in knockout models (potentiation of DEG/ENaC sodium channels by cold, (Askwith et al 2001), or play an important role as modulators of neuronal excitability in cold temperatures (Cold inhibition of leak potassium channels TREK-1/TRAAK, (Noel et al 2009) and potassium voltage-gated channel subfamily A (Kv1.) (Madrid et al 2009). The tetrodotoxin resistant

(TTXr) voltage-gated sodium channel (Nav) 1.8 is expressed in peripheral sensory neurons believed to be nociceptors. Nav. 1.8 was found to be resistant to cold-induced inhibition, while all other TTX sensitive (TTXs) Nav. channels were not (Zimmermann et al 2007). Thus, at painful low temperatures the Nav. 1.8 is responsible for the conduction of pain stimuli from the periphery.

Sarria, I

Besides menthol, a number of synthetic and natural chemicals have been shown to activate TRPM8 (Eid & Cortright 2009). Eucalyptol is another plant-derived chemical that also activates the channel, although less potently, whereas the synthetic chemical icilin is a much more potent TRPM8 agonist (McKemy et al 2002; Wei & Seid 1983).

TRPM8 Channel Characteristics

Similar to other TRP channels, TRPM8 is a non-descriminatory cation channel porous to both monovalent and divalent cations (McKemy et al 2002; Peier et al 2002). It has been reported that the values for the relative permeability for Ca2+ versus Na+ (PCa/PNa) range from

0.97 to 3.3 (McKemy et al 2002; Peier et al 2002). Alike other TRP channels, unitary TRPM8 channel events show an intermediate single-channel conductance of 40–83 picoSiemens (pS)

(Brauchi et al 2004; Hui et al 2005). Up to date, a detailed structure-function analysis of the

TRPM8 pore has not been presented, but in similarity with related TRP family members it is commonly assumed that TRPM8 functions as a tetramer (Hoenderop et al 2003; Owsianik et al

2006; Voets et al 2004b) and that the loop between transmembrane TM5 and TM6 make up the outer pore region and selectivity filter (Nilius et al 2005a; Owsianik et al 2006; Voets et al

2004b). Recently, Stewart et al (2010) have determined the subunit stoichiometry of the transient receptor potential of TRPM8 channel using atomic force microscopy (AFM) and report that the channel assembles as a tetramer.

TRPM8 activation is voltage dependent, similar to classical voltage-gated K+, Na+ and

Ca2+, but with severely weaker voltage sensitivity (Voets et al 2004a). At resting membrane

Sarria, I potentials of sensory neurons (~−70 mV), TRPM8 begins to conduct a considerable inward current upon cooling below ~25°C (McKemy et al 2002). This is because cooling allows a strong but graded shift of the voltage dependence of activation from strongly depolarized potentials towards the physiological potential range. Like cooling, menthol activates TRPM8 by shifting the voltage dependence of activation towards the physiological potential range (Voets et al 2004a). TRPM8 voltage sensitivity is conferred by positively charged amino acids in the segment (S) 4 and the 4-5 linker region, not unlike Kv. channels (Voets et al 2007). Furthermore, they showed that mutagenesis-induced changes in voltage sensitivity translated into altered thermal sensitivity. This group also showed that specific mutations in this region also affected menthol sensitivity and affinity, indicating a direct interaction between menthol and the TRPM8 voltage sensor. Although another group (Bandell et al 2006) suggested that menthol binding was influenced by S2, as mutations in this region strongly shifted the concentration dependence of menthol activation.

TRPM8 expression and physiological role

The ability to discriminate between cold temperatures in humans is highly sensitive. In controlled studies on human subjects, cooling the skin by as little as 1°C evoked a cooling sensation (Campero et al 2001). Additionally, TRPM8 senses cold in a very flexible manner being able to adapt to long-term variations in baseline temperature to sensitively detect small temperature changes. Generally, innocuous cool is defined as temperatures between 30°C and

15°C, whereas noxious cold is generally perceived as temperatures below 15°C (Davis & Pope

2002; Spray 1986). Interestingly, activation of TRPM8 starts occurring at a temperature

Sarria, I threshold of ~28°C, with currents increasing in magnitude down to 8°C (McKemy et al 2002;

Peier et al 2002), thus covering both innocuous cool to painful cold temperatures. In humans, the sensation of cold has been shown to be mediated by both myelinated Aδ- and unmyelinated C- fibers (Voets et al 2004a). Accordingly, the majority of TRPM8 channels are expressed on a subpopulation of small Aδ and C-polymodal DRG neurons that project to lamina I and IIo

(Takashima et al 2007) and respond to cool temperatures, but that do not express nociceptive markers. Nevertheless, around 10–20% of TRPM8-positive neurons also express TRPV1 in vivo

(Dhaka et al 2008; Takashima et al 2007) and up to 50% in culture (Babes et al 2004) as well as the nociceptive marker calcitonin gene related peptide (CGRP) (Caterina et al 1997; McNeill et al 1988; Takashima et al 2007). Consequently, neurons expressing TRPM8 could send nociceptive as well as cool signals to the spinal cord, depending on the context. Behavioral assays of mice with TRPM8 knockout further strengthen this point, these animals show severe deficits in sensing innocuous and painful cold and lack cold allodynia and analgesia, (Bautista et al 2007; Colburn et al 2007; Dhaka et al 2007). Thus the expression of TRPM8 in functionally distinct DRG neurons can account for multiple roles in somatosensation (Chung & Caterina

2007).

Cooling is known to lessen the effects of inflammation (Dhaka et al 2007; Proudfoot et al

2006) and activation of TRPM8 antagonizes capsaicin-induced nociception (Premkumar et al

2005). By the same token, tolerance to cooling is increased by activation of inflammatory mediators which activate TRPV1 and desensitize TRPM8 (Premkumar et al 2005).

Consequently, thermal activation of TRPV1 by inflammatory mediators could intensify thermal hyperalgesia by downregulating TRPM8 and reducing the cool and soothing sensation. In addition, (Xing et al 2007a) have shown that TRPM8 is involved in cold allodynia in rats with

Sarria, I chronic constrictive nerve injury (CCI), a neuropathic pain model exhibiting cold allodynia in hindlimbs. This is likely to occur through increased expression of TRPM8 in nociceptive neurons. Thus, depending on pain conditions, both TRPM8 receptor antagonists and agonists may be useful clinically for pain management.

Besides its role as a thermosensor in primary afferent fibers, TRPM8 has been shown to be expressed in a number of cell types in which there appear to be no thermosensory function such as: bladder, lungs, heart, and prostate (Sabnis et al 2008b; Stein et al 2004; Yang et al

2006). It is important to point out that, TRPM8 expression has been shown to be upregulated in a number of cancers including prostate, breast, skin, colorectal, lung, and bladder, thus making a plausible target for therapeutic treatments (Li et al 2009; Tsavaler et al 2001; Yamamura et al

2008).

Functional regulation of TRPM8

Calcium influx causes the decline of TRPM8 cold-activated currents as this is prevented by eliminating extracellular calcium or by chelating intracellular calcium (Okazawa et al 2002;

Reid et al 2001). Calcium-dependent modulation of TRPM8 is responsible for a feedback mechanism where opening of TRPM8 by cooling causes calcium influx, which diminishes

TRPM8’s cold sensitivity and causes it to close. Stronger cooling opens the TRPM8 channel again. Calcium influx thus provides a feedback mechanism that can explicate how cold adaptation can keep the threshold of TRPM8 just below the adapting temperature, ready to open on slight cooling.

Sarria, I

There is strong evidence supporting the role of phosphatidylinositol 4,5-bisphosphate

(PIP2) as a positive and direct modulator of TRPM8 channel’s sensitivity for cold or menthol in heterologous expression systems (Daniels et al 2009; Liu & Qin 2005; Rohacs et al 2005a). It was proposed that calcium influx through TRPM8 activated calcium dependent phospholipase C

(PLCδ1), and in turn this resulted in the depletion of cellular PIP2 levels and TRPM8 functional downregulation (Liu & Qin 2005). In these studies, PIP2 depletion prevented or highly impaired

TRPM8-mediated currents in whole-cell and inside out patches upon successive stimulation.

Furthermore, TRPM8 function was rescued by either inhibition of PIP2 hydrolysis or addition of

PIP2. Although, when TRPM8-mediated responses are carefully analyzed in these experiments, two temporally distinct types of Ca2+-dependent down-regulation can be identified, and inhibition of PIP2 hydrolysis or resynthesis by PI-kinases do not eliminate the faster desensitizing component (Daniels et al 2009; Liu & Qin 2005; Rohacs et al 2005a).

Protein phosphorylation has been established as a major regulatory mechanism of ion channels. The function of many thermo-TRPs is modulated by protein kinases, such as protein kinase C (PKC) and protein kinase A (PKA), and by protein phosphatases. In a way similar to

TRPV1 and other TRP channels, TRPM8 is strongly desensitized in a calcium-dependent manner by prolonged activation (McKemy et al 2002; Reid et al 2002; Reid & Flonta 2001b). In contrast to TRPV1 and TRPV4, protein kinases appear to function as negative regulators of TRPM8. In sensory neurons, application of the inflammatory agents bradykinin and prostaglandin E2 caused a reduction in the amplitude of TRPM8 responses to cooling and shifted the threshold temperature to colder values (Linte et al 2007). The reduction in amplitude was first suggested to be mediated by PKC apparently by an indirect action in which PKC activates a protein phosphatase (PP1) (Premkumar et al 2005). Although contrasting evidence has emerged from

Sarria, I more recent work which proposes direct downregulation of TRPM8 by PKC phosphorylation

(Abe et al 2006).

Another interesting kinase involved in TRP channel regulation is PKA. It has been well documented that PKA stimulation reduces TRPV1 desensitization (Lee et al 2005; Mohapatra &

Nau 2003; Schnizler et al 2008) and sensitizes TRPA1 (Schmidt et al 2009). De Petrocellis et al reported that PKA activation desensitized TRPM8 in HEK-293 cells (De Petrocellis et al 2007), although no study has shown if PKA has a role in regulating TRPM8 function in native cells.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is another calcium-activated protein kinase that regulates TRP channel activity, either positively as in the case for TRPV1 (Jung et al

2004) and TRPC6 (Shi et al 2004) or negatively as in the case of TRPM7 (Mishra et al 2009).

Interestingly, no known studies have been produced investigating the role this calcium-sensitive kinase might play in regulating TRPM8 activity.

Another important intracellular signaling molecule that mediates Ca2+-dependent regulation of Ca2+-permeable channels is calmodulin (CaM). CaM-mediated channel functional down-regulation has been observed in a number of TRP channels, including heat-sensing channel TRPV1 (Lishko et al 2007; Numazaki et al 2003; Rosenbaum et al 2004), warm temperature-sensing channel TRPV3 (Xiao et al 2008b), osmolarity-sensing channel TRPV4

(Strotmann et al 2003), the highly Ca2+-selective channels TRPV5 and TRPV6 (Lambers et al

2004; Niemeyer et al 2001), and TRPM4 (Nilius et al 2005b). Ca2+-dependent functional upregulation has also been observed in a number of TRP channels including TRPC5 (Ordaz et al

2005), TRPV3 (Xiao et al 2008b), TRPV6 (Lambers et al 2004) and TRPA1 (Doerner et al 2007;

Zurborg et al 2007) and TRPV4 (Strotmann et al 2010).

Sarria, I

Nerve growth factor (NGF) sensitizes TRPV1 and TRPA1 by acting on the high affinity nerve growth factor receptor (TrkA) receptor via the phosphatidylinositol 3 (PI3) kinase and p38 mitogen-activated protein kinase (MAPK) pathways respectively (Zhang et al 2005a). Similarly,

NGF upregulates TRPM8 expression, although the pathway for upregulation is unknown. When cultured in the presence of NGF, the number of DRGs that respond to menthol increases as well as the response’s intensity (Babes et al 2004). Moreover, (Xing et al 2007a) showed that after chronic constrictive nerve injury (CCI), a neuropathic pain model manifesting cold allodynia in hindlimbs, there was an increase in the percentage of nociceptive-like neurons expressing

TRPM8 and that menthol- and- cold responsiveness also increased in this population. NGF levels are known to become elevated under neuropathic conditions including chronic nerve injury (Funakoshi et al 1993; Herzberg et al 1997; Heumann et al 1987), thus it is attractive to suggest that NGF mediates the increase of TRPM8-mediated cold sensitivity on nociceptive afferent neurons, thus providing a mechanism for cold allodynia.

Research objectives and outline

The TRPM8 channel is a thermoreceptor expressed in primary sensory afferents and it serves as the principal detector of cold in mammals. Since its identification and cloning in 2001 as a cold transducer (McKemy et al 2002; Peier et al 2002) a lot of information has been uncovered about TRPM8, yet there are still gaps in our understanding of this channel. For example, one area of active research and a main focus of my graduate work has been the functional regulation of TRPM8 by secondary messengers. It is well established that calcium influx desensitizes TRPM8-mediated currents (McKemy et al 2002; Reid et al 2002; Reid &

Flonta 2001b). Scattered studies have suggested that several protein kinases downregulate

Sarria, I

TRPM8 function (De Petrocellis et al 2007), yet these studies looked at TRPM8 function using either heterologous expression systems, or functionally unidentified sensory neurons. TRPM8 is expressed in non-nociceptors as well as nociceptors, yet to my knowledge, no studies so far had looked at the role protein kinases might play in regulating TRPM8 function in these two functionally distinct DRG cells. I explore this in chapter II, by examining menthol- responsiveness and adaptation using calcium imaging.

To expand the initial findings on TRPM8 functional regulation by secondary messengers,

I analyzed cold and menthol activated currents using electrophysiological methods in DRGs and

HEK 293 cells stably expressing TRPM8. I noticed that cold and menthol-elicited inward currents showed two temporally distinct types of Ca2+-dependent down-regulation; one being a rapid reduction of TRPM8-mediated currents during a single agonist application and the other type of down-regulation being manifested by the reduction of inward currents to repeated agonist applications. Initially in chapter III, through pharmacological manipulation I explore how PIP2, protein kinases, and phosphatases regulate TRPM8 whole cell currents. Once the molecular targets that mediate the biphasic Ca2+-dependent down-regulation of TRPM8 are identified, I looked at the mechanisms of regulation by analyzing TRPM8 single channel recordings in inside out patches from HEK 293 cells expressing the channel.

In chapter II, I observed that TRPM8-expressing nociceptive-like and nonnociceptive-like

DRGs have different responses to menthol. Following the initial findings that TRPM8-mediated responses and adaptation were different in these two functionally distinct groups of cells, it is logical to think that conduction of cold stimulus (excitability) might also be different. It could be that cold-elicited action potentials in these two types of neurons expressing the cold transducer

Sarria, I would be distinct enough to account for the innocuous (28°C-15°C) and nociceptive (15°C and below) cold sensing range previously reported (Davis & Pope 2002; Spray 1986). Additionally, cold temperatures had been shown to have an effect on targets of neuronal excitability, such as potassium channels (Madrid et al 2009; Noel et al 2009) and the TTX sensitive and resistant (Nav

1.8) sodium channels (Zimmermann et al 2007). Thus, low temperatures might play a general role in modulating DRGs’ ability to fire action potentials depending on their respective neuronal conduction machinery i.e. specific Nav and Kv channel expression. Using dissociated rat DRGs and whole cell voltage- and current-clamp methods I set out to answer these questions in Chapter

IV.

Sarria, I

Chapter II:

TRPM8-mediated responses and adaptation are different in nociceptive-like vs. nonnociceptive-like neurons: role of protein kinases

Sarria, I

Abstract

Menthol-sensitive/capsaicin-insensitive neurons (MS/CI) and menthol- sensitive/capsaicin-sensitive neurons (MS/CS) are thought to represent two functionally distinct populations of cold-sensing neurons that use TRPM8 receptors to convey innocuous and noxious cold information respectively. However, TRPM8-mediated responses have not been well characterized in these two neuron populations. Using rat dorsal root ganglion neurons, here we show that MS/CI neurons had larger menthol responses with greater adaptation. In contrast,

MS/CS neurons had smaller menthol responses with less adaptation. All menthol-sensitive neurons showed significant reduction of menthol responses following the treatment of cells with the protein kinase C (PKC) activator PDBu (Phorbol 12,13-dibutyrate). PDBu-induced reduction of menthol responses was completely abolished in the presence of PKC inhibitors BIM

(bisindolylmaleimide) or staurosporine. When menthol responses were examined in the presence of protein kinase inhibitors, it was found that the adaptation was significantly attenuated by either BIM or staurosporine and also by the Ca2+/calmodulin-dependent protein kinase (CamKII) inhibitor KN62 (N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) in

MS/CI neurons. In contrast, in MS/CS neurons menthol response was not affected significantly by BIM, staurosporine or KN62. In both MS/CI and MS/CS neurons, the menthol responses were not affected by PKA activators forskolin and 8-Br-cAMP (8-Bromoadenosine- 3', 5'- cyclic monophosphate) or by protein kinase A (PKA) inhibitor Rp-cAMPs (Rp-Adenosine-3',5'-cyclic monophosphorothioate). Taken together, these results suggest that TRPM8-mediated responses are significantly different between non-nociceptive-like and nociceptive-like neurons.

Sarria, I

Introduction

Transient receptor potential M8 (TRPM8) receptor, first cloned by MacKemy and colleagues (McKemy et al 2002) as well as Peier and colleagues (Peier et al 2002) from primary afferent neurons of rats and mice, is a principal sensor for cold temperature and belongs to the transient receptor potential (TRP) protein family. Like most of other members in TRP family,

TRPM8 is a membrane ion channel that can allow positively charged ions (Na+, Ca2+, K+) to flow through cell membranes when the channel opens. The TRPM8 channel opens when temperature drops below 26 ± 2 oC, resulting in depolarizing membrane currents (McKemy et al

2002; Peier et al 2002; Tominaga & Caterina 2004). Membrane currents flowing through

TRPM8 channels increase with decreasing temperature and reach maximum response near 10 °C.

TRPM8 senses temperature changes in the range of both innocuous cold (28-15 oC) and noxious cold (<15 oC) (McKemy et al 2002; Peier et al 2002; Tominaga & Caterina 2004). Activation of

TRPM8 can result in a large increase of intracellular Ca2+ levels due to the high Ca2+ permeability of this channel (McKemy et al 2002; Peier et al 2002; Reid et al 2002; Tsuzuki et al

2004). TRPM8 can also be activated by menthol, an active ingredient of peppermint that produces a cooling sensation (McKemy et al 2002; Okazawa et al 2004; Peier et al 2002; Reid &

Flonta 2002).

TRPM8 receptors are expressed on 10-15% of the total trigeminal ganglion (TG) neuron population and 5-10% of dorsal root ganglion (DRG) neuron population (Abe et al 2005;

McKemy et al 2002; Okazawa et al 2004; Peier et al 2002). Consistently, the percentage of menthol-sensitive cells in acutely dissociated rat DRG neurons is similar to that of TRPM8- expressing DRG neurons (Xing et al 2007a; Xing et al 2006). Many TRPM8-expressing neurons

Sarria, I are found to lack nociceptive markers, suggesting that they are non-nociceptive cold sensing neurons (Peier et al 2002). However, studies have provided anatomical evidence showing

TRPM8 immunoreactivity on some TRPV1 (Transient receptor potential V1)-expressing afferent neurons (Abe et al 2005; Okazawa et al 2004). TRPV1-expressing neurons are believed to be nociceptive afferent neurons that transmit noxious signals to produce burning pain sensations

(Carlton & Coggeshall 2001; Caterina et al 2000; Caterina et al 1997). Using calcium imaging and patch-clamp recording techniques, Xing and colleagues (Xing et al 2006) have found that a subpopulation of menthol-sensitive neurons is also sensitive to capsaicin, a noxious stimulant that acts on TRPV1 receptors. Consistent with these observations, co-expression of TRPM8 and

TRPV1 have been directly visualized in mice engineered to express enhanced green fluorescent protein (EGFP) driven by a TRPM8 promoter (Dhaka et al 2008; Takashima et al 2007). Thus, menthol-sensitive neurons appear to consist of both non-nociceptive and nociceptive sensory neurons and may play roles in sensing innocuous and noxious cold respectively under physiological conditions (Xing et al 2007a).

TRPM8 can be regulated through second messenger systems (Liu & Qin 2005;

Premkumar et al 2005; Rohacs et al 2005b). A role for the PLC/PIP2 (Phospholipase C/ phosphatidylinositol (4,5) bisphosphate) second messenger pathway in regulating TRPM8 functions has been well established (Daniels et al 2009; Liu & Qin 2005; Rohacs et al 2005b). It has been suggested that Ca2+ influx through TRPM8 channels activates a Ca2+-sensitive phospholipase C and the subsequent depletion of PIP2 results in desensitization of TRPM8 channels (Daniels et al 2009; Liu & Qin 2005; Rohacs et al 2005b). Desensitization of TRPM8 channels could also be induced by inflammatory mediators that activate PLC to deplete PIP2

Sarria, I

(Linte et al 2007). In comparison with the PLC/PIP2 pathway, the roles of protein kinase pathways in regulating TRPM8 functions remain unclear. Premkumar and colleagues

(Premkumar et al 2005) showed in DRG neurons that PKC activators and bradykinin significantly reduced menthol responses. Using HEK293 cells expressing TRPM8, Abe and colleagues (Abe et al 2006) also showed that PKC activators reduced menthol responses. Other second message pathways such as PKA have also been suggested to play roles in regulating

TRPM8 functions (Bavencoffe et al 2010; De Petrocellis et al 2007). These previous studies on the regulation of TRPM8 functions were performed either using heterologous expression system or functionally unidentified sensory neurons. Therefore, it is unclear if the reduction of TRPM8 functions occurs in a similar manner across functionally distinct populations of neurons. In addition, previous studies did not test whether TRPM8-mediated responses were affected by different protein kinase inhibitors, a result that is essential for establishing the roles of protein kinases in modulating TRPM8 functions. In the present study, we addressed some of these issues by examining menthol-responsiveness and adaptation in menthol-sensitive/capsaicin- insensitive and menthol-sensitive/capsaicin-sensitive neurons.

Sarria, I

Methods

Adult Sprague Dawley rats (100–250 g, both genders) were used in all experiments.

Animal care and use conformed to National Institutes of Health guidelines for care and use of experimental animals. Experimental protocols were approved by the University of Cincinnati

Institutional Animal Care and Use Committee. DRG neuron cultures were prepared as described previously (Tsuzuki et al 2004). In brief, rats were deeply anesthetized with isoflurane (Henry

Schein, NY.) and sacrificed by decapitation. DRGs were rapidly dissected out bilaterally in

Leibovitz L-15 media (Fisher, GA) and incubated for 1 hour at 37 °C in minimum essential medium for suspension culture (S-MEM) (Invitrogen, Grand Island, NY) with 2% collagenase and 10% dispase and then triturated to dissociate neurons. The dissociated DRG neurons were then plated on glass coverslips pre-coated with poly-D-lysine (PDL, 12.5 µg/ml in distilled H2O) and laminin (20 µg/ml in Hank's Buffered Salt Solution HBSS, BD bio-science), and maintained in MEM (Invitrogen) culture medium that also contained nerve growth factor (2.5 S NGF; 10 ng/ml; Roche Molecular Biochemicals, Indianapolis, IN), 5% heat-inactivated horse serum (JRH

Biosciences, Lenexa, KS), uridine/5-fluoro-2’-deoxyuridine (10 µM), 8 mg/ml glucose, and 1% vitamin solution (Invitrogen). The cultures were maintained in an incubator at 37°C with a humidified atmosphere of 95% air and 5% CO2. Unless otherwise indicated, cells were used within 72 hours after plating.

For calcium imaging experiments, the calcium indicator Fluo-3 (Invitrogen) was loaded into DRG neurons on coverslips by incubation of cells with 5 µM Fluo-3-AM in normal bath solution at 37°C for 1 hour. Fluo-3-AM stock solution was made with 20% pluronic acid

(Molecular Probes) in dimethyl sulfoxide (DMSO) and the stock solution was diluted 1:200 with

Sarria, I

bath solution for final use. Normal bath solution contained (in mM) 150 NaCl, 5 KCl, 2 MgCl2,

2 CaCl2, 10 glucose, 10 HEPES, pH 7.3 adjusted with NaOH, and osmolarity 320 mOsm adjusted with sucrose. After dye loading, a coverslip was mounted on a 0.5-ml perfusion chamber and the chamber was then placed on the stage of an inverted Olympus IX70 microscope

(Lake Success, NY). Cells on the coverslip were continuously perfused with normal bath solution flowing at 1 ml/min. Fluo-3 was excited at 450 nm with a mercury lamp and fluorescence emission was collected at 550 nm, and the wave lengths of excitation and emission were achieved by a fluorescence filter set. Fluo-3 fluorescence in the cells was detected with a peltier-cooled charge-coupled device (CCD) camera (PentaMAX-III System, Roper Scientific,

Trenton, NJ) under a 10X objective. Images were acquired at one frame per second, 200 ms exposure time per frame, using the MetaFluor Imaging System software (Molecular Devices,

Downingtown, PA). Neurons were tested for their sensitivity to menthol (100 µM), AIT (allyl isothiocyanate, 100 µM), or capsaicin (0.5 µM) by applying these compounds for 10 seconds.

Adaptation of menthol responses was examined by a prolonged application of menthol (100 µM) for 5 min. Effects of protein kinases on menthol responses and adaptation were tested with 1 µM

PDBu (phorbol 12,13-dibutyrate), a protein kinase C (PKC) activator, 100 µM 8-Br-cAMP (8- bromoadenosine- 3', 5'- cyclic monophosphate) and 10 µM forskolin, two protein kinase A

(PKA) activators, 0.5 µM staurosporine, a broad spectrum protein kinase inhibitor, 1 µM BIM

(bisindolylmaleimide), a specific PKC inhibitor, 25 µM RP-cAMPs (Rp-adenosine-3',5'-cyclic monophosphorothioate), a specific PKA inhibitor, and 25 µM KN-62 (1-[N,O-bis(5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, Tocris), a specific

Ca2+/calmodulin-dependent protein kinase (CaMKII) inhibitor. Unless otherwise indicated, chemicals and compounds were purchased from Sigma (St. Louis, MO). Testing solutions were

Sarria, I rapidly applied to neurons through a glass tube (~500 µm ID) positioned 1.0 mm away from cells. Unless otherwise indicated, testing compounds were applied at an interval of 10 min and capsaicin was always tested last. All experiments where pharmacological agents were applied were done in separate dishes. All experiments were carried out at room temperature of ~24°C.

For most experiments, relative fluorescence intensity (ΔF/F0) was used to represent menthol responses and neurons with ΔF/F0 values of ≥ 0.2 (i.e., equal or above 20% baseline fluorescence intensity) were considered as responsive cells (Xing et al 2006). Percentages of maximal ΔF/F0 values were used as changes in menthol responses. In some experiments, intracellular Ca2+ concentrations ([Ca2+]) in cells were calibrated from the measured fluorescence signals. The following equation was used for the calibration of intracellular Ca2+ concentrations

(Eberhard & Erne 1989): [Ca2+] = Kd[(F-Fmin)/(Fmax-F)] where [Ca2+] is the concentration

(nM) of intracellular Ca2+, Kd is the dissociation constant of the dye, F is the fluorescence intensity, Fmin is the intensity at zero [Ca2+] and Fmax is the intensity at saturated [Ca2+].

Procedures for obtaining Fmax and Frnin caused damage to cells and were therefore carried out at the end of the experiments. Fmax was obtained first by adding the ionophore ionomycin (10

M), making the cell membrane permeable to Ca2+ and allowing the extracellular and intracellular Ca2+ to equilibrate. Following this, Fmin was obtained by adding EGTA [ethylene glycol bis(~-aminoethyl ether)-N,N,N',N'-tetraacetic acid; 20 mM] to chelate all Ca2+ inside and outside the cells. Then MnCl2 (30 mM) was added to quench the residual fluorescent signals due to autofluorescence (Roe et al 1990). Kd value of 404 nM was used according to a previous study (Lattanzio 1990). Changes in intracellular Ca2+ concentrations are calculated by

2+ 2+ 2+ 2+ 2+ Δ[Ca ]/[Ca ]0, where [Ca ]0 is basal intracellular Ca level and Δ[Ca ] is the difference

2+ 2+ between intracellular Ca concentrations at a given time point and [Ca ]0. Unless otherwise

Sarria, I indicated, data were presented as Mean ± SEM. Analysis of Variance (ANOVA) was applied for statistic analysis of unpaired data sets of multiple groups followed by Student-Newman-Keuls

Post Hoc Test. Student’s t-test was applied for paired data sets. Statistical significance was considered at the level of the p < 0.05.

Sarria, I

Results

Menthol responses and adaptation

Menthol-sensitive neurons were identified by calcium imaging following a brief application of 100 µM menthol for 10 seconds. These neurons consisted of 6.56% of total cells

(n = 1475) in our DRG neuron cultures, a result consistent with our previous study (Xing et al

2006). We tested these menthol-sensitive neurons with capsaicin (0.5 µM, 10 sec) and AIT (100

µM, 10 sec) in order to see if, in some of them, TRPM8 receptors were also co-expressed with

TRPV1 and TRPA1, two receptors believed to be expressed in nociceptive primary afferent neurons (Caterina et al 1997; Jordt et al 2004; Story et al 2003). Based on their sensitivity to capsaicin and AIT, menthol-sensitive neurons could be classified into four subpopulations: menthol-sensitive/capsaicin-insensitive (MS/CI, Fig. 2.1A), menthol-sensitive/capsaicin- sensitive (MS/CS, Fig. 2.1B), menthol-sensitive/AIT-insensitive (MS/AI, Fig. 2.1A), and menthol-sensitive/AIT-sensitive (MS/AS, Fig. 2.1B). Of 71 menthol-sensitive neurons tested with capsaicin, 58% (41/71) were MS/CI neurons and 42% (30/71) were MS/CS neurons (Fig.

2.1D). Of 30 menthol-sensitive neurons tested with AIT, 70 % (21/30) were MS/AI and 30%

(9/30) were MS/AS neurons (Fig.2.1E). Out of 7 neurons sensitive to menthol and AIT, 5 (71%) responded to capsaicin as well (Fig. 2.1B) and thereby belonging to MS/CS neuron population

(Jordt et al 2004; Story et al 2003). In AIT-sensitive neurons, we also analyzed menthol- sensitivity to see if menthol sensitivity and AIT sensitivity were correlated in rat DRG neurons

(Macpherson et al 2006)). We found that the majority of AIT sensitive neurons (80%, 34/42) were insensitive to menthol and only small percentage of AIT-sensitive neurons (20%, 8/42) was menthol-sensitive (Fig. 2.1F). Of 65 cells that responded to menthol and/or AIT, only 7 (11%) responded to both. The inverse correlation between menthol-sensitivity and AIT-sensitivity

Sarria, I suggests that TRPA1 is unlike to significantly account for menthol-induced responses in our rat

DRG neurons.

Menthol responses were analyzed and compared among subpopulations of menthol- sensitive neurons. Menthol responses were larger in MS/CI than in MS/CS (Fig. 2.1D).

Similarly, menthol responses were larger in MS/AI than in MS/AS (Fig. 2.1E). The peak responses to 100 µM menthol, expressed as increases of Fluo-3 fluorescence intensity (∆F/Fo), were 1.22 ± 0.13 (n = 40) for MS/CI and 0.70 ± 0.15 (n = 31, P < 0.05) for MS/CS (Fig. 2.1D);

1.11 ± 0.14 (n = 23) for MS/AI and 0.61± 0.14 (n = 7, P < 0.05) for MS/AS (Fig. 2.1E). We also analyzed AIT responses in both MI/AS and MS/AS neurons. Kinetics of AIT responses in both groups was much slower (Figure 1F) than that of menthol-responses (Figure 1D,E). The average peak response (∆F/Fo) to 100 µM AIT was 0.80 ± 0.72 (n = 35) for MI/AS neurons, significantly greater than that of that MS/AS neurons (0.43 ± 0.06, n=7) (Fig. 2.1D, P < 0.05).

Thus, AIT responsiveness also has inverse correlation with menthol-sensitivity.

Intrigued by the differences between MS/CI and MS/CS neurons in menthol responses to brief menthol applications, we examined whether there were also differences between these two subpopulations of neurons in their responses to prolonged menthol application (100 µM, 5 min).

Changes in intracellular Ca2+ concentrations ([Ca2+]) in MS/CI neurons following the prolonged application of menthol displayed adaptation, i.e., a gradual reduction of responses over time

2+ 2+ during prolonged menthol application (Fig. 2.2A,B). In our study, values of ∆[Ca ]/[Ca ]0 were in a good agreement with the values of ∆F/Fo when menthol responses were expressed as percent of maximal responses (Figure 2B). Therefore, we used ∆F/Fo values as menthol responses for most of experiments. During a 5-min menthol application, the peak response

Sarria, I

(∆F/Fo) of MS/CI neurons (1.1 ± 0.10, n = 22 cells, 10 different dishes) was significantly larger than that of MS/CS neurons (0.71 ± 0.10, n = 18 cells, 10 different dishes, P < 0.05) (Fig. 2.2C), a result similar to menthol response after brief menthol applications (Fig. 2.1D). In both MS/CI and MS/CS neurons, menthol responses showed adaptation. Interestingly, in the last thirty seconds of 5-min menthol application, the response (∆F/Fo) of MS/CI neurons was no longer larger than that of MS/CS neurons (Fig. 2.2C), indicating a different degree of adaptation between MS/CI neurons and MS/CS neurons. The rate of adaptation could be more clearly observed when menthol response at each time point was expressed as percent of peak response

(Fig. 2.2D). For example, at the end of 5-min menthol application, the relative menthol response was 44.5 ±4.6 % (n = 22) in MS/CI neurons and was significantly smaller than that in MS/CS neurons (63.8 ± 6.0%, n=18, P < 0.05). Taken together, these results indicated that MS/CI neurons had a greater adaptation rate in menthol responses than MS/CS neurons (Fig. 2.2D).

Given the difference in peak response and adaptation rate between MS/CI and MS/CS neurons, we next examined recovery of menthol response after adaptation. This was achieved by testing menthol response to brief menthol application (100 µM, 10 s) at 5 and 15 minutes after the end of prolonged menthol application (Fig. 2.3A). As shown in Figure 2.3B, C, menthol responses were 32.3 ±7.2% (∆F/Fo : 0.45 ± 0.13, n = 6 cells, 3 dishes) of peak responses in

MS/CI at the end of 5-min menthol application. After washing cells in normal bath solution for 5 min, menthol responses slightly increased, but were not significantly different from the responses at the end of 5-min menthol application. After washing cells in normal bath solution for 15 min, menthol responses recovered to 72.0 ± 10.2% (∆F/Fo: 1.02 ± 0.20, n = 6) of peak responses, significantly higher (p < 0.05) than the responses at the end of 5-min menthol application for

MS/CI neurons. For MS/CS neurons, recovery from adaptation reached 88.2 ± 8.9% (n = 8 cells,

Sarria, I

3 dishes) of peak responses after 15 min washing in normal bath solution. Brief menthol application (100 µM, 10 s) did not lead to any reduction of menthol response upon repeated menthol applications at 5 and 10 minute intervals in either MS/CI (n=12 cells, 7 dishes) or

MS/CS neurons (n = 11 cells, 7 dishes) (Fig. 2.4). Thus, a time interval of longer than 5 min during multiple brief menthol applications is a suitable paradigm for testing recovery of menthol responses (Fig. 2.3) and for some other experiments described below.

Effects of protein kinase activators

We examined whether in our MS/CI and MS/CS DRG neurons, direct activation of PKC with phorbol 12,13-dibutyrate (PDBu) significantly affected menthol-elicited responses. As shown in Figure 5, peak menthol response (∆F/Fo) was reduced significantly from 0.86 ± 0.13 in control to 0.25 ± 0.10 (n = 9 cells, 3 dishes, p < 0.01) after treatment of cells with 1 µM PDBu for 5 min (Fig. 2.5D). When expressed as percentage of peak response of control, menthol response was only 28.1± 9.8% of control following PDBu treatment (Fig. 2.5E). The inhibitory effect of PDBu was observed in every cell tested regardless whether they were MS/CI (29.8 ±

16.1%, n = 5) or MS/CS neurons (26.0 ± 11.8%, n = 4). The effect of PDBu was most likely due to its activation of PKC since PDBu had no significant effect on menthol-elicited responses when cells were incubated, prior to the application of PDBu, with either the potent protein kinase inhibitor staurosporine (0.5 µM, 10 min) (∆F/Fo: 0.88 ±0.22, 89.79 ± 16.3% of control, n = 6 cells, 2 dishes) or the specific PKC inhibitor BIM (1 µM, 10 min) (∆F/Fo: 0.82 ±0.15, 87.6 ±

14.9% of control, n=13 cells, 4 dishes) (Fig. 2.5D,E). Menthol-induced responses were not significantly altered when cells were only treated with BIM (1 µM, 10 min) (∆F/Fo: 1.19 ± 0.20,

Sarria, I

102.5 ± 10.0% of control, n=14 cells, 4 dishes) or staurosporine (0.5 µM, 10 min) (∆F/Fo: 1.22 ±

0.12, 123.0 ± 33.1% of control, n=24 cells, 7 dishes).

We examined whether PKA activation may have an effect on menthol response (Fig 2.6).

Menthol response was first tested by a brief menthol application (100 µM, 10 s) as control.

Subsequently, PKA activator 8-Br-cAMP (100 µM) or Forskolin (10 µM) was applied to the cells for 10 min. Following this treatment, menthol response was tested again by a brief menthol application (100 µM, 10 s) in the presence of 8-Br-cAMP (100 µM) or Forskolin (10 µM).

Menthol responses were 133.2 ± 30.3% (n = 8 cells, 3 dishes ) and 119.3 ± 13.8% (n = 6 cells, 2 dishes) of controls following the treatment of cells with 8-Br-cAMP (100 µM) or forskolin respectively, which were not significantly different from control group (Fig 2.6 C).

Effects of protein kinase inhibitors

We asked whether PKC, CaMKII, and PKA may play a role in shaping TRPM8-mediated responses during prolonged TRPM8 activation in both MS/CI and MS/CS neurons. This was achieved by testing effects of protein kinase inhibitors on menthol responses following prolonged menthol applications (Fig 2.7). In MS/CI neurons of control group for which cells were not treated with protein kinase inhibitors, menthol responses showed significant adaptation and the responses were reduced to 44.5 ± 4.6% (n = 22 cells, 10 dishes) of peak response at the end of 5- minute menthol application (Fig. 2.7A, also Fig. 2.2D). In the MS/CI neurons treated with staurosporine (0.5 µM, 10 min), PKC inhibitor BIM (1 µM, 10 min), or CaMKII inhibitor KN62

(25 µM, 10 minutes), the adaptation following prolonged menthol application was significantly attenuated (Fig. 2.7A). For example, at the end of 5-minute menthol application, menthol-

Sarria, I induced responses were 75.8 ± 4.3% (n=10 cells, 5 dishes) when cells were treated with staurosporine, 66.7 ± 5.2% (n=10 cells, 5 dishes) when cells were treated with BIM, 63.4 ± 4.9%

(n = 10 cells, 3 dishes) when cells were treated with KN62; the responses under these conditions were all significantly larger than that of control group (P < 0.05). In the MS/CI neurons treated with PKA inhibitor RP-cAMPs (25 µM, 10 minutes), no significant difference was observed in menthol responses at any time points between control group and RP-cAMPs-treated group (Fig.

2.7A). For example, menthol-evoked response was 48.6± 4.9% (n = 14 cells, 5 dishes) in RP- cAMPs-treated group at the end of 5-min menthol application, and was not significantly different from the control group (Fig. 2.7A).

In MS/CS neurons, menthol responses were not significantly affected by the treatment of cells with any of the above protein kinase inhibitors (Fig. 2.7B). For example, at the end of 5- minute menthol application, menthol responses were 61.4 ± 7.2% (n=10 cells, 4 dishes) in BIM- treated MS/CS group, 71.3 ± 5.0% (n=10 cells, 4 dishes) in staurosporine-treated MS/CS group,

65.4 ± 9.2% (n = 10 cells, 3 dishes) in KN62-treated group, and 70.4 ± 6.6% (n = 10 cells, 5 dishes) in RP-cAMPs-treated group; the responses under above conditions were not significantly different from controls (63.8 ± 6.0%, n=18, 10 dishes) (Fig. 2.7B).

Sarria, I

Discussion

In this study we demonstrated that menthol-sensitive/capsaicin-insensitive and menthol- sensitive/capsaicin-sensitive neurons had different degrees of responses and adaptation to menthol, that activation of protein kinase C, but not protein kinase A, resulted in a large reduction of menthol responses, and that protein kinase C and CamKII inhibitors had significant effects on menthol responses in MS/CI neurons but not in MS/CS neurons. These results reveal some new properties of TRPM8-mediated responses in both MS/CI and MS/CS neurons, the two neuron populations that most likely represent non-nociceptive cold-sensing neurons and nociceptive cold-sensing neurons respectively (Xing et al 2007b; Xing et al 2006).

We used menthol as a TRPM8 agonist in the present study. Menthol has been found to interact with TRPA1 in heterologous expression systems that express mouse and human TRPA1

(Macpherson et al 2006; Xiao et al 2008a), raising a possibility that menthol responses in some cells may be mediated by TRPA1 in our study. However, using DRG neurons from rats, we found inverse rather than positive correlation between menthol-sensitivity and AIT-sensitivity

(Fig. 2.1E,F). Consistent with our results, low incidence of co-activation of menthol and AIT has been previously reported in DRG cells by others (Jordt et al 2004; Munns et al 2007). The low incidence of co-sensitivity to menthol and AIT is unlikely due to the failure of mentioned agonists to cross-activate these two receptors most of the times.

Therefore, TRPA1 is unlikely to significantly account for menthol-induced responses in our rat DRG neurons. Menthol responsiveness was found to be higher in MS/CI neurons than in

MS/CS neurons, a result consistent with our previous study using both the calcium imaging and

Sarria, I patch-clamp recoding techniques on acutely dissociated DRG neurons (Xing et al 2007a; Xing et al 2006). Decay of menthol responses was found to be faster in MS/CI neurons than in MS/CS neurons, suggesting that MS/CI has faster adaptation and MS/CS has slower adaptation to menthol.

We showed that the PKC activator PDBu reduced menthol responsiveness in DRG neurons, and that this effect was abolished in the presence of staurosporine or BIM.

Staurosporine inhibits a number of protein kinases including PKC, CaMKII, and tyrosine kinase

(p60v-src), but BIM is a highly selective PKC inhibitor. The effect of these two inhibitors is consistent with a previous study that first suggested the involvement of PKC in regulating

TRPM8 function in sensory neurons (Premkumar et al 2005). We further showed that menthol- induced adaptation could be significantly attenuated by staurosporine and BIM in MS/CI neurons. The inhibitor experiments added an important supplement to strengthen the argument that PKC plays a role in the adaptation of menthol responses in MS/CI neurons. In addition to

PKC, we found that the selective CaMKII inhibitor KN62 attenuated adaptation of menthol responses in MS/CI neurons, suggesting that CaMKII may play a role in regulating TRPM8 functions in MS/CI neurons. In contrast to MS/CI neurons, menthol responses were not significantly affected by BIM, staurosporine, or KN62 in MS/CS neurons. We found that MS/CS neurons had smaller menthol response with weaker adaptation. It was initially thought that this property of MS/CS neurons might be a result of post-transcriptional regulation of TRPM8 function by protein kinases. However, the lack of the effects by protein kinase inhibitors on menthol responses in MS/CS neurons does not favor this idea. Alternatively, the smaller menthol responses in MS/CS neurons were due to the relatively lower TRPM8 expression as was proposed in our previous study (Xing et al 2006). The weaker adaptation observed in MS/CS

Sarria, I neurons could be due to the smaller menthol responses in these neurons. However, we did not observe a clear co-relation between the degree of adaption and menthol responsiveness.

Therefore, other factors might account for the differences in adaption rate between MS/CI and

MS/CS neurons. One possible factor is PIP2 because PIP2 plays an important role in regulating

TRPM8 functions and PIP2 hydrolysis accounts for menthol-induced desensitization (Liu & Qin

2005; Rohacs et al 2005b). It would be helpful in the future study to investigate whether PIP2 levels are significantly different between MS/CI and MS/CS neurons.

We explored potential involvement of PKA in regulating TRPM8 functions in rat DRG neurons by using both PKA activators and inhibitors. A previous study performed on HEK cells expressing TRPM8 showed that 8-Br-cAMP and forskolin, two PKA activators, inhibited

TRPM8 activity induced by menthol (De Petrocellis et al 2007). However, both compounds were not found to have any significant effect on menthol responses in our work as well as in another recent study (Bavencoffe et al 2010). The discrepancy could be due to the use of different cell types. Direct inhibition of PKA by Rp-cAMP-S did not significantly affect menthol responses in our study, suggesting that there is no direct connection between PKA activity and TRPM8 functions. However, our result does not exclude the possibility that TRPM8 activity could be regulated indirectly through PKA pathway (Bavencoffe et al 2010).

The differences in TRPM8-mediated responses and adaptation in nociceptive and non- nociceptive neuron populations may have physiological significances. Behavioral responses to innocuous and noxious cold stimuli are different and TRPM8-mediated adaptation may contribute to the differences. Mammals capably adapt to innocuous cold. On the other hand, noxious cold is poorly adapted, which perhaps is a conserved biological trait of mammalian

Sarria, I sensory systems for animals to be aware of a harmful cold environment. TRPM8-mediated responses and adaptation in nociceptive and non-nociceptive neuron populations may only partially contribute to behavioral cold adaptation because other receptor molecules such as

TRPA1 have been reported to also serve as cold sensors (Karashima et al 2009; Story et al 2003).

It would be interesting in future research to directly demonstrate differences to cold adaptation between nociceptive- and non-nociceptive cold sensing neurons and determine if such differences contribute to behavioral cold responses and adaptation.

Sarria, I

Tables and Figures

Figure 2.1. Responses to menthol, capsaicin and AIT in dorsal root ganglion neurons of rats. A, B, C) Images show Fluo-3 fluorescence intensity (ΔF/F0) before (baseline) and following the sequential applications of menthol (100 μM, 10 s), AIT (100 μM, 10 s), and capsaicin (0.5 μM, 10 s) with a time interval of 10 min between each application. Traces alongside with each set of images are respective time-course of fluorescence intensity (ΔF/F0) changes. The cell in A was a menthol-sensitive/AIT-insensitive/capsaicin-insensitive neuron and in B was a menthol-sensitive/AIT-sensitive/capsaicin-sensitive neuron. The cell in C was a menthol-insensitive/AIT sensitive/capsaicin-sensitive neuron. D, E) Pie graphs in each panel show percentage distribution in total menthol-sensitive neurons of menthol-sensitive/capsaicin-

Sarria, I insensitive neurons (MS/CI, n = 40) and menthol-sensitive/capsaicin-sensitive (MS/CS, n = 31) neurons (D) as well as in total menthol-sensitive neurons of menthol-sensitive/AIT-insensitive neurons (MS/AI, n = 23) and menthol-sensitive/AIT-sensitive (MS/AS, n = 7) neurons. (E). Traces in D show menthol responsiveness (ΔF/F0) in MS/CI and MS/CS neurons. Traces in E show menthol responsiveness (ΔF/F0) in MS/AI and MS/AS neurons. F) Pie graph shows percentage distribution in total AIT-sensitive neurons of menthol-insensitive/AIT-sensitive neurons (MI/AS, n = 35) and menthol-sensitive/AIT-sensitive (MS/AS) neurons. Two traces show AIT responsiveness (ΔF/F0) in MI/AS and MS/AS neurons. Scale bar in each image is 20 μm. The horizontal bar in each trace indicates the 10-sec drug applications. Data are mean ± SEM in C, D, and E.

Sarria, I

Figure 2.2. Menthol responses in menthol-sensitive/capsaicin-insensitive and menthol- sensitive/capsaicin-sensitive neurons following prolonged menthol application. A) Time course of the changes of intracellular Ca2+ concentrations ([Ca2+]) in MS/CI neurons (n = 22) following the application of 100 μM menthol for 5 min. B) Menthol responses in MS/CI neurons expressed as percentages of maximal Δ[Ca2+]/[Ca2+]0 values or maximal ΔF/Fo values. C) Time course of menthol responses (ΔF/Fo) in MS/CI (n = 22) and MS/CS (n = 18) neurons during 5-min application of 100 μM menthol. D) Same as C except menthol responses at each time point are expressed as percent of peak menthol responses. In each experiment, menthol was continuously applied for 5 min. Capsaicin-sensitivity for each cell was tested 10 min after the termination of menthol applications. The horizontal bar in each figure indicates 5-min menthol application. Data are mean ± SEM; *P < 0.05.

Sarria, I

Figure 2.3. Recovery after menthol-induced adaptation in menthol-sensitive/capsaicin- insensitive and menthol-sensitive/capsaicin sensitive neurons. A) Sample traces show menthol responses and adaptation in a MS/CI neuron and a MS/CS neuron following 5-min menthol application (left panel), recovery for 5 min (middle panel) and 15 min (right panel) in normal bath. The end of 5-min menthol application is indicated. Recovery was tested with short menthol application (10 sec). Menthol concentration was 100 μM for both prolonged and short applications. B) Pooled results show menthol responses (ΔF/F0) in MS/CI (n = 6) and MS/CS neurons (n = 8) at the end of 5-min menthol application, 5 min and 15 min recovery in normal bath. C) Similar to B except menthol responses were expressed as percent of maximum menthol responses observed during 5-min menthol application. Data are mean ± SEM in B and C; *P < 0.05.

Sarria, I

Figure 2.4. Responses to multiple brief applications of menthol. A, B) Sample traces of responses (ΔF/F0) to three brief applications of menthol (100 μM) in MS/CI (n = 12) and MS/CS (n = 11) neurons. Time interval was 5 min between first application and second application and was 10 min between second application and third application. Menthol was applied for 10 s each time. C) Summary of menthol responses. Data are mean ± SEM

Sarria, I

Figure 2.5. Effect of PKC activator PDBu on menthol responses. A) Sample images (left) and the trace to the right show fluorescence intensity in a cell at baseline, during menthol application, following treatment with 1 μM PDBu, and co-application of menthol with PDBu. B) Similar to A except BIM was applied together with PDBu and the test was on a different cell. C) Similar to B except staurosporine (Sta) was tested instead and the test was on a different cell. D, E) Pooled results show menthol responses before and after treatment with PDBu (n = 9), with PDBu in the presence of BIM (n = 13), with PDBu in the presence of Sta (n = 6), with BIM alone (n = 14), and with Sta alone (n = 24). Menthol responses were expressed as ΔF/F0 (D) and percent of menthol responses before treatment (E). In all cases 100 μM menthol was applied for

Sarria, I

10 seconds. The PKC inhibitors BIM (1 μM) and Sta (0.5 μM) were pre-applied for 10 min and then co applied with menthol. Maximum fluorescence intensity was used for menthol responses presented in D and E. Scale bars are 20 μm. Data are represented as mean ± SEM; *P < 0.05.

Sarria, I

Figure 2.6. Lack of effect by PKA activators on menthol responses. A, B) Traces show menthol responses in a cell before and after treatment with 10 μM forskolin (10 min) (A) and in another cell before and after treatment with 100 μM 8-Br-cAMP (10 min) (B). Line breaks in the time axis are equivalent to 10 minutes. The cell in B was subsequently treated with 100 μM 8- Br-cAMP plus 1 μM PDBu. C) Summary of menthol responses before and after treatment with forskolin and 8-Br-cAMP. Menthol responses were expressed as percent of menthol responses before treatment (control). Menthol was applied for 10 seconds and peak fluorescence intensity was used. Data are represented as mean ± SEM.

Sarria, I

Figure 2.7. Effect of protein kinase inhibitors on menthol responses in MS/CI and MS/CS neurons. A) Responses of MS/CI neurons to prolonged menthol applications (100 μM, 5 min) in control group (red, n = 22) and groups treated with 1 μM BIM (orange, n = 10), 0.5 μM Sta. (green, n = 10), 25 μM KN62 (blue, n = 10), and 25 μM Rp-cAMP (purple, n = 14), P < 0.05. B) Responses of MS/CS neurons to prolonged menthol applications (100 μM, 5 min) in control group (red, n = 18) and groups treated with 1 μM BIM (orange, n = 10), 0.5 μM Sta. (green,

Sarria, I n = 10), 25 μM KN62 (blue, n = 10), and 25 μM Rp-cAMP (purple, n = 10). In both A and B, menthol responses were expressed as percent of peak responses. The data for control groups in both A and B are taken from those in Figure 2D. The horizontal bar in each panel indicates duration of 5-min menthol application. Data are mean ± SEM.

Sarria, I

Notes to Chapter 2

Progress: Published in Molecular Pain (2010) 6:47, doi: 10.1186/1744-8069-6-47

Authors: Ignacio Sarria1,2 and Jianguo Gu1

Affiliations: 1 Department of Anesthesiology, University of Cincinnati College of Medicine, PO

Box 670531, 231 Albert Sabin Way, Cincinnati, OH 45267-0531, USA

2 Graduate Program in Neuroscience, University of Cincinnati College of Medicine, PO Box

670531, 231 Albert Sabin Way, Cincinnati, OH 45267-0531, USA

Acknowledgements: We thank Joanne Anderson and Jennifer Ling for their technical assistance and Dr. Mark Baccei for providing thoughtful comments on the manuscript. This work was supported by a NIH grant DE018661 to J.G.G

Sarria, I

Chapter III:

TRPM8 acute desensitization is mediated by calmodulin and

requires PIP2: Distinction from tachyphylaxis

Sarria, I

Abstract

The cold-sensing channel TRPM8 features Ca2+-dependent down-regulation, a cellular process underlying somatosensory accommodation in cold environments. The Ca2+-dependent down- functional regulation of TRPM8 is manifested with two distinctive phases, acute desensitization and tachyphylaxis. Here we show in rat dorsal root ganglion neurons that TRPM8 acute desensitization critically depends on phosphatidylinositol 4,5-bisphosphate (PIP2) availability rather than PIP2 hydrolysis, and is triggered by calmodulin activation. Tachyphylaxis on the other hand, is mediated by phospholipase hydrolysis of PIP2 and protein kinase C/phosphatase

1,2A. We further demonstrate that PIP2 switches TRPM8 channel gating to a high open probability state with short closed times. Ca2+-calmodulin reverses the effect of PIP2, switching channel gating to a low open probability state with long closed times. Moreover, Ca2+- calmodulin and PIP2 inversely shift the voltage-dependent gating of TRPM8. Thus, through gating modulation, Ca2+-calmodulin provides a mechanism to rapidly regulate TRPM8 functions in the somatosensory system.

Sarria, I

Introduction

Transient receptor potential melastatin 8 (TRPM8) channel, the principal sensor of cold temperatures, is expressed in the somatosensory system and used to detect a broad range of cooling temperatures (Colburn et al 2007; Dhaka et al 2007; Kobayashi et al 2005; McKemy

2005; McKemy et al 2002; Nealen et al 2003; Peier et al 2002; Stucky et al 2009). It may be also involved in pathological pain sensations under disease conditions (Colburn et al 2007; Levine &

Alessandri-Haber 2007; Xing et al 2007b). Interestingly, TRPM8 has been identified in respiratory, visceral, vascular and cancer tissues where it is implicated in respiratory and visceral disorders, and cancer development (Cho et al 2010; Hayashi et al 2009; Knowlton & McKemy

2010; Mukerji et al 2006; Sabnis et al 2008a; Tsavaler et al 2001; Xing et al 2008; Yang et al

2006; Zhang & Barritt 2004). Thus, TRPM8 functional states and its regulation may have broad biological significances.

An important feature of TRPM8 channel is Ca2+-dependent functional down-regulation following activation and subsequent increases of intracellular Ca2+ level (McKemy et al 2002;

Peier et al 2002). This down-regulation is manifested by a substantial reduction of TRPM8- mediated responses when cooling temperatures or TRPM8 agonists activate the channel (Daniels et al 2009; Liu & Qin 2005; Premkumar et al 2005; Rohacs et al 2005b). TRPM8 functional down-regulation in the somatosensory system is believed to play a role in sensory adaptation to cold environments (Daniels et al 2009; Liu & Qin 2005; Premkumar et al 2005; Rohacs et al

2005b). One question about Ca2+-dependent down-regulation of TRPM8 is what intracellular mechanisms are downstream of Ca2+. The answer to this question has significance in that physiological and pathological conditions may affect cold temperature sensitivity through the

Sarria, I downstream mechanisms. Previous studies have shown that phosphatidylinositol 4,5-bisphosphate

(PIP2) hydrolysis following phospholipase C (PLC) activation results in TRPM8 down-regulation

(Daniels et al 2009; Liu & Qin 2005; Rohacs et al 2005b). It has been suggested that Ca2+- dependent down-regulation of TRPM8 may be partially due to the activation of Ca2+-sensitive

PLC, which results in the hydrolysis of PIP2 and subsequent TRPM8 functional down-regulation

(Daniels et al 2009). In addition, protein kinase C (PKC) and protein phosphatases have also been demonstrated to down-regulate TRPM8 function (Abe et al 2006; Premkumar et al 2005). It has been suggested that Ca2+ entry following TRPM8 activation may activate Ca2+-sensitive PKC and protein phosphatases, which in turn results in Ca2+-dependent down-regulation of TRPM8 (Abe et al 2006; Premkumar et al 2005). When TRPM8-mediated responses are carefully analyzed, two temporally distinct types of Ca2+-dependent down-regulation can be identified. One is a rapid reduction of TRPM8-mediated response during a single application of agonist, and desensitization is a proper term for this type of functional down-regulation. The other type of down-regulation is manifested by the reduction of responses to repeated agonist applications, and tachyphylaxis is an appropriate term for this functional change of TRPM8. The presence of these two types of

TRPM8 down-regulation raise a new question as what specific roles PIP2/PLC and PKC/protein phosphatase may play in each type of down-regulation. In addition, most of previous studies on

TRPM8 regulation were performed in heterologous expression systems. It is necessary to directly examine TRPM8 regulation in cold-sensing sensory neurons because there are cellular and molecular differences between heterologous expression systems and sensory neurons.

Calmodulin (CaM) has been recognized to be an important intracellular signaling molecule to mediate Ca2+-dependent regulation of cation channels that are Ca2+-permeable on plasma membranes. These cation channels often become functionally down-regulated when Ca2+-

Sarria, I calmodulin binds to the channels. The functional down-regulation of Ca2+-permeable cation channel by Ca2+-calmodulin serves as a negative feedback mechanism to prevent excessive Ca2+ influx into the cells to cause cell toxicity. CaM-mediated channel down-regulation has been observed in a number of TRP channels, including heat-sensing channel TRPV1 (Lishko et al

2007; Numazaki et al 2003; Rosenbaum et al 2004), warm temperature-sensing channel TRPV3

(Xiao et al 2008b), osmolarity-sensing channel TRPV4 (Strotmann et al 2003), the highly Ca2+- selective channels TRPV5 and TRPV6 (Lambers et al 2004; Niemeyer et al 2001), and TRPM4

(Nilius et al 2005b). Although Ca2+-CaM may be a common mechanism to down-regulate TRP channels function, the degree and kinetics of down-regulation for each TRP channel may be substantially different. For example, TRPM4 and TRPM5, which are both activated directly by

Ca2+, show a slow, Ca2+‑dependent desensitization (Ullrich et al 2005; Zhang et al 2007). In addition to down-regulation, Ca2+-dependent functional up-regulation has also been observed in a number of TRP channels including TRPC5 (Ordaz et al 2005), TRPV3 (Xiao et al 2008b),

TRPV6 (Lambers et al 2004) and TRPA1 (Doerner et al 2007; Zurborg et al 2007) and TRPV4

(Strotmann et al 2010). For TRPV4, a direct binding of Ca2+-CaM to the intracellular domain of the channel is shown to be critical for Ca2+-dependent functional up-regulation (Strotmann et al

2010). Thus, Ca2+-CaM-mediated regulation of TRP channels may result in distinct functional changes for different TRP channels and the differences could be further amplified by cell type differences. Although it is very likely that TRPM8 channels may be regulated by Ca2+-CaM, no study so far has provided direct experimental evidence to validate this idea. If TRPM8 channels are indeed regulated by Ca2+-CaM, there are questions that need to be further addressed. These questions include whether and how Ca2+-CaM may be involved in acute desensitization and/or tachyphylaxis, and to what extent Ca2+-CaM may change the kinetics and magnitudes of TRPM8-

Sarria, I mediated responses in sensory neurons. The answers to these questions will provide insights into cellular and molecular mechanisms of sensory processing of cold stimuli under physiological and pathological conditions. In the present study, we show in somatosensory neurons that multiple intracellular signaling pathways have distinct roles in acute desensitization and tachyphylaxis of

TRPM8.

Sarria, I

Materials and Methods

Cell preparations- For DRG neurons, adult Sprague Dawley rats (100–250 g, both genders) were used. Animal care and use conformed to National Institutes of Health guidelines for care and use of experimental animals. Experimental protocols were approved by the

University of Cincinnati Institutional Animal Care and Use Committee (ID: Gu 09-07-01-01).

DRG neuron cultures were prepared as described previously (Tsuzuki et al 2004). In brief, rats were deeply anesthetized with isoflurane (Henry Schein, NY.) and sacrificed by decapitation.

DRGs were rapidly dissected out bilaterally in Leibovitz-15 media (Mediatech Inc. VA) and incubated for 1 hour at 37 °C in minimum essential medium for suspension culture (S-MEM)

(Invitrogen, Grand Island, NY) with 0.2% collagenase and 0.5% dispase and then triturated to dissociate neurons. The dissociated DRG neurons were then plated on glass coverslips pre-coated with poly-D-lysine (PDL, 12.5 µg/ml in distilled H2O) and laminin (20 µg/ml in Hank's Buffered

Salt Solution HBSS, BD bio-science), and maintained in MEM culture medium (Invitrogen) that also contained nerve growth factor (2.5 S NGF; 10 ng/ml; Roche Molecular Biochemicals,

Indianapolis, IN), 5% heat-inactivated horse serum (JRH Biosciences, Lenexa, KS), uridine/5- fluoro-2’-deoxyuridine (10 µM), 8 mg/ml glucose, and 1% vitamin solution (Invitrogen). The cultures were maintained in an incubator at 37°C with a humidified atmosphere of 95% air and

5% CO2. Unless otherwise indicated, cells were used within 72 hours after plating.

Human embryonic kidney (HEK) 293 cell line was obtained from ATCC (Manassas,

Virginia). The cells were cultured in DMEM (Hyclone) supplemented with 10% FBS (Hyclone) and 0.5 % Penicillin-Streptomycin in culture flasks. Wild type mouse TRPM8 was stably transfected into the HEK293 cells using the protocol described in a previous study (Hu et al

Sarria, I

2004). For electrophysiology recordings, cells were plated on PDL-coated glass coverslips one day before using for experiments.

Electrophysiology- Patch-clamp recordings were performed on menthol-sensitive DRG neurons and HEK 293 cells stably expressing TRPM8. Menthol-sensitive neurons were pre- identified with Ca2+ imaging (Xing et al 2007a) and the cells that showed strong Ca2+ responses following menthol application (100 µM, 5 s) were used for recordings. Cells were perfused at room temperature of 24 oC with normal bath solution containing (in mM) 150 NaCl, 5 KCl, 2

MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.3 adjusted with NaOH, and osmolarity 320 mOsm adjusted with sucrose. In some experiments, calcium was omitted from the bath solution and 5 mM EGTA was added for extracellular Ca2+-free experiments. For whole-cell recordings, cells were voltage-clamped at -70 mV in the whole-cell configuration using a Multiclamp 700A amplifier (Axon Instruments) and signals were sampled at 5 kHz and filtered at 2 KHz using pCLAMP 9.0 (Axon Instruments). Electrode internal solutions contained (in mM) 110 Cs2SO4,

2.4 MgCl2, 10.0 Hepes, 5.0 Na2ATP, 0.33 GTP-Tris salt, pH was adjusted to 7.35 with NaOH and osmolarity 320 mOsm was adjusted with sucrose. In some experiments to test intracellular Ca2+ buffer effects, electrode internal solutions also contained 5 mM EGTA or 5 mM BAPTA.

Recording electrode resistance was around 5.0 MΩ. Whole-cell membrane currents were evoked by rapidly applying menthol solution (100 µM) or cold bath solution (12 ± 0.5 °C) to cells through a glass tube (500 µm ID) positioned 1.0 mm away from cells. The on-time for menthol solution application to a cell under recording was usually less than 500 ms. In order to achieve rapid cooling on recorded cells, bath solution was pre-cooled and maintained at a constant low temperate inside the temperature controlling head stage of the TCM-1 temperature controlling system (Warner Instruments). The cold bath solution was rapidly applied (2 ml/min) to the cells

Sarria, I from a short tube (0.2 cm L, 500 µm ID). With this setting, the on-time to reach stable cooling of

12 ± 0.5 °C usually took less than 3 second, much faster that the time of temperature drop (> 30 s) in most of previous studies (Daniels et al 2009; McKemy et al 2002; Peier et al 2002). Unless otherwise indicated, other testing compounds were directly introduced into the cell by mixing them in the electrode internal solutions.

For cell-attached and inside-out experiments, external bath and electrode internal solutions were ionically symmetrical and contained (in mM) 140 Na-gluconate, 10 NaCl, 10.0 Hepes, pH was adjusted to 7.35 with NaOH and osmolarity 320 mOsm adjusted with sucrose. Recording electrode resistance was around 10 MΩ. In inside-out experiments where cold response was examined, perfusing solution was maintained at 15 °C. For experiments where testing agents were applied, excised membranes were quickly transferred to a separate chamber at 24 °C.

Experiments were performed with membrane potential held from -100 mV to 100 mV using an

Axopatch 200B amplifier. Signals were sampled at 10 KHz and filtered at 2 KHz. Data were collected using the Clampex 9 software.

Phosphatidylinositol-4,5-bisphosphate C-8 (sodium salt) (PIP2) was purchased from

Cayman Chemichals, it was dissolved in electrode internal solution, flash frozen and then stored at -20 °C, and used within 48 hours. Calmodulin (porcine) was purchased from A.G Scientific

Inc. Ophiobolin A was purchased from Santa Cruz Biotechnology. Okadaic acid, U73122,

Go6976, and KN-62 were purchased from Tocris. Menthol, AITC, BIM, BAPTA, and EGTA were purchased from Sigma. Final concentrations of compounds used were: PIP2 (50 µM)

(Rohacs et al 2005b), calmodulin (10 µM) (Black et al 2004), Ophiobolin A (100 µM) (Leung

Sarria, I et al 1985), okadaic acid (100 nM) (Schonthal 1998) KN-62 ( 25 µM), menthol (100 µM), AITC

(100 µM), BIM (1 µM) (Sarria & Gu 2010), Go6976 (1 µM) (Gschwendt et al 1996).

Unless otherwise indicated, these compounds were included in the electrode internal solution and applied into cells through recording electrodes. Recordings were performed after 4 min equilibrium of intracellular contents with electrode internal solutions.

Data analysis- Clampfit 9 was used for analysis of whole-cell and unitary currents, least square fittings, and to create figures. For acute desensitization, percentage desensitization was obtained by dividing the current reduction at 30 seconds by the peak current value. For tachyphylaxis, percentage tachyphylaxis was obtained by dividing current reduction between peaks of two stimuli by peak current values of the first stimulus. For cell-attached and inside-out recordings, channel openings and closings were determined off-line by using a 50% crossing threshold. The probability of channel opening (Po) was calculated as the ratio of the total channel- open time to total time. For single channel recordings, fitting of the closed and open dwell time components were obtained by using the variable metric-maximum likelihood method. The usefulness of adding exponential components was assessed by using an F test (De Koninck &

Mody 1994) and also by visual inspection. Unless otherwise specified data in all figures are presented as Mean ± SEM. Analysis of Variance (ANOVA, one way) were used for statistic analyses of data sets of multiple groups and/or treatments followed by Student-Newman-Keuls

Post Hoc Test for all pairwise comparisons or Dunnet Post Hoc Test for comparison against control group. Student’s t-test was to evaluate the significance of changes in mean values between two groups. Statistical significance was considered at the level of the p < 0.05.

Sarria, I

Results

Acute desensitization and tachyphylaxis of TRPM8 in rat DRG neurons. We performed whole-cell recordings on rat DRG neurons that were voltage-clamped at -70 mV and bathed in an external solution containing 2 mM Ca2+ at the room temperature of 24 oC. When menthol (100

µM, 30 s) was applied for the first time, inward currents showed acute desensitization (Fig. 3.1a, left) with current amplitude reduced by 43.8 ± 5.9 % (n = 14) at the end of 30-s menthol application (Fig. 3.1c). For tachyphylaxis, menthol was applied a second time after a 10-minute interval and maximal inward currents were reduced by 69.0 ± 3.9% (n = 12, Fig. 3.1a right; Fig.

3.1d) in comparison with the peak currents of the first menthol application. Similar to menthol, both acute desensitization and tachyphylaxis were also observed when a cold bath solution was rapidly applied to menthol-sensitive DRG cells pre-identified with Ca2+ imaging. A rapid application of cold bath solution (12 ± 0.5 °C) for 30 s resulted in acute desensitization by 58.6.3

± 9.3 % (n = 9, Fig. 3.1b, left; Fig. 3.1c) at the end of cold bath application. A rapid temperature drop rather than slow temperature ramp was found to be essential to observe cold-induced acute desensitization. In this experiment, temperature dorp from 24 oC to 12 oC was less than 3 s at recording sites, more than 10 times faster than previous studies using temperature ramp (McKemy et al 2002; Thut et al 2003). Repeated cold bath applications with a 10-min interval resulted in tachyphylaxis by 62.2 ± 11.8 % (n = 7, Fig. 3.1b, right; Fig. 3.1d) for the second stimulation.

We determine whether both acute desensitization and tachyphylaxis occur in a similar manner when DRG cells were bath perfused at a more physiological temperature (32 oC) before the application of menthol and cold stimulation. Under this condition, acute desensitization occurred with peak current amplitude being reduced by 57.79 ± 7.43 % (n = 10) at the end of 30-s

Sarria, I menthol application (Fig. 3.2a, left; Fig. 3.2c). Tachyphylaxis occurred with maximal inward currents of the second menthol application being reduced by 68.28 ± 7.25 % (n = 8, Fig. 3.2a, right; Fig. 3.2d) in comparison with the peak currents of the first menthol application. Similar to menthol, both acute desensitization and tachyphylaxis were also observed when a cold bath solution was rapidly applied to menthol-sensitive DRG cells pre-identified with Ca2+ imaging. A rapid application of cold bath solution (12 ± 0.5 °C) for 30 s resulted in acute desensitization by

48.21 ± 6.39 % (n = 12, Fig. 3.2b, left; Fig. 3.2c) at the end of cold bath application. Repeated cold bath applications with a 10-min interval resulted in tachyphylaxis by 71.86 ± 4.2 % (n = 8,

Fig. 3.2b, right; Fig. 3.2d) for the second stimulation. Thus, both acute desensitization and tachyphylaxis occurred in a similar manner under the conditions when cells were bath-perfused either at room temperature or at more physiological temperature.

Similar to DRG neurons, HEK 293 cells expressing mouse TRPM8 displayed an acute desensitization to menthol application (100 µM, 30 s) in which peak currents were reduced by

47.56 ± 4.61 %. Tachyphylaxis occurred with maximal inward currents of the second menthol application being reduced by 71.25 ± 5.34 % (n = 8). When cold (12 ± 0.5 °C, 30s) was used as an agonist, acute desensitization and tachyphylaxes were 63.30 ± 3.33 % and 65.77 ± 8.57 % respectively (n = 13). We have shown that TRPM8 rather than TRPA1 accounts for strong responses evoked by 100 µM menthol in rat DRG neurons (Sarria & Gu 2010). Nevertheless, menthol was shown to have a promiscuous relationship with TRPA1 receptors in expression systems (Macpherson et al 2006). Consistently with our previous finding, TRPA1 agonist allyl isothiocyanate (AITC) did not evoke detectable inward currents in menthol- and cold-sensitive cells in this study (100 µM, n = 5/5).

Sarria, I

PIP2 attenuates tachyphylaxis but not acute desensitization of TRPM8. Do acute desensitization and tachyphylaxis have the same or distinctive molecular mechanisms? To answer this question, we set out to examine how PIP2 availability affected acute desensitization and tachyphylaxis in DRG neurons since PIP2 has been indicated to play an important role in regulating TRPM8 function (Liu & Qin 2005; Rohacs et al 2005b). When the PLC inhibitor

U73122 (25 µM) was included in recording internal solution to block PIP2 hydrolysis, acute desensitization of menthol-evoked currents was 36.6 ± 8.0 % (n = 9, Fig. 3.3b&d) and was as strong as the control, while tachyphylaxis was significantly attenuated to 40.2 ± 9.4 % (n = 9,

Fig. 3.3b&e). A possible explanation for the lack of effect by U73122 on acute desensitization could be due to incomplete block of PLC. To test this possibility we next examined whether direct application of PIP2 (50 µM) into cells would prevent acute desensitization along with tachyphylaxis. To our surprise, direct application of PIP2 into cells did not prevent acute desensitization at all. On the contrary, the acute desensitization became even more substantial at the degree of 70.0 ± 6.7 % (n = 9) and also reproducible upon repeated menthol applications (Fig.

3.3c&d). Tachyphylaxis on the other hand, was greatly attenuated by PIP2 to only 32.7 ± 10.7 %

(n = 9, Fig. 3.3c&e). Neither U73122 nor PIP2 had significant effect on the peak current amplitude evoked by the first menthol application (Fig. 3.3f). The opposite effects of PIP2 on acute desensitization and tachyphylaxis suggest that the two regulatory processes may be mechanistically different.

Both acute desensitization and tachyphylaxis are Ca2+-dependent but they have different

Ca2+-sensitivity. Because both acute desensitization and tachyphylaxis are activity-dependent, we next tested whether both processes are Ca2+-dependent and if so, whether the two processes have different Ca2+-sensitivity. We compared menthol-evoked currents in DRG cells under the

Sarria, I following conditions: 1) without including a Ca2+ buffer in the internal solution, 2) including a slow Ca2+ buffer EGTA (5 mM), or 3) a fast Ca2+ buffer BAPTA (5 mM) in the internal solution, or 4) including BAPTA (5 mM) in the internal solution together with an extracellular Ca2+-free condition. In the presence of 5 mM EGTA, acute desensitization (Fig. 3.4a&d) was 42.4 ± 5.6%

(n = 11) and tachyphylaxis (Fig. 3.4a&e) was 56.9 ± 6.9% (n = 8), not significantly different from those without a Ca2+ buffer (Fig. 3.3d&e). Interestingly, 5 mM BAPTA in the internal solution significantly attenuated tachyphylaxis (28.0 ± 6.8%, n = 9, Fig 4b&e) but had no significant effect on acute desensitization (31.1 ± 7.6% (n = 11, Fig. 3.4b&d). Further removal of extracellular

Ca2+, however, significantly attenuated both acute desensitization (7.7 ± 3.6%, n = 10, Fig.

3.4c&d) and tachyphylaxis (18.4 ± 4.8%, n = 7, Fig. 3.4c&e). There was no significant difference in peak currents of the first menthol-evoked responses under different Ca2+ buffering conditions

(Fig. 3.4f). Thus, while both acute desensitization and tachyphylaxis are Ca2+-dependent, acute desensitization is more sensitive to Ca2+ in comparison with tachyphylaxis as is evidenced by the differential effect of the fast Ca2+ buffer BAPTA.

Inhibiting protein kinase C or protein phosphatase 1,2A attenuate tachyphylaxis, but not acute desensitization. Previous studies showed that activation of either protein kinases C or protein phosphatases down-regulated TRPM8 function (Abe et al 2006; Premkumar et al 2005).

We set out to determine whether acute desensitization in DRG cells may be mediated by protein kinase C/protein phosphatases. To test this possibility, we examined Ca2+-dependent acute desensitization in the presence of protein kinase C inhibitor BIM or in the presence of protein phosphatase 1,2A (PP1,2A) inhibitor okadaic acid. These experiments were conducted under the condition when intracellular Ca2+ was not buffered so that tachyphylaxis was maximized. Under this condition and in the presence of 1 µM BIM (Fig. 3.5b&g) or 100 nM okadaic acid (Fig.

Sarria, I

3.5c&g), tachyphylaxis was 39.7 ± 8.6% (n = 9) and 40.7 ± 8.1% (n = 9), respectively, significantly less than the control without the inhibitors (Fig. 3.5a&g). On the other hand, acute desensitization remained strong and was not significantly affected by either inhibitor in the first menthol application (Fig. 3.5b,c&f). Acute desensitization reoccurred in subsequent menthol application when PKC or protein phosphatase 1,2A was blocked (Fig. 3.5b&c in comparison with

Fig. 3.5a). Acute desensitization in second menthol application was 52.0 ± 7.8 % (n = 8) in the presence of BIM and 44.5 ± 11.5 % (n = 7) in the presence of okadaic acid, not significantly different from the degree of acute desensitization during the first menthol application (43.8 ± 5.9

%, n = 14). Thus, inhibition of PKC and protein phosphatase 1,2A did not prevent acute desensitization although it attenuated tachyphylaxis.

Blocking calmodulin diminishes acute desensitization. Calmodulin has been shown to directly regulate functions of some TRP channels (Nilius et al 2005b; Numazaki et al 2003; Zhu

2005). We asked whether calmodulin is involved in regulating TRPM8 in DRG cells, and if so, whether acute desensitization and/or tachyphylaxis is/are mediated by calmodulin. To test this idea, we determined whether ophiobolin A, a specific calmodulin inhibitor, affected acute desensitization of TRPM8. In the presence of 100 µM ophiobolin A, acute desensitization was substantially attenuated to 21.2 ± 4.7% (n = 8, Fig. 3.5d&f), while tachyphylaxis remained strong

(62.33 ±12.31 %, n = 8) and was not significantly attenuated (Fig. 3.5d&g). Since PIP2 is essential for the appearance of desensitization current upon repeated stimulation (Fig. 3.3c), we included both ophiobolin A (100 µM) and PIP2 (50 µM) in the recording solutions. Under this condition both acute desensitization and tachyphylaxis were greatly diminished to 19.6 ± 6.5% (n

= 10) and 11.3 ± 11.2% (n = 8), respectively (Fig. 3.5e&f&g). Acute desensitization was unlikely mediated by CaMKII or PKD since CaMKII inhibitor KN62 (25 µM) or PKD inhibitor Go6976

Sarria, I

(1 µM) did not significantly attenuate acute desensitization (KN62: 39.01 ± 6.42 %, n = 6,

Go6976: 49.45 ± 7.34 %, n=6, vs. control: 43.8 ± 5.9 %, n = 14). These results support our hypothesis that calmodulin is a primary cause of acute desensitization and PIP2 hydrolysis on the other hand, is an important cause of tachyphylaxis.

PIP2 and calmodulin inversely modulate TRPM8 channel gating. To provide further insights into mechanisms by which PIP2 and calmodulin regulate TRPM8 channels, we performed single channel recordings from membranes of HEK 293 cells that stably express

TRPM8 channels and studied effects of PIP2 and calmodulin on TRPM8 gating properties. In inside-out patches, unitary currents were observed in TRPM8-expressing HEK293 cells (Figure

3.6a) when 100 µM menthol was included in patch electrodes or temperature dropped to 15 oC.

Unitary currents of membrane patches showed a lack of outward rectification and single channel conductances were 59.0 ± 2.9 pS with menthol (n = 5-10) and 62.0 ± 3.1 pS (n = 5) measured at

15 °C (Fig. 3.6b). TRPM8 channel shows voltage-dependent increases in open probability with either 100 µM menthol (n=5-10) or 15 °C (n=5) (Fig. 3.6c). The voltage-dependent change of channel open probability mirrors the outward rectification property observed in whole-cell

TRPM8 currents described previously (McKemy et al 2002). With either menthol (n=5-10) or cold stimulus (n=5), there was a slight increase in the mean open time as voltage increased (Fig.

3.6d). The unitary currents were not observed in control HEK 293 cells that did not express

TRPM8. In cell-attached configuration before excising the patched membranes, the channel open probability (Po) was high at 0.24 ± 0.04 and 0.15 ± 0.05 when pipette potentials were 100 mV or

70 mV, respectively (n = 6, Fig. 3.7a,b&c ). After membranes were excised and inside-out configuration was acquired, channel open probability at 70 mV was significantly reduced to 0.02

± 0.01 (n = 6, Fig. 3.7a,b&c). When PIP2 (50 µM) was added in the solution, the open

Sarria, I probability increased to 0.20 ± 0.05 (n = 6), restored to the cell-attached values (Fig. 3.7a,b&c).

Exposure of the excised membrane to PIP2 did not significantly change the amplitude of unitary currents (n = 7, Fig. 3.5d). Channel conductance calculated from unitary currents was 53.0 ± 4.7 pS (n = 6) without PIP2 and 56.0 ± 1.8 pS (n = 7) with PIP2. Mean open time was also not affected (Fig. 3.7e), but mean closed time was significantly shortened from 107.9 ± 19.4 ms (n =

6) in the absence of PIP2 to 7.2 ± 1.6 ms when PIP2 was present (Fig. 3.7f). While PIP2 increased channel open probability to 0.24 ± 0.07 in yet another group of cells, addition of calmodulin (10 µM) along with 40 µM Ca2+ reversed the effects caused by PIP2, resulting in a significant reduction of the open probability to 0.05 ± 0.02 (n = 7, Fig. 3.8a,b,c). Addition of calcium (40 µM) alone did not have significant effects. Ca2+-calmodulin did not have a significant effect on the amplitude of unitary currents measured at 70 mV (Fig. 3.8e) and the calculated conductance of 54 ± 2.6 pS (n = 7) was not significantly different from that in the absence of Ca2+-calmodulin. When PIP2 or Ca2+-calmodulin was added, mean open time was not significantly different from the control group (Fig. 3.8f). However, mean closed time was significantly shortened from 96.6 ± 14.5 ms (n = 7) in the control to 10.16 ± 3.15 ms (n = 7) with

PIP2. Addition of Ca2+-calmodulin reversed the mean closed time to 86.7 ± 25.6 ms (Fig. 3.8f, n

= 7). Dwell time of channel open could be best fitted into a two exponential model and closed time was best fitted into three components for control, with PIP2, and PIP2 plus calmodulin (Fig.

3.8g). There were no changes of relative portions of open time for either term (Fig. 3.8g&h left).

On the other hand, the portions of closed time showed a significant shift from longer closing (3rd term) to shorter closing (1st and 2nd terms) when PIP2 was added (Fig. 3.8f right & 8g right).

Addition of Ca2+-calmodulin reversed the channel closed-time distribution and shifted it back to the pre-PIP2 values with longer closings occupying 45 ± 11 % of the time vs. 17.1 ± 1.8 % when

Sarria, I only PIP2 was present (n = 7, Fig. 3.8f &g right). Thus, PIP2 and Ca2+-calmodulin inversely regulate TRPM8 gating.

Sarria, I

Discussion

In the present study we have investigated the two phases of Ca2+-dependent down- regulation of TRPM8 channels. We have shown for the first time that acute desensitization is triggered by Ca2+-calmodulin and that PIP2 availability is essential for the appearance of acute desensitization. We have provided clear evidence that PIP2 hydrolysis and PKC/PP1,2A activity are two main causes of tachyphylaxis. We have also demonstrated that Ca2+-calmodulin and PIP2 inversely modulate TRPM8 gating to confer functional states of TRPM8 channel for acute desensitization and tachyphylaxis.

The degree of acute desensitization and tachyphylaxis to consecutive TRPM8 activations has had large variations among different studies (Malkia et al 2009; McKemy et al 2002; Rohacs et al 2005b; Thut et al 2003). Several factors may account for the large variations, including on- time speed of cooling, TRPM8 agonists applied to cells, techniques used for measuring TRPM8 activation, and cell types. High on-time speed of an agonist is critical for revealing rapid desensitization of any ion channel. We have noticed that the phase of acute desensitization would be small or even unnoticeable when menthol or cold solutions were not applied fast enough (on- time speed is slow). The on-time speed for cold application in most of the previous studies was probably too slow (> 30 s) to reveal acute desensitization and its kinetics (McKemy et al 2002;

Thut et al 2003). Previous studies using Ca2+-imaging technique also yielded the appearance of slow TRPM8 desensitization/tachyphylaxis (Malkia et al 2009; Thut et al 2003) because of its technical limitation. Different cell types may also contribute to the variations in the degree of acute desensitization and tachyphylaxis. The kinetics of acute desensitization for TRPM8 appeared to be slower in very big cells such as oocytes (McKemy et al 2002) probably because

Sarria, I agonist on-time could not be achieved fast and uniformly for very big cells. Substantial recovery of TRPM8 from tachyphylaxis was reported in oocytes when the cells were held at 32 oC (Daniels et al 2009). It was thought that physiological temperature might favor PIP2 re-synthesis, thereby allowing a faster recovery of TRPM8 from functional down regulation. In our study on DRG neurons and under whole-cell recording configuration, tachyphylaxis of TRPM8 currents was profound with little recovery in 10 min when cells were held at either room temperature or 32oC.

The lack of recovery from tachyphylaxis in our DRG neurons could be due to a severe dialysis of some intracellular components that are essential for PIP2 re-synthesis. Consistent with this idea, tachyphylaxis of TRPM8 was largely prevented when PIP2 was included in our recording solution. Dialysis of other intracellular components (Andersson et al 2007; Zakharian et al 2009) may also account for some degree of TRPM8 tachyphylaxis and poor recovery. It is conceivable that TRPM8 tachyphylaxs in sensory neurons may be less severe and its recovery may be faster in intact cells under physiological conditions.

An intriguing finding in our study is that inhibiting PLC to reduce PIP2 metabolism or directly introducing PIP2 into reduces tachyphylaxis. Previous studies have indicated that PIP2 is essential for maintaining functions of TRP channels including TRPM8 (Karashima et al 2008; Liu

& Qin 2005; Liu & Liman 2003; Lukacs et al 2007; Rohacs et al 2005b; Zhang et al 2005b). Our study indicates that PIP2 maintains TRPM8 in sensory neurons at a high functional state that is manifested by a high open probability due to the promotion of short channel closings. This is consistent with a recent study of TRPM8 gating by PIP2 using planar lipid bilayers (Zakharian et al 2010). The high functional state is a prerequisite for TRPM8 to undergo gating switch to the low functional state, which is characterized by low open probability dominated by long closed

Sarria, I times in single channel activities. In acute desensitization, Ca2+-calmodulin plays a key role in mediating such a gating switch (Fig. 3.7).

Acute desensitization of TRPM8 is highly sensitive to Ca2+ near the channel pore as evidenced by its resistance to fast intracellular Ca2+ buffering by BAPTA. In contrast, tachyphylaxis may require Ca2+ diffusion as it can be effectively attenuated by intracellular

BAPTA. The different sensitivity to Ca2+ buffering may be due to differences in calcium affinities between calmodulin and PLC (Chin & Means 2000; Wiesner et al 1996), which would favor a sharper acute desensitization by calmodulin and a kinetically slower tachyphylaxis through PIP2 hydrolysis by PLC. The proximity of their regulatory sites to the channel pore may be another factor underlining different Ca2+ buffering sensitivity. Our results suggest that the site involved in acute desensitization either has a very high Ca2+ affinity or it is located on or very close to intracellular termini near the pore of the TRPM8 channels. In our quest to find if acute TRPM8 desensitization is directly mediated by Ca2+-calmodulin or through other linked intracellular signaling pathways, we observed that neither PKC or phosphatase inhibitors attenuated acute desensitization. Additionally, the degree of acute desensitization was not affected by either

CaMKII inhibitor KN-62 39.01 ± 6.42 % (n=6) or PKD inhibitor Go6976 49.45 ± 7.34 % (n=6) when compared to control 43.8 ± 5.9 % (n = 14). These results together with direct effects of calmodulin on TRPM8 gating in inside-out recordings point to a direct action of calmodulin during acute desensitization. This is consistent with kinetics and high Ca2+-sensitivity of acute desensitization. Binding sites for calmodulin has been identified in several other TRP channels including TRPM4 and TRPV1 and Ca2+-calmodulin binding at these sites are suggested to confer

Ca2+-dependent regulation of these channels (Nilius et al 2005b; Numazaki et al 2003). A calmodulin binding site has been located at amino acid 145-198 of the N-terminus of TRPM8

Sarria, I using a pull-down assay (Qin & Flores 2007). This raises the possibility that the binding of calmodulin at this site may cause the channel to enter the low functional state, resulting in acute desensitization (Fig. 3.9). Future studies using point mutation are needed to further pinpoint the sites that are critical for calmodulin binding and acute desensitization.

Different from acute desensitization, we found that tachyphylaxis was significantly attenuated by inhibiting PIP2 hydrolysis or directly providing PIP2. This result is consistent with the idea that PIP2 is essential in maintaining TRPM8 at the high functional state and also identifies the specific role of PIP2 hydrolysis in TRPM8 regulation. PIP2 hydrolysis through

Ca2+-dependent PLC can cause a gradual reduction of PIP2 availability, which in turn switches the channel to the low functional state and thus tachyphylaxis (Fig. 3.9). At the single channel level, the low functional state has low open probability with a predominance of long closed times.

This is detectable upon excising the patches from cell-attached to inside-out configuration. PIP2 prevents the channel from entering the low functional state and thereby prevents tachyphylaxis.

Tachyphylaxis and the events that cause tachyphylaxis (e.g. PIP2 hydrolysis) may start occurring along with the acute desensitization phase as evidenced by the slow and mild current decay in the presence of calmodulin inhibitor ophiobolin A (Fig. 3.5d). However, tachyphylaxis is kinetically much slower in comparison with acute desensitization and thereby is less significant for the initial reduction of TRPM8 responses. The events that lead to tachyphylaxis appear to have a profound effect such that it is continuous even after TRPM8 activation is terminated. While we identify that

PLC/PIP2 is a main pathway leading to tachyphylaxis, we also identify that block of either PKC or protein phosphatase 1,2A reduced tachyphylaxis, a result delineates the temporal phase of the effects mediated by PKC and protein phosphatase 1,2A in down-regulation of TRPM8. A previous study suggested that PKC activation caused dephosphorylation rather than

Sarria, I phosphorylation of TRPM8, which lead to TRPM8 down-regulation (Premkumar et al 2005). It is possible that dephosphorylation of TRPM8 either directly by phosphatase 1,2A or indirectly following PKC activation may affect PIP2 availability or the affinity the TRPM8 channels have for PIP2, which could contribute to tachyphylaxis (Fig. 3.9). We showed that the inhibition of

PKC or protein phosphatase 1,2A resulted in a re-appearance of the acute desensitization in response to each repeated menthol application, a result similar to the experiments with PLC inhibitor or with the intracellular addition of PIP2. This raises the possibility that PKC or protein phosphatase 1,2A activity may affect PIP2 availability or affinity between PIP2 and TRPM8 protein and thereby changing channel functional states.

In summary, calmodulin and PIP2 inversely switch channel gating to regulate the functional states of TRPM8. Although both acute desensitization and tachyphylaxis are the results of the gating switches from a high functional state maintained by PIP2 to a low functional state, the two processes are different with the former requiring Ca2+-calmodulin and the latter dependent on PLC, PKC and protein phosphatases. Thus, through multiple intracellular signaling pathways,

TRPM8 functions in sensory neurons can be regulated in the forms of acute desensitization and tachyphylaxis.

Sarria, I

Tables and Figures

Figure 3.1. Menthol-and cold-evoked currents in DRG neurons bath-perfused at room temperature. (a) Sample traces show whole-cell currents recorded from a rat DRG neuron following 2 applications of 100 µM menthol. (b) Sample traces show whole-cell currents recorded from a rat DRG neuron following 2 applications of a cold bath solution. Two traces under the currents show the temperature step that produces rapid cooling from room temperature of 24 oC to 12 oC. In both (a) and (b), each application was 30 s and intervals between each

Sarria, I application was 10 min. (c) Pooled results of acute desensitization during the first application of 100 µM menthol (n = 9) or 12 oC cold bath solution (n = 7). (d) Pooled results of tachyphylaxis following the consecutive application of 100 µM menthol (n = 9) or cold 12 oC cold bath solution (n = 7). In all experiments cells were held at -70 mV. Data represent Mean ± SEM.

Sarria, I

Figure 3. 2. Menthol-and cold-induced responses in DRG neurons bath-perfused at 32°C (a) Sample traces show whole-cell currents recorded from a rat DRG neuron following 2 applications of 100 µM menthol. The menthol solution was at the room temperature of 24 oC. (b) Sample traces show whole-cell currents recorded from a rat DRG neuron following 2 applications of a cold bath solution. Two traces under the currents show the temperature step that produces rapid cooling from 32oC to 12oC. In both (a) and (b), each application was 30 s and intervals between each application was 10 min. (c) Pooled results of acute desensitization during the first application of 100 µM menthol (n = 8) or 1 oC cold bath solution (n = 10). (d) Pooled results of tachyphylaxis following the consecutive application of 100 µM menthol (n = 8) or 12oC cold bath solution (n = 10). In all experiments cells were held at -70 mV. Data represent Mean ± SEM.

Sarria, I

Figure 3.3. PIP2 attenuates tachyphylaxis and confers TRPM8 functional state for acute desensitization. Sample traces show menthol-evoked whole-cell currents from rat DRG neurons of untreated (a, control), PLC blocker U73122-treated (b), and PIP2-treated cells (c). In each group, menthol (100 µM) was applied twice at an interval of 10 min and the duration of each application was 30 s. (d) Summary of desensitization from peak (p) to the end (d) of first menthol application in untreated (n = 12), U73122 (n = 9) and PIP2 treated (n = 9) cells. (e) Summary of tachyphylaxis from the peak current (p) of the first menthol application to the current maximum of the second menthol application (t) for untreated (n = 12), U73122 (n = 9) and PIP2 (n = 9) treated cells. (f) Average peak current of the first menthol application for the

Sarria, I three groups, n is the same as in (d) and (e). U73122 (25 µM) and PIP2 (50 µM) were included in the internal electrode solution, which in all cases was allowed to dialyze for 3-4 minutes before applying menthol (100 µM). All experiments were carried out at 24 oC with cells held at - 70 mV and bath solution contained 2 mM Ca2+. Data represent Mean ± SEM, *P < 0.05 vs. control, one way ANOVA and Dunnet post-hoc.

Sarria, I

Figure 3.4. Both acute desensitization and tachyphylaxis are Ca2+-dependent but show different Ca2+-sensitivity. Sample traces show menthol-evoked currents from rat DRG neurons under following intracellular Ca2+ buffering conditions: 5 mM EGTA (a), 5 mM BAPTA (b), or 5 mM BAPTA in a Ca2+-free (0 Ca2+) bath solution (c). In each group, menthol (100 µM) was applied twice at an interval of 10 min and the duration of each application was 30 s. (d) Desensitization from the peak current (p) to the end (d) of the first menthol application for

Sarria, I

EGTA (n = 11), BAPTA (n = 10) and BAPTA/0 Ca2+ (n = 10) groups. (e) Average tachyphylaxis from the peak current of the first menthol application (p) to the maximum current of the second menthol application (t) in EGTA (n = 8), BAPTA (n = 9) and BAPTA/0 Ca2+ (n = 7) groups. (f) Average peak current amplitude of the first menthol application for the three groups, cell numbers are the same as in (d). All experiments were carried out at 24oC with cells held at -70 mV. Calcium chelators were introduced into the cells via the recording internal solution and allowed to dialyze for 3-4 minutes before menthol application. Data represent Mean ± SEM, *P < 0.05 vs. control, one way ANOVA and Dunnet post-hoc.

Sarria, I

Figure 3.5. Inhibition of calmodulin attenuates acute desensitization, blocking PKC and protein phosphatase 1,2A reduces tachyphylaxis . Sample traces show menthol-evoked currents from rat DRG neurons under the following conditions: control (a), 1 µM PKC blocker BIM (b), 100 nM protein phosphatase 1,2A blocker okadaic acid (OA, c), 100 µM calmodulin blocker ophiobolin A (Oph, d), and 100 µM ophiobolin A plus 50 µM PIP2 (e). (f)&(g) Summary of acute desensitization (f) and tachyphylaxis (g) in control (n = 14), with BIM (n = 9), OA (n = 9), Oph (n = 8), and Oph plus PIP2 (n = 10). In each group, menthol (100 µM) was applied twice at an interval of 10 min and the duration of each application was 30 s. Data

Sarria, I represent Mean ± SEM, *P < 0.05 vs. control, one way ANOVA and Dunnet post-hoc. In all experiments, currents were recorded at holding potential of -70 mV in bath solution containing 2 mM Ca2+ and temperature of 24°C. Blockers and PIP2 were introduced into the cells via the recording internal solution and allowed to dialyze for 3-4 minutes before applying menthol (100 µM).

Sarria, I

Figure 3.6. Single channels characteristics of TRPM8. (a). Unitary currents recorded from an inside-out patch with either electrode containing 100 µM menthol (left 3 traces) or in a cold bath solution (15oC, no menthol, right 3 traces). Pipette potentials were -70, 70 and 100 mV. Closed (c) and open (o) states are indicated. (b) I-V relationship of TRPM8 unitary currents and calculated conductance (G) (c). Open probability (Po) at different holding potentials. (d) Dwell time of channel open at different holding potentials. In (b), (c) and (d), solid circles represent tests of menthol and open circle tests of cold, n = 5-10 cells for each holding potential. HEK 293 cells stably expressing TRPM8 were used.

Sarria, I

Figure 3.7. Modulation of TRPM8 channel gating by PIP2. (a) Unitary currents recorded from a single patch under the following conditions, from top to bottom: cell-attached configuration (C/A) with pipette’s potentials of 70 mV and 100 mV and in inside-out configuration (I/O) with pipette’s potentials of 70 mV before and after addition of 50 µM PIP2. Open (o) and closed (c) states are indicated in the first trace. (b) Time course of TRPM8 channel open probability (Po) for the recording illustrated in (a). Time bins: 2 s. Breaks: 15 s. (c) Mean open probability (Po). (d) Mean unitary current amplitude. (e) Mean open time. (f) Mean closed time. In all experiments, HEK 293 cells stably expressing mouse TRPM8 were used and 100 µM menthol was included in the recording electrodes. Data represent Mean ± SEM, n = 6 for each group, *P < 0.05 vs. control, one way ANOVA and post-hoc STK pairwise comparisons.

Sarria, I

Figure 3.8. Differential modulation of TRPM8 gating by PIP2 and calmodulin. (a) Unitary currents recorded from an inside-out patch with pipette potentials at 70 mV before (control; upper 2 traces), during application of 50 μM PIP2 (middle 2 traces) and after addition of 10 μM calmodulin (CaM) plus 40 μM Ca2+ (bottom 2 traces). (b) Time course of TRPM8 open probability (Po) for the recording illustrated in (a). Time bin: 2 s; breaks: 15 s. (c) Mean Po at 70 mV in control (n = 7), following PIP2 (n = 7), and after Ca2+-calmodulin (n = 7). (d) Inverse shift of voltage-dependence of TRPM8 open probability by PIP2 and Ca2+-CaM in the recording illustrated in (a). (e) Mean unitary current amplitude. (f) Mean open and closed time. (g) Dwell histograms of open and closed states in control (top), following PIP2 (middle), and addition of

Sarria, I

Ca2+-CaM (bottom). The curves show the variable metric-maximum likelihood fit; n is the number of events. There were two open components and three closed components. τ, time constant; percentage values indicate relative areas of each component. In average, τ1 = 0.37 ± 0.05 ms and τ 2 = 1.7 ± 0.26 ms for open time. For closed time, τ1 = 0.68 ± 0.05 ms, τ2 = 6.3 ± 0.92 ms, and τ3 = 117 ± 31 ms. (h) Summary of the relative areas occupied by each component in the open state (left) and closed state (right) during control, PIP2, and PIP2 + Ca2+-CaM. Data represent Mean ± SEM, *P < 0.05 vs. control, one way ANOVA and post-hoc STK pairwise comparisons. HEK 293 cells stably expressing TRPM8 were used and 100 μM menthol was included in recording electrodes.

Sarria, I

Figure 3.9. Schematic diagram of Ca2+-dependent modulation of TRPM8 channels Ca2+ entry through TRPM8 channels or other sources can activate calmodulin, which leads to a switch of TRPM8 channels from a high to a low activity state and thereby causing acute desensitization at the macroscopic current level. Calmodulin binding sites may be located at N- terminus of TRPM8 channels. Calmodulin-mediated acute desensitization may not be observed if TRPM8 channels are already at low activity state due to other regulatory mechanisms. One of the other regulatory mechanisms is PIP2 hydrolysis following the activation of Ca2+-dependent PLC, which also result in a switch of TRPM8 channels from a high to a low activity state. However, because there are multiple steps that are involved in PIP2 hydrolysis following Ca2+ entry and also because PIP2 on membranes is abundant, the consumption of PIP2 is likely to be a slower process in comparison with calmodulin activation by Ca2+. Therefore, the switch of TRPM8 channels from a high to a low activity state following PIP2 hydrolysis is also likely to be a slower process or tachyphylaxis at the macroscopic current level. TRPM8 channels may be

Sarria, I dephosphorylated directly by protein phosphatase1,2A or indirectly following PKC activation, which may lower the affinity between TRPM8 channels and PIP2 and thereby affecting the activity state of TRPM8 channels.

Sarria, I

Notes to Chapter 3

Progress: Articles in Press. J Neurophysiology (September 7, 2011). doi:10.1152/jn.00544.2011

Authors: Ignacio Sarria1, Jennifer Ling1, Michael X. Zhu2, Jianguo G. Gu1

Affiliations: 1 Department of Anesthesiology and the Graduate Program in Neuroscience, The

University of Cincinnati College of Medicine, PO Box 670531, 231 Albert Sabin Way,

Cincinnati, OH 45267-0531, USA.

2 Department of Integrative Biology & Pharmacology, The University of Texas Health Science

Center at Houston, 6431 Fannin Street, Houston, Texas 77030, USA.

Acknowledgements: We would like to give out thanks to Joanne Anderson for technical assistance: This work was supported by a NIH grant DE018661 to J.G.G.

Sarria, I

Chapter IV:

Cold differentially modifies sensory neuron action potential firing properties: Contribution to sensory distinction between innocuous and noxious cold

Sarria, I

Abstract

Cooling temperatures not only directly generate pleasant cooling and painful sensations, but also affect other sensory modalities such as touch, itch and pain probably through modifying sensory neuron excitability. Here we investigated whether and how cooling temperatures modify membrane and action potential firing properties in two functionally distinct groups of rat dorsal root ganglion (DRG) neurons, Tetrodotoxin-sensitive (TTXs) Na+ channel-expressing neurons and Tetrodotoxin-resistant (TTXr) Na+ channel-expressing neurons.

We found that multiple action potential firing responding to prolonged membrane depolarization was strongly suppressed in TTXs neurons but was maintained or facilitated in

TTXr-neurons when cooling temperatures were applied to these cells. We further found that cooling temperatures strongly inhibited A-type K+ currents (IA) and TTXs Na+ channels but had less inhibitory effects on TTXr Na+ channels. For TTXs cells, strong inhibition of TTXs Na+ channels by cooling temperatures was due to the promotion of voltage-dependent slow inactivation, which contributed to the impairment of action potential firing at cooling temperatures. The inhibition of IA currents by cooling temperatures impaired the rapid and full membrane repolarization after an action potential firing, which is a cause of the failure of repeated firing of TTXs action potentials. For TTXr neurons, voltage-dependent slow inactivation was not affected by cooling temperatures, which allowed these neurons to fire TTXr action potentials at noxious cold temperatures. Moreover, IA in TTXr neurons served as a brake to oppose membrane depolarization, and the inhibition of IA currents by cooling temperatures released the brake and thereby facilitating multiple TTXr action potentials. Finally, in DRG neurons that expressed cold transducer TRPM8 channels we show that the effects of cooling

Sarria, I temperatures on IA currents, TTXs and TTXr Na+ channels contributed to sensory distinction between innocuous and noxious cold stimuli.

Sarria, I

Introduction

Psychophysical studies in normal humans show that cooling temperatures in the range of

30°C to 15°C are innocuous while below 15°C provokes painful sensations that often described as multiple modalities including burning, stinging, tingling, and pressing (Morin & Bushnell

1998). Under pathological conditions such as complex regional pain syndrome, innocuous cold can induce painful sensations that are clinically called cold allodynia (Tahmoush et al 2000).

Recent studies have indicated that TRPM8 is a principal transducer for cold stimuli in sensory primary afferents (Bautista et al 2007; Colburn et al 2007; Dhaka et al 2007; McKemy et al

2002; Peier et al 2002; Reid et al 2002) although other molecules such as TRPA1 (Story et al

2003) and TREK1 channels (Reid & Flonta 2001a; Viana et al 2002b) are also proposed to be candidates of cold transducers. TRPM8 has been shown to be able to detect cooling temperatures between 28°C to 10°C (McKemy et al 2002; Peier et al 2002) and the cold sensor is expressed in a subpopulation of nociceptive and non-noceptive sensory neurons at different abundance

(Dhaka et al 2008; Takashima et al 2007; Xing et al 2007b; Xing et al 2006), suggesting that this single molecule may be involved in both innocuous cooling and noxious cold. However, it is still a mystery how mammalian sensory system can differentiate between innocuous and noxious cooling stimuli with this single transducer.

Interestingly, in addition to the sensory modality of cold, cooling temperatures affect other sensory modalities such as touch (Phillips & Matthews 1993), itch (Fruhstorfer et al 1986) and pain (Meeusen & Lievens 1986; Oosterveld & Rasker 1994). For example, touch sensations become less acute and itch can be relieved at cooling temperatures (Fruhstorfer et al 1986;

Phillips & Matthews 1993). Effect of cooling temperatures on pain is more intriguing. Cooling

Sarria, I temperatures are often used to relieve acute pain such as using an ice pack to relieve pain after ankle sprain (Meeusen & Lievens 1986). However, chronic pain such as arthritic joint pain can become exacerbated in cold weather (von Mackensen et al 2005), yet cold therapy is sometimes used to relieve chronic pain (Oosterveld & Rasker 1994). The broad range in modification of sensory modalities by cooling temperatures suggests that a fundamental neuronal process rather than a specific cold transducer may be involved, and one possibility is the effect of cooling temperatures on sensory afferent excitability and action potential firing properties. This hypothesis stems from an early study in giant axon of squid showing that action potentials were significantly affected by cooling temperatures (Hodgkin & Katz 1949).

Voltage-gated Na+ channels and voltage-gated K+ channels are two main ion channels that determine properties of an action potential firing and the firing patterns of action potentials.

Adult somatosensory neurons express several types of voltage-gated Na+ channels, including

NaV1.1, NaV1.6, NaV1.7, NaV1.8 and NaV1.9 (Dib-Hajj et al 2010; Wang et al 2011). Of them,

+ NaV1.8 and NaV1.9 are resistant to the block by tetrodotoxin (TTX-resistant Na channels) and selectively expressed on small diameter nociceptive sensory neurons to conduct nociceptive information (Akopian et al 1996; Akopian et al 1999; Lai et al 2002); the remaining Na+ channels are highly sensitive to the block by TTX and expressed on both non-nociceptive and nociceptive neurons (Dib-Hajj et al 2010). Cells that predominantly expressed TTX-sensitive

Na+ channels were most likely to be non-nociceptive neurons although differentiation between non-nociceptive and nociceptive neurons cannot be simply determined by TTX-sensitivity

(Connor et al 2005) . It has been shown that TTX-sensitive Na+ channels and TTX-resistant Na+ channels are two important molecular determinants for the thresholds of action potential firing in

Sarria, I non-nociceptive and nociceptive neurons, respectively (Yoshimura et al 1996). One important property of voltage-gated Na+ channels is their slow inactivation, which occurs near resting membrane potentials, is voltage-dependent, and can significantly influence sensory neurons action potential firing (Blair & Bean 2003; Rush et al 1998). A recent study showed that cooling temperatures shift slow inactivation of TTX-sensitive Na+ channels to more hyperpolarized membrane voltages but had little effects on the slow inactivation of TTX-resistant Na+ channels, which reveals an underlying mechanism for nociceptive afferent to be able to conduct sensory impulses at nociceptive cold temperatures (Zimmermann et al 2007).

Cooling temperatures may also affect sensory neuron excitability and action potential firing properties through effects on K+ channels. It has been shown in a small population of sensory afferents that cooling temperatures close leak K+ conductance to cause membrane depolarization (Reid et al 2002; Viana et al 2002b). Closing leak K+ conductance by cooling temperatures also could increase sensory neuron input resistance, which was thought to contribute to the increased excitability of some nociceptive afferents (Zimmermann et al 2007).

Studies have also associated A-type K+ currents (IA currents) with the high temperature threshold in a subpopulation of cold-sensing trigeminal neurons (Madrid et al 2009). IA currents are mediated by voltage-gated K+ channels that are characterized as voltage-dependent inactivation and are expressed on most DRG neurons in a variable abundance. IA currents are involved in modulating the action potential shape, threshold, and the regulation of the inter-spike interval (Yoshimura et al. 1996; Yost 1999). Several subtypes of IA currents have been identified in different functional groups of sensory neurons, consistent with the expressing of multiple subunits of voltage-gated K+ channels in these DRG neurons (Gold et al 1996; Rasband et al 2001;

Sarria, I

Yoshimura et al 1996) In nociceptive neurons, IA currents have been proposed to function as a brake to counteract membrane depolarization and thereby restricting nociceptive neurons excitability (Sculptoreanu et al 2004; Vydyanathan et al 2005). Inhibiting or enhancing IA currents pharmacologically increased or suppressed C-fiber afferent neuron excitability respectively (Sculptoreanu et al 2004). Down-regulation of IA currents occurred following nerve injury, which is thought to contribute to the increases of nociceptive neuron excitability that leads to neuropathic pain (Chien et al 2007; Tan et al 2006). It is currently unknown whether cooling temperatures may modify sensory neuron excitability and firing properties through affecting voltage-gated K+ channels functions. In the present study, we show that both voltage- gated Na+ channels and voltage-gated K+ channels are molecular determinants for cold effects on sensory neuron excitability and action potential firing properties, which contribute to sensory distinction between innocuous cold and noxious cold in neurons that expressed the cold sensor

TRPM8.

Sarria, I

Methods

Cell preparations: For DRG neurons, adult Sprague Dawley rats (100–250 g, both genders) were used. Animal care and use conformed to National Institutes of Health guidelines for care and use of experimental animals. Experimental protocols were approved by the

University of Cincinnati Institutional Animal Care and Use Committee (ID: Gu 09-07-01-01).

DRG neuron cultures were prepared as described previously (Tsuzuki et al 2004). In brief, rats were deeply anesthetized with isoflurane (Henry Schein, NY.) and sacrificed by decapitation.

DRGs were rapidly dissected out bilaterally in Leibovitz-15 media (Mediatech Inc. VA) and incubated for 1 hour at 37 °C in minimum essential medium for suspension culture (S-MEM)

(Invitrogen, Grand Island, NY) with 0.2% collagenase and 0.5% dispase and then triturated to dissociate neurons. The dissociated DRG neurons were then plated on glass coverslips pre- coated with poly-D-lysine (PDL, 12.5 µg/ml in distilled H2O) and laminin (20 µg/ml in Hank's

Buffered Salt Solution HBSS, BD bio-science), and maintained in MEM culture medium

(Invitrogen), 5% heat-inactivated horse serum (JRH Biosciences, Lenexa, KS), uridine/5-fluoro-

2’-deoxyuridine (10 µM), 8 mg/ml glucose, and 1% vitamin solution (Invitrogen). The cultures were maintained in an incubator at 37°C with a humidified atmosphere of 95% air and 5% CO2.

For the experiments on cold- and menthol-responsive cells, cells were used within 24hrs after plating. For the rest of the experiments cells were used within 36 hours after plating.

Electrophysiology: Coverslips with cultured neurons were placed in a 0.5-ml microchamber, mounted on an inverted microscope (Olympus IX70), and continuously perfused with a bath solution (see below) at 2 ml/min. Small-sized DRG neurons were chosen for patch- clamp recordings. These cells were pre-identified as menthol- and cold-insensitive cells (MIS)

100

Sarria, I and menthol- and cold-sensitive cells (MS) by using the Ca2+ imaging method which we have previously described (Sarria & Gu 2010). Patch-clamp recordings were then performed on these

MIS and MS cells to study effects of cooling temperatures on their membrane excitability and action potential firing properties under current clamp configuration and voltage-gated Na+ channels and voltage-gated K+ channels under voltage-clamp confirmation. For current-clamp experiments, cells were perfused with a normal bath containing (in mM) 150 NaCl, 5 KCl, 2

MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.3 and ~330 mOsm. Unless otherwise indicated, patch-clamp electrode internal solution contained (in mM) 135 K-Gluconate, 5 KCl, 2.4 MgCl2,

0.5 CaCl2, 5 EGTA, 10.0 Hepes, 5.0 Na2ATP, 0.33 GTP-Tris salt, pH was adjusted to 7.35 with

NaOH and osmolarity was adjusted with sucrose to 320 mOsm. In experiments performed on

MS cells, 5 EGTA was replaced with 20 Bapta to prevent TRPM8 channels from tachyphylaxis

(Sarria et al 2011). Whole-cell voltage-clamp recordings were performed using different bath solutions and internal solutions in order to isolate currents of interests. For isolating sodium currents under whole-cell voltage clamp, bath contained (in mM) 70 NaCl, 50 Choline-Cl, 20

TEA-Cl, 1 MgCl2, 0.01 CaCl2, 10 glucose, 10 HEPES, pH 7.3 and ~330 mOsm. Patch-clamp electrode internal solution contained (in mM) 110 Cs2SO4, 2.4 MgCl2, 0.5 CaCl2, 5 EGTA, 10.0

Hepes, 5.0 Na2ATP, 0.33 GTP-Tris salt, pH was adjusted to 7.35 with NaOH and osmolarity 320 mOsm was adjusted with sucrose. For potassium currents isolation, bath contained in (mM) 130

Choline-Cl, 5 KCL, 1 MgCl2, 10 Glucose, 10 Hepes 10, pH 7.3 and ~330 mOsm. Patch-clamp electrode internal solution contained (in mM) 135 K-Gluconate, 5 KCl, 2.4 MgCl2, 0.5 CaCl2, 5

EGTA, 10.0 Hepes, 5.0 Na2ATP, 0.33 GTP-Tris salt, pH was adjusted to 7.35 with NaOH and osmolarity 320 mOsm was adjusted with sucrose. Junction potentials between baths and electrode solutions were calculated and corrected for in the data analysis. Recording electrode

101

Sarria, I resistance was 4-6 MΩ, and membrane access resistance at whole-cell configuration was under

20 MΩ and was not compensated. Current and voltage signals were recorded with an Axopatch

200B amplifier (Axon Instruments) and signals were sampled at 10 kHz and filtered at 2 KHz using pCLAMP 9.0 (Axon Instruments).

Current-clamp protocols: Menthol- and Cold-insensitive cells were current clamped and a series of square pulse currents were injected to the cells at a rate of 1 Hz and duration of 600 ms for cells expressing TTXs Na+ channels and 4 s for cells expressing TTXr Na+ channels. The current-clamp protocols were applied at 29°C, 24°C, 15°C, and 10°C. Cells that had a resting membrane potential higher than -40 mV were discarded. Cells with an increase in passive leak current during cooling were also discarded. For menthol- and cold-sensitive cells, square pulse currents (600 ms) were tested at 24°C first. Then a slow ramp of cooling temperature from 29°C to 10°C was applied to cells to evoke cold-induced membrane depolarization and action potential firing.

Voltage-clamp protocols: TTX (500 nM) was used to determine if a cell had only TTX- sensitive voltage-gated Na+ channels (TTXs cell) or had TTX-resistant voltage-gated Na+ channels (TTXr cell) as well. This was achieved by applying a series of voltage steps (10 mV each step) from the holding potentials of -80 mV (from -90 to 10 mV) in the absence and presence of presence 500 nM TTX.

Isolated Nav currents were activated by test pulses to 10 mV (TTX-sensitive) or 0 mV

(TTX-resistant) from holding potentials (Vh) of -120 mV or -80 mV, respectively. For steady- state fast inactivation, 50-ms prepulses from -140 mV (TTXs) or -120mV (TTXr) in steps of 10

102

Sarria, I mV were used, followed by a 50-ms test pulse to 10mV (TTXs) or 0mV (TTXr) (Vh= -80). For steady-state slow inactivation, 30-s pre-pulses from 120mV to 0mV in steps of 10mV were used, followed by a 100-ms pulse to -120mV to remove fast inactivation, and then a 50-ms test pulse to 10mV (TTXs) or 0mV (TTXr) (Vh= -80 mV).

Pure voltage-gated potassium (Kv) currents were activated with by 480-ms depolarizing voltage steps to potentials ranging between -35 and +45 mV in 10 mV increments preceded by a

2-second pre-pulse voltage step to either – 100 mV (for whole current) or -10 mV (non- inactivating (IK) current only). The steady-state inactivation protocol consisted of a 2-second pre-pulse voltage step to potentials ranging between - 110 and 0 mV followed by a depolarizing voltage step to +45 mV.

Application of cooling temperatures and Drugs: The temperatures of bath solutions were controlled by a Peltier cooling device (model TCM-1), which were delivered to recorded cells from a short tube (0.2 cm L, 500 µm ID) with the outlet 500 µm from the recorded cells. The temperatures at the recording sites were continuously recorded with a thermal probe that attached to the controller of the Peltier cooling device. Drugs were delivered by another short tube same as the one described, but without temperature control; thus they were delivered and tested at room temperature ~24°C. For voltage- and current-clamp experiments, a targeted temperature of

29°C, 24°C, 15°C, or 10°C was reached first and then voltage and/or current protocols were applied to determine changes in currents mediated by voltage-gated Na+ and K+ channels, and membrane properties respectively. For current clamp experiments of cold-sensing cells in which cold was used to drive depolarization, membrane potentials and action potential firing were

103

Sarria, I continuously recorded while temperature of perfusion bath solution dropped from 29~10°C. The cooling temperature was controlled to have a slow ramp that took 3 min from 29 to 10°C.

Data Analysis: Whole-cell recordings from voltage and current clamp experiments were analyzed using Clampfit 9 software. The inactivating/transient potassium current (A-type) was obtained by digital subtraction of the records obtained when pre-pulsing to -10mV from the currents obtained from the -100mV pre-pulse potential. The 4-Aminopyridine (4-AP) susceptible current (A-type) was obtained by digital subtraction of the records to the same potential before and after application of 100 μM 4-AP for 2 minutes. To obtain the midpoint of activation and steady-state fast and slow inactivation for Nav channels, test-pulse-evoked peak currents were measured and normalized to conductance (G) by dividing the evoked current by the driving force, according to the following equation: G=I /(Vm-Vrev), where Vm is the testing potential and Vrev is the theoretical reversal potential of the current determined from the Nernst equation for Na+ ion. Subsequently, the data were plotted and fitted with a Boltzmann function: G =

Gmax/ (1+ exp[(V1/2 - V)/s]), where G is the observed whole-cell conductance, Gmax is the fitted maximal conductance, V1/2 is the potential for half-maximal activation, and s is the slope factor. Data are reported as mean ± SEM. Statistical significance (p < 0.05) was assessed by

Student’s t test for two groups or by Analysis of Variance (ANOVA, one way) for data sets of multiple treatments followed by Student-Newman-Keuls Post Hoc Test for all pairwise comparisons or Dunnet Post Hoc Test for comparison against control group.

104

Sarria, I

Results

We chose small-sized DRG cells with diameters in a relatively narrow range (around 20 to 30 µm) so that passive cell membrane parameters are comparable among the cells included in this study. Cells were classified into TTX-sensitive (TTXs) and TTX-resistant (TTXr) cells based on their sensitivity to the block by 500 nM TTX of voltage-gated Na+ channels and/or action potentials firing. TTXs cells were those cells whose voltage-evoked Na+ channel currents were completely inhibited by 500 nM TTX. TTXr cells were the cells whose voltage-evoked

Na+ channel currents retained at least 10% of total current in the presence of 500 nM TTX. TTXr cells usually also expressed TTXs currents in a variable abundance. Our TTXs cells had membrane capacitance of 23.6 ± 1.4 pF (n = 59) and was not significantly different from the membrane capacitance of our TTXr cells (22.0 ± 1.5 pF, n = 48).

We tested effects of cooling temperatures on TTXs and TTXr cells that did not express cold sensors first. These are the cells that did not respond to menthol and cold (menthol- and cold-insensitive cells) in our experiments using Ca2+ imaging technique for pre-identification

(Sarria & Gu 2010). The reason to choose these neurons is to avoid the complications by the transducer activation, which allowed us to study the contribution of non-transducer molecules to neuronal excitability and action potential firing under better controlled membrane voltages.

Figure 4.1 shows examples of the effects of cooling temperatures on action potential firing patterns in TTXr and TTXs cells. At 24°C, upon maintained membrane depolarization for 4 sec with current steps at 3X rheobase, TTXr cells (n = 37) showed several action potential firing patterns, including tonic (n=11, Fig. 4.1E), adaptive (n =6, Fig. 4.1D), irregular (n = 5, Fig.

4.1C), long-delayed (n = 4, Fig. 4.1B), and phasic (n = 11, Fig. 4.1A) firing patterns. Twenty five

105

Sarria, I

(25) of them were tested with cooling temperatures of 15°C, the same current steps increased firing frequency in 11 out 25 cells (11/25) identified as irregular (3/5), delayed (2/4), phasic

(4/7), tonic (2/6), and adaptive (0/3) firing cells at 24oC. The firing frequency of the remaining

14 cells tested were either maintained at the same level (n = 8) or reduced slightly (n = 6) in comparison with 24oC. It was noticed that the types of cooling effects were associated with the relative abundance of TTXr Na+ currents in total Na+ currents and also associated with the amplitude of outward inactivating K+ currents. A total of 9 TTXr cells that responded to cooling with increased frequency were tested with TTX and they showed a higher fraction of TTXr currents (77.4 ± 6.9 %, n = 9) and larger inactivating K+ currents (30.0 ± 3.9 pA/pF, at -10 mV, n=9). Out of the 14 cells that did not show increased action potential frequency by cooling, 11 were tested with TTX and these cells had a lower fraction of TTXr currents (30.2 ± 5.2 %, n =

11) and smaller inactivating K+ currents (15.2 ± 6.6 pA/pF at -10 mV, n=11) P<0.05.

Accordingly, we subdivided TTXr cells in to high fraction TTXr Na+ currents (TTXrh) and low fraction TTXr Na+ currents (TTXrl) groups. TTXs cells (n =22) also showed tonic (n=11, Fig.

4.1F), adaptive (n=8, Fig. 4.1G), and phasic (n=3, Fig. 4.1H), at 24oC, but no long-delayed firing pattern was observed. In contrast to TTXr cells, cooling temperatures of 15oC always converted multiple action potential firing to a single all-or-none phasic firing (n = 12) either with or without subsequent aborted potentials (Fig. 4.1).

A series of temperatures at 29°, 24°, 15°, and 10oC were tested on TTXrh cells (Fig.

4.2A). At 15 and 10oC, action potential frequency was much higher than that of 29°C and 24oC at different current steps. For example, at a current pulse of 0.08-0.09 nA, action potential numbers per 4s were significantly higher at 15oC and 10oC (8.3 ± 1.7, n =9 and 9.0 ± 2.1, n=9) than those at 24oC and 29oC (3.4 ± 0.8, n = 9 and 3.1 ± 2.0, n = 6, P< 0.05). Cooling

106

Sarria, I temperatures had little effects on action potential height. First and last action potentials elicited by a current pulse of 0.08-0.09 nA, were not different across any temperature (29°C, 24°C, 15°C, and 10°C), P>0.05, (Fig. 4.2E). For example action potentials’ height were 63.3 ± 3.6 mV (1st), and 61.6 ± 5.1 mV (last) at 24oC, n= 7 and were 65.0 ± 2.0 mV (1st), and 59.0 ± 2.2 mV (last) at

10oC, n=7. Action potential latency change was a prominent effect by cooling temperatures on the TTXrh cells that had long-delayed latency. The latency to action potential threshold was decreased by 88% as temperature was dropped from 24°C to 15°C (3226 ± 675 ms and 388 ± 88 ms respectively, n = 4, P <0.05).

We examined membrane parameters of TTXrh cells (Table 4.1), including resting membrane potentials (RMP), rheobase, action potential threshold, action potential width, action potential hyperpolarization (AHP) peak and amplitude, input resistance, and latency to action potential threshold. Except resting membrane potentials, which was not affected by cooling temperatures, all other parameters were altered by cooling temperatures. Rheobase lessened with decreasing temperatures. Action potential threshold increased (became less negative) at lower temperatures. Action potential width (w1/2) increased with increasing cold, and AHP decreased

(less negative) with decreasing temperatures. Latency to action potential threshold slightly increased as temperature dropped (in cells without delayed firing).

For TTXrl cells, action potential firing properties were examined at 24°C and 15oC (Fig.

4.2C). Overall, these cells fired action potentials at a higher frequency than TTXrh cells at 24°C.

For example, at a current step of 0.08-0.09 nA, TTXrl cells fired 18.8 ± 3.8 APs/4s, n=11, while

TTXrh cells only fired 3.4 ± 0.8 APs/4s, n = 9, P<0.05. When action potential frequency of

TTXrl cells at 24 and 15°C was compared, we see that they were similar at lower current steps

107

Sarria, I and the frequency became lower only at higher current injections (≥ 0.08-0.09 nA). As temperature lowered from 24°C to 15°C, TTXrl input resistance, action potential threshold, width and latency increased, while AHP peak decreased and resting membrane potential, rheobase and AHP did not change significantly (Table 4.1).

In contrast to TTXr, cooling temperatures suppressed firing of multiple action potentials in TTXs cells (Fig. 4.2b). Even a change of temperatures from 29oC to 24oC affected action potential firing pattern in TTXs cells, and turned tonic firing to slightly adaptive firing and significantly reduced action potential firing frequency (21.2 ± 2.7 AP/600ms at 29°C, n=8 vs. 13

± 2.0 spikes/600ms at 24°C, n= 12, at a current pulse of 0.08, P<0.05). At 15°C and 10, usually only a single all-or-none full sized action potential could be elicited, which sometimes was followed by several aborted potentials. Action potential height was significantly reduced at cooling temperatures (Fig. 4.2E), indicating increased adaptation and failure of action potentials at cooling temperatures. The height of final action potentials (including aborted) elicited by a current pulse of 0.08-0.09 nA (600ms), were different across temperatures (29°C, 24°C, 15°C, and 10°C). For example, final action potentials’ height were 48.8 ± 5.3 mV at 29°C (n=8), 38.4 ±

4.6 mV at 24°C (n=12), 28.8 ± 4.0 mV at 15°C n=7, and 26.6 ± 6.1 mV at 10°C n=5, P<0.05.

We examined membrane parameter of TTXs cells (Table 4.1), including resting membrane potentials (RMP), rheobase, action potential threshold, action potential width, AHP peak and amplitude, input resistance, and latency to action potential threshold. Resting membrane potentials were not affected by cooling temperatures. Rheobase was higher at 24°C, but was the same at 29°C, 15 and 10°C. Action potential threshold increased (became less negative) at lower temperatures. Action potential width (w1/2) increased with increasing cold, and AHP decreased

108

Sarria, I

(less negative) with decreasing temperatures. Latency to action potential threshold increased as temperature dropped.

When these parameters are compared between TTXr and TTXs cells at each temperature

(Table 4.1), TTXr cells have slightly more negative resting membrane potentials, higher rheobase, comparable input resistance, much higher action potential threshold, longer latency to action potential threshold, and broader action potential width at 29°C and 24°C but less broad than TTXs at 10°C. Membrane parameters of TTXrh and TTXrl cells (Table 4.1) were compared at 24 °C and we found that TTXrl cells have similar resting membrane potentials, rheobase, input resistance, action potential width (w1/2), and latency to action potential threshold, but lower action potential threshold and smaller AHP. However, cooling temperatures at 15°C turned all membrane parameters of TTXrl cells similar to those of TTXrh cells.

The differential effects of cooling temperatures on action potential firing patterns could be associated with differential effects of cooling temperatures on TTXs and TTXr channel functions (Zimmermann et al 2007). We examined voltage-dependent activation and inactivation of TTXs and TTXr Na+ channels to analyze how membrane potentials affect Na+ channels availability at different temperatures. Both TTXr and TTXs currents’ amplitude were progressively reduced when temperatures lowered from 29°C to 24°C, 15°C, and 10oC (Fig. 4.3 a&b). When compared to 29°C, cold at 15°C induced a right-shift in the voltage-dependent activation of TTXs Na+ conductance, V1/2 = -30.1 ± 2.9 mV vs. -20.7 ± 1.7 mV (n = 10, P <

0.05), respectively. In contrast, the same temperature change did not significantly shift the voltage-dependent activation of TTXr Na+ channels, V1/2 = -19.5 ± 2.0 mV and -16.5 ± 2.1 mV at 29°C and 15°C respectively (n=10, P > 0.05, Fig. 4.3C). The degree of inhibition at each

109

Sarria, I cooling temperatures was much less severe for TTXr than TTXs Na+ channels (Fig. 4.3b&c).

The differential inhibition by cooling temperatures of TTXs and TTXs currents were not due to steady state fast inactivation since the steady state fast inactivation of TTXr was almost identical at 29°C and 10°C and steady state fast inactivation of TTXs was also very similar at the two temperatures (Fig. 4.3d). However, TTXr underwent fast inactivation at more depolarized potentials (V1/2: -33.4 ± 0.5 mV at 29 oC, -33.5 ± 0.5 mV at 10 oC, n =6) and TTXs at more hyperpolarized membrane potentials (V1/2: -59.6 ± 1.2 mV at 29 oC, -54.2 ± 2.5 mV at 10oC, n

=6). Thus, if cooling temperatures affect membrane repolarization, it would have more profound effects on TTXs than on TTXr Na+ channels. We examined the steady-state slow inactivation of

TTXs and TTXr channels (Fig. 4.3E, F). At 24oC, Steady state slow inactivation of both TTXr and TTXs channels had similar V1/2 (TTXr: -50.5 ± 1.0 mV, n=6; TTXs: -50.5 ± 2.2, mV, n=6).

Cooling temperature at 10oC did not significantly affect TTXr slow inactivation (V1/2= -49.8 ±

0.5 mV) but shifted TTXs slow inactivation to much more hyperpolarized potential with V1/2 of

-87.5 ± 2.1 mV. At 10oC and resting membrane potentials near -50 mV, the fraction of remaining current was about 20% for TTXs and about 60% for TTXr. Thus, if cooling temperatures affect membrane repolarization, the effects would be integrated into the voltage dependent slow inactivation which would have bigger influence on TTXs cells that TTXr cells at cooling temperatures.

The increases of action potential width and decreases of AHP amplitude suggested that membrane repolarization was severely hampered by cooling temperatures after action potential firing (Fig 2, Table 4.1). Since A-type K+ currents have been indicated to be involved in membrane repolarization and AHP in sensory neurons, we directly examined effects of cooling temperatures on IA currents in both TTXs and TTXr cells. For the voltage protocol used in this

110

Sarria, I study, we encountered two types of IA currents (Fig. 4.4), IAf (fast inactivating IA) and IAs

(slow inactivating IA). Most of cells had IAs either with or without IAf. Of 18 TTXr cells examined, 7 were IAs only, 7 were a mixture of IAs and IAf, and 4 were IAf only. Of 15 TTXs cells examined, 9 were IAs only, 4 were a mixture of IAs and IAf, and 2 were IAf only. In all these cells there was always a non-inactivating IK current. Figure 4.5 shows the isolated IA and

IK currents in TTXr (n=8) and TTXs (n=8) cells. In the cells included in this study, the amplitude of IA was comparable between TTXs and TTXr (Fig. 4.5a,d). IK currents were also comparable between the two types of cells (Fig. 4.5b,e). IA currents were examined at 29°C,

24°C, 15°C and 10°C (Fig. 4.6a,d). Maximum IA current (120 ± 13 pA/pF, n=16) was obtained at 29°C with an activating pulse of 45 mV. At 24°C, IA had already been significantly reduced with 68 % (82 ± 10 pA/pF, n=13) peak current remaining and was further inhibited at temperatures of 15°C and 10°C with 26 % (31 ± 5 pA/pF, n=11) and 12 % (14 ± 3 pA/pF, n=11) current remaining respectively. In contrast, IK was only slightly reduced by cooling temperatures as even at 10°C 82 % (55 ± 11 pA/pF, n= 7) of current still remained from maximum current obtained at 29°C (67 ± 8 pA/pF, n=10, P>0.05, Fig. 4.6b,e). Steady-state inactivation protocol showed that inhibition of inactivating Kv currents by cold (10°C) was largely voltage independent, as pre-pulsing to negative potentials (-100 mV, 2s) before an activating pulse of 45 mV resulted in just 32 % (55 ± 7 pA/pF, n=11) of peak current remaining from the maximum current obtained at 29°C (170 ± 14 pA/pF, n=16, (Fig. 4.6c,f). IA currents were selectively inhibited by 100 uM 4-AP by 46 % (92 ± 26/164 ± 30 pA/pF, n=5). 100 uM 4-AP did not affect

IK at all.

If the inhibition of IA currents accounted for the changes of the excitability and action potential firing patterns at cooling temperatures, pharmacological block of IA currents should

111

Sarria, I mimic the effects of cooling temperatures. Therefore, we examined effects of 100 uM 4-AP, which selectively inhibited IA currents at this concentration, on both TTXs and TTXr cells.

Indeed, at 24oC 4-AP increased action potential firing numbers of TTXr cells (Fig. 4.7A, C left).

For example, at a 0.15 nA current step, action potential firing numbers were 4.4 ± 2.0 APs/4s

(n=6) before 4-AP and increased to 10.2 ± 2.8 APs/4s (n=6, P<0.05) after 4-AP. On the other hand, in TTXs cells, 4-AP converted high-frequency tonic firing into adaptive firing with a reduction of action potential firing numbers. (Fig. 4.7B,C right). With a current pulse of 0.15 nA, action potential firing numbers were 14.2 ± 3.0 APs/600ms (n=6) before 4-AP and decreased to

6.3 ± 2.6 APs/600 ms (n=6, P<0.05) after 4-AP. Similar to cooling temperatures, 4-AP increased action potential width decreased AHP peak and rheobase for both TTXs and TTXr cells (Table

4.2). It had little effect on resting membrane potentials, action potential threshold, and input resistance in both TTXs and TTXr cells. These results supported the idea that inhibition of IA currents by cooling temperatures can differentially affect action potential firing patterns of TTXs and TTXr cells.

Effects of cooling temperatures on sensory neuron excitability and action potential firing properties should shape action potential firing patterns of cold-sensing neurons responding to innocuous and noxious cold stimuli. To test this hypothesis, we determined effects of cooling temperatures on TTXs and TTXr neurons that expressed the cold transducer TRPM8 channel.

TRPM8-expressing cells were pre-identified as cold- and menthol-sensitive neurons by using

Ca2+ imaging method. TTX sensitivity was then tested with 500 nM TTX. We first examined membrane properties and action potential firing patterns of these cells in responding to step current injections at 24oC (Table 4.3). Most membrane parameters and action potential firing patterns of TTXs/TRPM8 cells (n= 12) were similar to TTXs cells that did not respond to cold

112

Sarria, I and menthol (Table 1). For TTXr/TRPM8 cells, membrane parameters and action potential firing patterns were similar to TTXrl cells (Table 4.1). Under voltage clamp configuration,

TTXr/TRPM8 had a lower fraction of TTXr currents from total Na+ currents, 28.1 ± 4.7%, n=11.

We examined the effects of cooling temperatures on voltage-activated inward currents (mainly

Na+ currents) and outward currents (mainly K+ currents). These currents were not isolated because we intended to keep this set of experiments under normal conditions so that the cells could still be used for the subsequent test of cold-induced action potential firing. Similar to the cells that did not respond to menthol and cold, cooling temperatures (10°C) inhibited TTXs Na+ currents more severely than TTXr Na+ currents, sparring 18.1 ± 3.1%,(1996 ± 362 pA, n=11) of

TTXr and 4.9 ± 1.0%, (3636 ± 621 pA, n=10) of TTXs Na currents elicited at 24°C, (P<0.05).

Effects of cooling temperatures on IA were estimated by examining the effects of cool temperatures on voltage-activated outward currents (Figure 4.8). For both TTXs/TRPM8 and

TTXr/TRPM8 cells, changing temperatures from 24oC to 10oC significantly inhibited outward currents. We have shown that cooling temperatures preferentially inhibit IA over IK currents in

TTXs and TTXr cells (Fig. 4.6), thus the results indicate that there were substantial amount of peak outward currents at 24°C that were IA, which were inhibited at 10oC. For example, the amount of peak outward current inhibited in TTXr cells by 10°C was 74.2 ± 4.4 % (28.9 ± 6.8 /

111.9 ± 11 pA/pF, n=11, P<0.05)

We examined action potential firing of TTXs/TRPM8 and TTXr/TRPM8 cells as they responded to slow ramps of cooling temperatures from 29oC to 10oC. TTXs/TRPM8 cells responded to a cooling ramp with membrane depolarization that started at 27.6 ± 0.4oC (n = 9), but action potential firing started at 26.6 ± 0.7 oC (n = 9, Fig. 4.9A). Action potential firing became adapted with a rapid reduction of action potential amplitudes (from voltage threshold to

113

Sarria, I peak) as temperatures decreased, and action potentials were completely aborted at 18.3 ± 0.9oC

(n =9). Spike frequency changed during cooling, but there was no consistent pattern in spike frequency over the cooling temperatures. Spike frequency was at its highest at 26oC with 6.0 ±

1.3 Hz (n = 9, Fig. 4.10A). The reduction of spike amplitude (action potentials and abortive potentials) followed linear decay with a slope of -8.0 ± 1.4 mV/oC (n = 9, Fig. 4.10B). The AHP peak of the first action potential was -55.6 ± 1.9 mV, and was reduced linearly during cooling with a slope of (-3.3 ± mV/oC, n = 9, Fig. 4.10C). The width of the first action potential was 3.3

± 0.2 ms (n = 9) and was increased exponentially during cooling with a constant of 2.35 ± 0.3 ms

(n = 9, Fig. 4.10D). Membrane depolarization increased during cooling reaching a maximum membrane potential of -27.2 ± 2.5 mV (n = 9, Fig. 4.10E) before the final spike was seen. Thus, at temperatures where action potentials were aborted, membrane potential remained depolarized at values above action potential threshold, suggesting that the adaptation is not related to a possible TRPM8 desensitization. The adaptation of cold sensitive TTXs cells shared the similar characteristics with the adaptation seen at cooling temperatures for the TTXs cells that did not express TRPM8.

In contrast to TTXs/TRPM8 cells, TTXr/TRPM8 cells responded to cooling temperatures with membrane depolarization at the temperature of 21.8 ± 0.5oC (n = 7) and started to fire action potentials only when temperatures reduced to 17.9 ± 0.7oC (n = 7, Fig. 4.9B). While some of these TTXr/TRPM8 cells stopped action potential firing before the end of cooling temperature of 10oC (n =4), other cells continued all-or-none action potentials at 10oC (n = 3), the lowest temperatures used in this study. The average temperature for these cells to stop firing action potentials was 12 ± 0.4oC (n = 7). Action potentials displayed a reduction in amplitude with decreasing temperatures and the reduction of spike amplitude followed a linear decay with a

114

Sarria, I slope of -5.1± 0.8 mV/oC (n = 7, Fig. 4.10G). Spike frequency increased during cooling with action potential frequency peaking at 3.4 ± 0.8 Hz (n= 7, Fig. 4.10F) at 15oC. The threshold of first action potential was -11.2 ± 1.8 mV, significantly higher than the action potentials evoked by current inject at 24oC (-19.3 ± 2.0 mV, n=11, P<0.05). The AHP peak of the first action potential was -41.8 ± 1.8 mV (n =7), and was further reduced linearly during cooling with a slope of -4.0 ± 0.6 mV/oC (Fig. 4.10H). The width of the first action potential was broadened to

16.6 ± 4.4 ms (n = 7), and was further increased exponentially during cooling with a constant of

3.5 ±0.9 ms (n = 7, Fig. 4.10I). The changes in both AHP peak and AP width were consistent with a significant inhibition of IA currents at the temperatures of the first action potentials and

APs afterward (Fig. 4.6). Membrane depolarization increased during cooling reaching a maximum membrane potential of -14.0 ± 2.3 mV (n = 7, Fig. 4.10J) before the final spike was seen.

115

Sarria, I

Discussion

We show that cooling temperatures have differential effects on TTXs and TTXr DRG neuron excitability and action potential firing properties. The effects are attributed to the inhibition of A-type K+ currents and different voltage-dependent Na+ channel activation and inactivation at cold temperatures, which shapes action potential firing properties of TTXs and

TTXr sensory neurons in an integrative manner. The differential effects of cooling temperatures on TTXs and TTXr neurons may contribute to sensory distinction between innocuous and noxious cold stimuli, and also provide insights into how cooling temperatures may modify other sensory modalities.

We found that cooling temperatures preferentially inhibited IA currents but have less effects on IK currents in both TTXs and TTXr cells. IA currents in our DRG neurons showed a significant reduction from 29oC to 24oC, were strongly inhibited at 15 oC and almost completely inhibited at 10oC. Several types of IA currents have been identified in both nociceptive and non- nociceptive DRG neurons (Gold et al 1996; Yoshimura et al 1996). In our small-sized TTXs and

TTXr cells, most of them showed either IAs currents only or a mixture of IAs and IAf currents.

Inhibition of IAs by cooling temperatures was seen clearly in the cells that expressed only IAs.

We did not isolate IAf from IAs in this study, but IAf was most likely also inhibited by cooling temperatures since both IAf and IAs components were found to be reduced in the cells with a mixture of IAf and IAs currents. The inhibition of IA currents by cooling temperatures was due to the inhibition of its activation because the inhibition occurred even at very negative holding potentials that prevented steady-state inactivation. Previous studies have shown that the roles of

IA currents in neuronal excitability and action potential firing can be very different in different

116

Sarria, I types of cells (Hess & El Manira 2001; Sculptoreanu et al 2004; Vydyanathan et al 2005), which seems to be also the case for our TTXs and TTXr DRG neurons.

In a previous study a cooling temperature of 10oC reduced the sizes of abortive potentials in DRG neurons of NaV1.8 Knockout mice (Zimmermann et al 2007). In our study, TTXs cells showed all-or-none action potential firing with several different firing patterns at 24oC. Cooling temperatures resulted in the change of action potential firing properties from multiple firing

(tonic or adaptive) to severe adaptive and phasic firing at 15°C and 10oC respectively. Similar to the previous study (Zimmermann et al 2007), we showed that cooling temperatures shifted steady-state slow inactivation curve to more hyperpolarizing voltages, which resulted in substantial slow inactivation near resting membrane potentials. At noxious cold temperatures of

15 and 10 oC, we showed that the remaining TTXs channels were still able to generate a single all-or-none action potential firing in our small-sized TTXs DRG neurons. However, the subsequent action potential firing was aborted partially or completely. The failure of subsequent action potential firing could be due to the reduction of TTXs channel availability following the first action potential firing. Almost all TTXs channels undergo voltage-dependent inactivation at the peak of an action potential (Blair & Bean 2002). Therefore, TTXs Na+ channel availability after an action potential firing is critically dependent on membrane repolarization, which recovers TTXs Na+ channels from inactivation (Hess & El Manira 2001). Membrane repolarization during an action potential in our TTXs cells was significantly contributed by IA currents. By inhibition of IA, cooling temperatures impaired membrane repolarization after an action potential firing as is evidenced by the broadened TTXs action potentials and reduced

AHP. The impaired membrane repolarization and the shift of voltage-dependent inactivation of

TTXs together were the causes of aborted action potentials at noxious cold temperatures.

117

Sarria, I

Consistently, low concentrations of 4-AP, which inhibit IA currents in TTXs cells, also changed tonic firing to adaptive or phasic firing in these cells. Thus, non-nonciceptive neurons that express only TTXs Na+ channels can be effectively silenced at nociceptive cooling temperatures.

Different from TTXs cell, neither steady-state slow inactivation nor fast inactivation of

TTXr Na+ channels were significantly affected by cooling temperatures, a result consistent with a previous study in mouse DRG neurons (Zimmermann et al 2007). This result supported the idea that TTXr channels retained ability to conduct sensory impulses under nociceptive cold temperatures (Zimmermann et al 2007). In a sub-group of TTXrh cells which displayed delayed action potential firing, cooling temperatures reduced latency of action potential firing. Unlike

TTXs cells, multiple action potential firing was maintained and even facilitated at cooling temperatures in TTXr cells. Increases in membrane input resistance by cooling temperatures have been suggested to account for increased excitability of nociceptors in a skin-nerve preparation (Zimmermann et al 2007). However, in our phasic firing cells identified at 24oC, injecting high amount of currents that were electrically equivalent to the increases of input resistance was unable to change action potential firing pattern but noxious cold converted their phasic firing to multiple action potential firing, arguing against that the changes in input resistance by cooling temperatures is the main factor to increase action potential firing frequency in TTXr cells. TTXr cells in our study were found to express IA currents. The cells that showed increased action potential frequency were those expressed significantly high level of IA currents and those cells also had bigger AHP. IA currents in TTXr cells were inhibited significantly by cooling temperatures, which may account for the reduction of AHP and action potential broadening by cooling temperatures in these cells. In TTXr cells, a large IA current would be turned on before membrane potential reached action potential threshold because these cells had a

118

Sarria, I much higher threshold for action potential firing. Thus, IA in TTXr cells mainly served as a brake to oppose membrane depolarization and limit action potential firing. Inhibition of IA by cooling temperatures would release the brake to allow rapid membrane depolarization and facilitate TTXr action potential firing. Consistent with this idea, we showed that pharmacologically inhibited IA currents by 4-AP, which did not significantly affect membrane input resistance, increased action potential firing frequency, a result consistent with previous studies in nociceptive-like small-sized DRG neurons (Vydyanathan et al 2005).

We studied TTXs and TTXr cells that expressed cold-transducer TRPM8 channels. We showed that TTXs/TRPM8 cells fired action potentials in innocuous cooling temperature range from 26.5oC to 18oC, suggesting that these are non-nociceptive neurons specific for sensing innocuous cold. Our TTXs/TRPM8 cells had temperature threshold for membrane depolarization of 27.6 oC, which is consistent with the previous studies of LT-CS (low threshold cold-sensing) cells using Ca2+ imaging method (Madrid et al 2009; Nealen et al 2003; Thut et al 2003). For

TTXr/TRPM8 cells, we found that they fired action potentials in uncomfortable and noxious cold temperature range (<18oC). This temperature threshold of action potential firing is consistent with noxious cold sensations in human psychophysical studies (Morin & Bushnell 1998).

TTXr/TRPM8 neurons had temperature thresholds of 22oC for membrane depolarization, which was similar to the HT-CS (high threshold cold-sensing) trigeminal neurons observed using Ca+- imaging technique (Madrid et al 2009; Nealen et al 2003; Thut et al 2003). Our previous study showed that TTXr/TRPM8 cells also expressed TRPV1 and P2X receptors (Xing et al 2007b;

Xing et al 2006). Thus, TTXr/TRPM8 cells are most likely to be polymodmal nociceptors that detect noxious cold and other nociceptive stimuli, similar to those nociceptors described by others (Madrid et al 2009; Zimmermann et al 2007).

119

Sarria, I

The temperature threshold to start action potential firing in our TTXs/TRPM8 cells and

TTXr/TRPM8 cells are determined by at least two factors, the level of expression/functional regulation of TRPM8 and the voltage threshold for action potential firing. We have previously shown that TRPM8-mediated responses in TTXs cells were higher than in TTXr cells (Xing et al

2007b; Xing et al 2006). Corroboratively, TRPM8-mediated responses were higher in LT-CS

(low threshold-cold sensing) cells than in HT-CS (high threshold-cold sensing cells) (Madrid et al 2009). The second factor is action potential firing threshold, the voltage at which action potential start to fire in an all-or none fashion, which was significantly lower in TTXs/TRPM8 than in TTXr/TRPM8 cells (Table 4.3). These two factors favor TTXs/TRPM8 cells to start action potential firing at higher temperatures (low threshold). However, cooling enhances the voltage-dependent slow inactivation of TTXs Na+ channels, while it also decreases their voltage- dependent activation. Low temperatures also impair membrane repolarization due to the inhibition of IA. The combination of these cooling effects on TTXs cells lead to action potentials that are committed to be aborted near noxious cold and thereby set a range of innocuous cold sensing for TTXs/TRPM8 cells. In contrast, because TTXr/TRPM8 cells display lower TRPM8-mediated responses and their action potential threshold is higher, they require much colder temperatures to depolarize the membrane enough to fire action potentials.

Moreover, the lack of effects by cooling on voltage-dependent steady state activation or slow inactivation of TTXr Na+ channels permits TTXr/TRPM8 cells to generate action potentials at nociceptive cooling temperatures. Cooling temperatures significantly inhibited IA currents in

TTXr/TRPM8 cells. IA in HT-CS cells have been proposed to be a brake to prevent cells from being excited by cold temperatures (Madrid et al 2009). Interestingly, our results indicated that it is the noxious cold itself that releases this brake by inhibiting IA.

120

Sarria, I

Tables and Figures

Table 4.1. Comparison of membrane parameters between TTXrh, TTXrl, and TTXs DRGs at different temperatures.

RMP Rheobase Input R Threshold AP width AHP AHP peak Latency (mV) (pA) (M) (mV) (ms) mV mV ms

29 oC TTXrh n=6 -54±2 88±12† 465±50 -10.4±3.2† 3.7±0.4† 40.9±.2.2† -49.9±2.6 NA TTXs n=8 -49±2 15±3 728±137 -29.5±1.3 2.9±0.2 24.0±2.8 -53.5±2.4 NA 24 oC TTXrh n=9 -52±2 58±8*† 825±106* -4.7±2.0† 5.3±0.4*† 40.1±1.6† -44.9±1.9 352 ±33† TTXrl n=11 -50.5±4 41±9 740±115 -11.3±2.0 5.3±0.6 32.1±1.8 -43.4±1.6 319 ±47 TTXs n=12 -50±1 26±5* 742±106 -25.1±0.9* 3.4±0.2* 23±1.3 -47.9±1.1* 101 ±5 15 oC TTXrh n=9 -53±3 33±4*† 1206±155* -3.7±1.8† 13.1±1.1* 35.1±1.4*† -38.8±1.5* 397 ±33*† TTXrl n=11 -51.2±4 31±7 1500±247* -4.2±1.3* 14.3±2.0* 34.2±1.9 -38.4±2.0* 395 ±58* TTXs n=12 -50±1 13±2 1725±254* -18.8±1.2* 15.8±1.7* 18.7±1.4 -38.3±1.0* 159 ±14* 10 oC TTXrh=9 -52±3 32±4*† 2050±334* -1.4±1.6*† 15.3±1.5*† 35.0±2.3† -36.4±2.5* NA TTXs n=8 -47±1 14±2 1803±282* -17.2±1.7* 24.1±3.3* 18.1±1.2 -35.3±1.6* NA

Cells were current-clamped and given a series of current steps for a period of 600ms (TTXs) or 4s (TTXrh, TTXrl) at 1Hz at 29°C, 24°C, 15°C, 10°C. * P<0.05, Same cell type, different temperature (control is the highest temperature tested in group), † P<0.05, Same temperature, different cell type. NA, did not perform test at this temperature. TTXs sensitivity was assessed under voltage-clamp by remaining inward Na+ current to depolarizing potentials in the presence of 500 nM TTX.

121

Sarria, I

Table 4.2. Effects of 4-AP on membrane parameters of TTXr and TTXs cells

RMP (mV) Rheobase Input R Threshold AP width AHP AHP peak (pA) (M) (mV) (ms) mV TTXr n=6 Control 53.4±2.9 84.2±14.2 645±130 -12.2±2 5.9±0.6 39.0±2.2 -49.7± 1.2

4-AP 53.0±3.2 49.2±14.9* 658±198 -10.8±1.4 9.6±1.3* 32.9±1.8* -45.3 ±1.5*

TTXs n=6 Control -48.8±1.2 28.6±5.1 794±165 -24.3±1.0 3.28±0.9 22.6±2.0 -46.8±1.6

4-AP -47.7±0.9 15.7±4.3* 989±239 23.4±1.5 5.1±0.4* 19.3±2.4 -42.7±2.1*

Membrane parameters were recorded before (control) and after adding IA blocker 4-AP (100 µM, 2-minute incubation). 4-AP was delivered externally mixed with the bath solution. Experiments were performed at 24°C. Current pulses were delivered as described in Table 1. * P<0.05, Control vs. 4-AP within same cell type.

122

Sarria, I

Table 4.3. Comparison of membrane parameters between cold sensing TTXs and TTXr cells

RMP (mV) Rheobase Input R Threshold AP width AHP AHP peak (pA) (M) (mV) (ms) (mV) mV TTXs n=12 -51.7 ± 0.7 31 ± 9 437 ± 71 -30.9 ± 1.4† 3.5 ± 0.2† 15.4 ±1.0† -46.2 ± 1.1 TTXr n= 11 -51.0 ± 1.3 37 ± 9 535 ± 35 -19.3 ± 2.0 4.9 ± 0.4 25.8 ± 1.5 -45.1 ± 1.4

DRGs were classified as cold/menthol by calcium imaging. Experiments were performed at 24°C. Current pulses were delivered as described in Table 1. † P<0.05, TTXs vs. TTXr.

123

Sarria, I

Fig. 4.1. Action potential firing patterns of TTXr and TTXs DRGs at different temperatures. Under current-clamp, current pulses were given to TTXr (4s at 1Hz, left) and TTXs cells (600 ms at 1Hz, right) at 24°C followed by 15°C. At 24°C, firing of TTXr cells was classified as phasic (A), delayed (B), irregular (C), adaptive (D), or tonic (E). The same current step at 15°C allowed multiple AP and often increased firing in TTXr DRGs. At 24°C, firing of TTXs cells was classified as tonic (F), adaptive (G), or phasic (H). Almost always, cooling temperature of 15° allowed a single but not full multiple APs in TTXs DRGs.

124

Sarria, I

Fig 4.2. Effect of temperature on action potential firing of TTXrh, TTXrl, and TTXs cells. Cells pre-identified as TTXrh (a), TTXs (b) and TTXrl (c) were given a series of current pulses at 29°C, 24°C, 15°C, and 10°C. (d) Summary of action potential firing frequency, TTXrh (29°C n=6, 24°C n=9, 15°C n=9, 10°C n=9). TTXs, (29°C n=8, 24°C n=12, 15°C n=12, 10°C n=8). TTXrl (24°C n=11, 15°C n=11). (e) Summary of the effect of temperatures on action potential height for TTXrh and TTXs cells, n is same as in (d) for both groups. First and last action potentials (including abortive) were selected from the lowest current step that yielded maximum AP frequency in each cell. Data represent mean ± SEM. * P<0.05, a=abortive action potential.

125

Sarria, I

Fig. 4.3. Low temperatures differentially affect TTXs and Nav 1.8 TTXr VGSN. a, Representative traces of TTXs (right) and TTXr (left) sodium currents in DRG neurons recorded at 29, 24, 15 and 10°C, held at -80 mV. Currents were activated by 500 ms long test pulses from - 90 to +10 mV in steps of 10 mV. b, Summary of peak currents normalized by cell capacitance TTXs (empty symbols, n=10) and TTXr (filled symbols, n=10) (left), and percentage of maximum current I at 29°C (right). c. Voltage-dependence activation of TTXs (empty symbols, n=10) and TTXr (filled symbols, n=10) conductance at 29°C and 15°C. (d) Steady-state fast

126

Sarria, I inactivation of TTXs (open circles, n=6) and TTXr (filled squares, n=6) currents in DRGs, Red, 29°C; blue, 10°C. Steady-state slow inactivation of TTXs (n=6) (e) and TTXr, (n=6) (f), Red, 24 °C; blue, 10°C. TTXs sensitivity was assessed by remaining inward Na+ current in the presence of 500 nM TTX. A-803467 is a Nav 1.8 channel specific blocker (100 μM). Data represent Mean ± SEM. *P<0.05.

127

Sarria, I

Fig. 4.4. Identification of Kv potassium currents in DRGs. a, Traces of whole outward currents from three separate cells evoked by voltage steps (shown below the traces) from a 2- second pre-potential of -100 mV (a, middle and bottom) or -60 mV (a, top). b, Same activation steps as in a, but preceded by a 2-second pre-pulse to -10 mV, spared some potassium currents that had no transient component (IK), this current was not inactivated by high voltage. c, Subtraction of non-inactivating from whole-currents revealed isolated fast inactivating (IAf) (c top), slow inactivating (IAs) (c middle), and mixture of both IAf and IAs (c bottom) Kv currents in DRGs.

128

Sarria, I

Fig. 4.5. Kv currents in TTXr and TTXs DRGs. Representative traces of IA (a) and IK (b) currents from a TTXr (left) and a TTXs (right) cell. Summary of peak IA (d) and IK (e) currents plotted versus activating potentials from both cell types. c, Traces showing the steady state inactivation of whole Kv currents in a TTXr (left) and a TTXs cell (right). Steady state inactivation protocol consisted of a 2-second pre-pulse voltage step to potentials ranging between - 110 and 0 mV followed by a depolarizing step to +45 mV. f, Summary of peak currents from TTXr and TTXs cells plotted vs. pre-pulse potentials from the steady state inactivation protocol. Current amplitude was normalized by cell capacitance. TTXr n=8, TTXs n=8. Data are mean ± SEM.

129

Sarria, I

Fig. 4.6. Low temperatures inhibit activation of IA Kv currents. Representative traces obtained from a single DRG showing activation of Isolated IA (a) and IK (b) currents as well as steady state inactivation of whole Kv currents (c) at 29°C, 24°C, 15°C, and 10°C. Summary of maximum IA (d) and IK (e) currents from respective activation protocols and steady state inactivation of whole Kv currents (f) at 29°C (n=16), 24°C (n=13), 15°C (n=11), and 10°C (n=11). Current amplitude was normalized by cell capacitance. Data are mean ± SEM.

130

Sarria, I

Fig 4.7. Transient A-type Kv channel blocker 4-AP enhances AP firing in TTXr, but not in TTXs cells. Single traces showing action potential firing of a TTXr (a) and a TTXs (b) cell before (left) and after (right) a two-minute incubation of 100 µM 4-AP. c, Action potential firing frequency of TTXr (left) and TTXs (right) cells before and after exposure to 4-AP. d, Effect of 4-AP on action potential height of TTXr (left) and TTXs (right) cells. n=6 TTXr, n=6 TTXs, f=first and l=last action potentials. * P<0.05, data are mean ± SEM.

131

Sarria, I

Fig. 4.8. Effect of cold temperature on IA in TRPM8/cold sensing cells. Total IA currents were estimated by examining the effects of cool temperatures on voltage-activated outward currents. Cooling preferentially inhibits IA over IK currents (Fig.4.6), thus at 10°C a fraction of

Kv current (mostly IK, blue) remains from the total outward current obtained at 24°C (IA+IK, black), with a substantial amount of IA (red) inhibited by cold. TTXs (A), n=10, TTXr (B), (n=11). Cold sensing cells were identified with calcium imaging before voltage-clamping. Data are mean ± SEM.

132

Sarria, I

Fig. 4.9. Cold sensitive TTXr and TTXs DRGs fire action potentials at different temperature ranges. Cold sensitive cells were identified using calcium imaging with a rapid cold bath application (10 °C, 5 seconds) and TTX resistance was determined by remaining inward currents under voltage clamp with application of 0.5 uM TTX. Simultaneous recording of membrane potentials (top traces) and bath temperature (bottom traces) during cooling from a TTXs neuron (A) and a TTXr neuron (B). In a and b, left insets shows temperatures that elicited first action potentials, and right inset shows temperatures at which action potentials were abortive (A, TTXs) and or at which last one was recorded (B, TTXr). (C) Summary data showing that action potential firing in TTXs (n= 9) and TTXr (n=7) cells occur at different temperature ranges. Data represent mean ± SEM, *P < 0.05.

133

Sarria, I

Fig 4.10. Membrane and cold-induced action potential firing properties of TTXs/TRPM8 and TTXr/TRPM8 DRGs. Membrane depolarization was elicited by cooling temperatures from a slow dropping ramp starting at 29oC and ranging down to 10oC. Top panel are plotted data from 9 individuals TTXs cells. Lower panel are plotted data from 7 individual TTXr cells. Lines are best fit for each category from one TTXr and one TTXs cell chosen to best represent each group. Cold sensing cells were identified with calcium imaging before current-clamping.

134

Sarria, I

Notes to Chapter 4

Progress: In preparation for submission to peer review journal

1,2 1 Authors: Ignacio Sarria Jennifer Ling1, and Jianguo Gu

Affiliations: 1 Department of Anesthesiology, University of Cincinnati College of Medicine, PO

Box 670531, 231 Albert Sabin Way, Cincinnati, OH 45267-0531, USA

2 Graduate Program in Neuroscience, University of Cincinnati College of Medicine, PO Box

670531, 231 Albert Sabin Way, Cincinnati, OH 45267-0531, USA

Acknowledgements: This work was supported by a NIH grant DE018661 to J.G.G

135

Sarria, I

Chapter V:

Summary

136

Sarria, I

Results from chapter II show that the TRPM8 specific agonist menthol, elicited responses in nociceptors and nonnociceptors-like (MS/CI and MS/CS) DRGs. MS/CS cells in general had smaller responses to menthol with weaker adaptations than MS/CI cells and blocking protein kinases (PKC, CaMKII, but not PKA) attenuated adaptation in the MS/CI group. In the discussion we hypothesized that the difference of menthol responses and adaptation in MS/CS cells could be due to lower expression of TRPM8 because of the lack of effect by protein kinase inhibitors. We also mentioned PIP2 levels as another possible factor contributing to the differences in response intensity and adaptation rate between MS/CI and MS/CI cells, although we did not examine this in the study. In the next study (chapter III) we saw that calcium induces

PIP2 hydrolysis through PLC and that pip2 is necessary for high TRPM8 functional activity, as it increases the channel open probability and combats tachyphylaxis. Although we did not test

PIP2 differences in these two functionally distinct cells, we did test sensitivity to noxious agent capsaicin, the TRPV1 receptor agonist. In the periphery, nociceptive DRGs express receptors/channels that have calcium-permeability and are activated by pain- causing/inflammatory agents, pH, heat, chemical agents and even calcium itself, i.e. TRPV1,

TRPA1, P2X2,4 , NK-1, PGE2 receptor EP1 and bradykinin receptors (Burgard et al 1999;

Caterina et al 1997; Li & Zhao 1998; Lin et al 2006; McNamara et al 2007; Vellani et al 2004).

Thus prior to activation, TRPM8 may already be more desensitized in DRG nociceptors

(MS/CS) due to higher transient calcium levels that lead to higher PIP2 hydrolysis than in non- nociceptors (MS/CI). This might be particularly true in settings where there is an injury, trauma, disease of the PNS. This offers a possible explanation to why kinase inhibitors reduced adaptation in MS/CI, but not significantly so in MS/CS cells, as these cells may have lower levels of membrane-bound PIP2, rendering TRPM8 channels in an already low functional state.

137

Sarria, I

It would be interesting to investigate if TRPM8 expression levels are different in nociceptors and non-nociceptors and account for the functional differences observed between these two cell types. Also interesting would be to discover the exact target by which kinases exert their effect on TRPM8 function, be it the channel itself, interfering with PIP2-TRPM8 binding at the C- terminal, or activating another downstream molecule.

Although blocking PKC and CaMKII diminished adaptation after a prolonged menthol application in MS/CI cells, an obvious initial sharp reduction in fluorescence was usually seen shortly after agonist application. This initial reduction in response intensity was usually followed by a prolonged and less steep signal reduction. In voltage-clamp experiments, these two distinct phases became even more apparent. Results presented in chapter III of this dissertation identify two phases of Ca2+-dependent down-regulation of TRPM8 channels. It was discovered that that acute desensitization is triggered by Ca2+-calmodulin and that PIP2 is essential for the appearance of acute desensitization. Clear evidence was provided showing that PIP2 hydrolysis and

PKC/PP1,2A activity are two main causes of tachyphylaxis. Lastly it was also revealed that the functional states underlining acute desensitization and tachyphylaxis are set forth by Ca2+- calmodulin and PIP2 inversely modulating TRPM8 gating. Thus, through multiple intracellular signaling pathways, cold-elicited currents in sensory neurons can be regulated in the forms of acute desensitization and tachyphylaxis, underlining the phenomenon of cold adaptation in mammals.

It is possible that binding of calmodulin at the the N-terminus (amino acid 145-198) of

TRPM8 may cause the channel to enter the low functional state, resulting in the acute desensitization of whole-cell currents described in chapter III. We tested this hypothesis by

138

Sarria, I applying menthol and cold stimuli to HEK 293 cells expressing the TRPM8 mutant that had the calmodulin binding site (K153-K175) deleted, but we failed to elicit any whole-cell or inside-out currents. This was in sharp contrast to cells with wild type TRPM8 for which both menthol and cold elicited desensitizing currents (Fig. 5.1, 5.2). Thus, this particular mutation in which the entire calmodulin binding side was deleted might have resulted in non-functional TRPM8 channels. Future studies using site-directed mutagenesis of single or in combinations of a few amino acids are needed to reveal the sites that are critical for calmodulin binding and acute desensitization.

The experiments described in Chapter II and III aimed at understanding the mechanisms of functional downregulation of the cold transducer and analyzing TRMP8-mediated responses in nonnociceptors and nociceptor-like cells. In chapter IV I looked at how temperature affects neuronal excitability and how TRPM8-positive cells conduct impulses when stimulated by cooling temperatures. Briefly, the results in chapter IV show that cooling temperatures have differential effects on TTXs and TTXr DRG neuron excitability and action potential firing properties. The effects are attributed to the inhibition of A-type K+ currents and different voltage-dependent Na+ channel activation and inactivation at cold temperatures, which shapes action potential firing properties of TTXs and TTXr sensory neurons in an integrative manner.

The differential effects of cooling temperatures on TTXs and TTXr neurons may contribute to sensory distinction between innocuous and noxious cold stimuli, and also provide insights into how cooling temperatures may modify other sensory modalities.

Finally, I looked at how neuronal excitability in cold-sensing primary sensory neurons may be affected by incubation with NGF. Results of those experiments are presented in this

139

Sarria, I section as I felt that initial findings need corroboration and may not match well with the general flow of chapter IV, and thus it would be better to present them separately. When cultured in the presence of NGF, the number of DRGs that respond to menthol increases as well as the response’s intensity (Babes et al 2004). Moreover, (Xing et al 2007a) showed that after chronic constrictive nerve injury (CCI), a neuropathic pain model manifesting cold allodynia in hindlimbs, there was an increase in the percentage of nociceptive-like neurons expressing

To test this general idea, DRGs were cultured in the presence of NGF (10ng/ml) for 24-

72 hours and response of TRPM8 positive cells to a slow cooling ramp were analyzed (Fig. 5.3).

The temperature range at which action potentials started and failed did not change in NGF-TTXs

(26.4°C±0.8°C--18.2°C±0.6, n=10) vs. control-TTXs cells (26.5°C±0.7--18.3±0.8, n=9, P>0.05).

There was no change in the temperature at which last action potentials were recorded in NGF-

TTXr (11.4°C±0.3, n=13) vs. control-TTXr cells (12.0°C±0.5°C, n=7, P>0.05). Interestingly the temperatures at which action potentials began firing in NGF-TTXr cells (21.6°±0.7°C, n=13,

P<0.05) were warmer than in control-TTXr cells (17.9°C±0.7°C, n=7) (Fig. 5.3). This suggests that NGF might be responsible for the cold allodynia observed in models of neuropathic pain, by allowing TRMPM8-nociceptors to conduct impulses at higher (more innocuous) temperatures.

Futures studies should look into how NGF mediates this effect, whether it is by causing TRPM8 overexpression and or TRPM8 functional upregulation.

140

Sarria, I

Altogether, the findings presented in this dissertation outline a multifaceted scheme where TRPM8, secondary messengers, conduction machinery, and temperature itself interplay and serve as the molecular mechanisms underlining cold sensing in primary sensory neurons.

Novelty, importance, and perspective of study’s findings

The importance of the work presented in this dissertation lays in that it reveals new information about the mechanisms of cold sensing, functional regulation of the TRPM8 channel, and neuronal excitability of mammalian primary sensory neurons. Specifically and succinctly the major findings were: 1-Functionaly distinct primary sensory neurons have different TRPM8- mediated responses as well as calcium-mediated regulation by protein kinases. 2- Discovery of calmodulin as a regulator of TRPM8 channel function, and clarification of calcium-mediated regulation of TRPM8 function. 3- Discovery of the effect temperature has on the excitability of putative primary sensory nociceptors and non-nociceptors and how this contributes to sensory distinction between innocuous and noxious cold. In a broader context, these findings fit and expand the current knowledge on thermosensation, functional regulation of TRP channels, and neuronal excitability of mammalian primary sensory neurons.

The contributions from these studies might be helpful even beyond somatosensation and primary sensory neurons. For example, TRPM8 might play an active role in cancer development as it is found to be upregulated on cancerous prostate tissue. In this scenario, clear knowledge of the channel’s sensitization and desensitization pathways could help it become a target for anti- cancerous drugs. Additionally, the founding member of the TRPM family, TRPM1, has been recently identified as the sole channel responsible for the depolarization of ON bipolar cells in

141

Sarria, I the retina, and absence or mutation of TRPM1 results in night blindness in humans, mice, and certain horse breeds. Interestingly, TRPM1-mediated responses on ON bipolar cells have been recently shown to be desensitized by calcium and upregulated by phospholipids. I think it would be very interesting and worthy to investigate if the targets and mechanisms of TRPM8 regulation put forth in this manuscript might also regulate TRPM1 function.

Although the results from my work might be helpful beyond thermosensation, its main importance is that it shows in vitro that TRPM8 is an ion channel which endows varying cold sensitivity to different neuronal subtypes, its function is regulated by calmodulin, PIP2 and PKC, probably differently in nociceptors, and that it is expressed in primary sensory neurons along with functionally distinct Nav and Kv channels. Together, these events likely help set up the resulting perceptual outcome of TRPM8 activation.

142

Sarria, I

Figures

Fig. 5.1. Whole-cell currents of wildtype and nonfunctional TRPM8 mutant without calmodulin binding site. Whole-cell currents were elicited by 100 μM menthol (a) and 15°C (b) from wild type mouse TRPM8 expressed in HEK 293 cells. 100 μM menthol (c) and 15°C (d) failed to elicit whole-cell currents from mutant mouse TRPM8 that had the calmodulin binding site (K153-K175) deleted. (e). Summary results of maximum current elicited for each group, WT-menthol n=8, WT-cold n=14, Mutant-menthol n=6, Mutant-cold n=6. Data are mean ± SEM

143

Sarria, I

Fig 5.2. Single-channel currents of wildtype and nonfunctional TRPM8 mutant without calmodulin binding site. Inside-out recordings of currents elicited by 100 μM menthol (a) and 15°C (b) from wild type mouse TRPM8 expressed in HEK 293 cells. 100 μM menthol (c) and 15°C (d) failed to elicit any channel activity from mutant mouse TRPM8 that had the calmodulin binding site (K153-K175) deleted. (e). Summary of channel open probability calculated for each group. WT-menthol n=6, WT-cold n=5, Mutant-menthol n=4, Mutant-cold n=4. Data are mean ± SEM

144

Sarria, I

Fig 5.3. NGF lowers excitability threshold in nociceptive-cold sensing DRGs. DRGs were incubated with NGF (10ng/ml) for 24-72 hours. Cold sensitive cells were identified using calcium imaging with a rapid cold bath application (10 °C, 5 seconds) and TTX resistance was determined by remaining inward currents under voltage clamp with application of 0.5 uM TTX. Simultaneous recording of membrane potentials (top traces) and bath temperature (bottom traces) during cooling in a TTXs neuron (A) and a TTXr neuron (B). In A (TTXs) and B (TTXr), arrows (from left to right) show temperatures at which first and last action potential spikes were recorded respectively. For TTXr cells, last action potential recorded does not mean abortive. Summary data showing the effects of NGF on the temperature range of action potential firing in TTXs-NGF, n=10 vs. Control-TTXs n=9 (C), and TTXr-NGF n=13 vs. Control-TTXr n=7 (D). Data represent mean ± SEM, * P<0.05.

145

Sarria, I

References

Abe J, Hosokawa H, Okazawa M, Kandachi M, Sawada Y, et al. 2005. TRPM8 protein localization in trigeminal ganglion and taste papillae. Brain Res Mol Brain Res 136:91-8 Abe J, Hosokawa H, Sawada Y, Matsumura K, Kobayashi S. 2006. Ca2+-dependent PKC activation mediates menthol-induced desensitization of transient receptor potential M8. Neurosci Lett 397:140-4 Akopian AN, Sivilotti L, Wood JN. 1996. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379:257-62 Akopian AN, Souslova V, England S, Okuse K, Ogata N, et al. 1999. The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat Neurosci 2:541-8 Andersson DA, Nash M, Bevan S. 2007. Modulation of the cold-activated channel TRPM8 by lysophospholipids and polyunsaturated fatty acids. J Neurosci 27:3347-55 Askwith CC, Benson CJ, Welsh MJ, Snyder PM. 2001. DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature. Proc Natl Acad Sci U S A 98:6459-63 Babes A, Zorzon D, Reid G. 2004. Two populations of cold-sensitive neurons in rat dorsal root ganglia and their modulation by nerve growth factor. Eur J Neurosci 20:2276-82 Bandell M, Dubin AE, Petrus MJ, Orth A, Mathur J, et al. 2006. High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol. Nat Neurosci 9:493-500 Bautista DM, Siemens J, Glazer JM, Tsuruda PR, Basbaum AI, et al. 2007. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448:204-8 Bavencoffe A, Gkika D, Kondratskyi A, Beck B, Borowiec AS, et al. 2010. The transient receptor potential channel TRPM8 is inhibited via the alpha2A adrenoreceptor signaling pathway. J Biol Chem 285(13):9410-9 Black DJ, Tran QK, Persechini A. 2004. Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+. Cell Calcium 35:415-25 Blair NT, Bean BP. 2002. Roles of tetrodotoxin (TTX)-sensitive Na+ current, TTX-resistant Na+ current, and Ca2+ current in the action potentials of nociceptive sensory neurons. J Neurosci 22:10277-90 Blair NT, Bean BP. 2003. Role of tetrodotoxin-resistant Na+ current slow inactivation in adaptation of action potential firing in small-diameter dorsal root ganglion neurons. J Neurosci 23:10338-50 Brauchi S, Orio P, Latorre R. 2004. Clues to understanding cold sensation: thermodynamics and electrophysiological analysis of the cold receptor TRPM8. Proc Natl Acad Sci U S A 101:15494-9 Burgard EC, Niforatos W, van Biesen T, Lynch KJ, Touma E, et al. 1999. P2X receptor-mediated ionic currents in dorsal root ganglion neurons. J Neurophysiol 82:1590-8 Campero M, Serra J, Bostock H, Ochoa JL. 2001. Slowly conducting afferents activated by innocuous low temperature in human skin. J Physiol 535:855-65

146

Sarria, I

Carlton SM, Coggeshall RE. 2001. Peripheral capsaicin receptors increase in the inflamed rat hindpaw: a possible mechanism for peripheral sensitization. Neurosci Lett 310:53-6 Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, et al. 2000. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288:306-13 Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389:816-24 Chien LY, Cheng JK, Chu D, Cheng CF, Tsaur ML. 2007. Reduced expression of A-type potassium channels in primary sensory neurons induces mechanical hypersensitivity. J Neurosci 27:9855-65 Chin D, Means AR. 2000. Calmodulin: a prototypical calcium sensor. Trends Cell Biol 10:322-8 Cho Y, Jang Y, Yang YD, Lee CH, Lee Y, Oh U. 2010. TRPM8 mediates cold and menthol allergies associated with mast cell activation. Cell Calcium 48:202-8 Chung MK, Caterina MJ. 2007. TRP channel knockout mice lose their cool. Neuron 54:345-7 Clapham DE. 2003. TRP channels as cellular sensors. Nature 426:517-24 Colburn RW, Lubin ML, Stone DJ, Jr., Wang Y, Lawrence D, et al. 2007. Attenuated cold sensitivity in TRPM8 null mice. Neuron 54:379-86 Connor M, Naves LA, McCleskey EW. 2005. Contrasting phenotypes of putative proprioceptive and nociceptive trigeminal neurons innervating jaw muscle in rat. Mol Pain 1:31 Daniels RL, Takashima Y, McKemy DD. 2009. Activity of the neuronal cold sensor TRPM8 is regulated by phospholipase C via the phospholipid phosphoinositol 4,5-bisphosphate. J Biol Chem 284:1570-82 Davis KD, Pope GE. 2002. Noxious cold evokes multiple sensations with distinct time courses. Pain 98:179-85 De Koninck Y, Mody I. 1994. Noise analysis of miniature IPSCs in adult rat brain slices: properties and modulation of synaptic GABAA receptor channels. J Neurophysiol 71:1318-35 De Petrocellis L, Starowicz K, Moriello AS, Vivese M, Orlando P, Di Marzo V. 2007. Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids. Exp Cell Res 313:1911-20 Dhaka A, Earley TJ, Watson J, Patapoutian A. 2008. Visualizing cold spots: TRPM8-expressing sensory neurons and their projections. J Neurosci 28:566-75 Dhaka A, Murray AN, Mathur J, Earley TJ, Petrus MJ, Patapoutian A. 2007. TRPM8 is required for cold sensation in mice. Neuron 54:371-8 Dib-Hajj SD, Cummins TR, Black JA, Waxman SG. 2010. Sodium channels in normal and pathological pain. Annu Rev Neurosci 33:325-47 Doerner JF, Gisselmann G, Hatt H, Wetzel CH. 2007. Transient receptor potential channel a1 is directly gated by calcium ions. Journal of Biological Chemistry 282:13180-9 Eberhard M, Erne P. 1989. Kinetics of calcium binding to fluo-3 determined by stopped-flow fluorescence. Biochem Biophys Res Commun 163:309-14 Eid SR, Cortright DN. 2009. Transient receptor potential channels on sensory nerves. Handb Exp Pharmacol:261-81 Fruhstorfer H, Hermanns M, Latzke L. 1986. The effects of thermal stimulation on clinical and experimental itch. Pain 24:259-69

147

Sarria, I

Funakoshi H, Frisen J, Barbany G, Timmusk T, Zachrisson O, et al. 1993. Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve. J Cell Biol 123:455-65 Gold MS, Shuster MJ, Levine JD. 1996. Characterization of six voltage-gated K+ currents in adult rat sensory neurons. J Neurophysiol 75:2629-46 Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, Johannes FJ. 1996. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett 392:77-80 Hayashi T, Kondo T, Ishimatsu M, Yamada S, Nakamura K, et al. 2009. Expression of the TRPM8-immunoreactivity in dorsal root ganglion neurons innervating the rat urinary bladder. Neurosci Res 65:245-51 Hensel H, Zotterman Y. 1951. The effect of menthol on the thermoreceptors. Acta Physiol Scand 24:27-34 Herzberg U, Eliav E, Dorsey JM, Gracely RH, Kopin IJ. 1997. NGF involvement in pain induced by chronic constriction injury of the rat sciatic nerve. Neuroreport 8:1613-8 Hess D, El Manira A. 2001. Characterization of a high-voltage-activated IA current with a role in spike timing and locomotor pattern generation. Proc Natl Acad Sci U S A 98:5276-81 Heumann R, Lindholm D, Bandtlow C, Meyer M, Radeke MJ, et al. 1987. Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regeneration: role of macrophages. Proc Natl Acad Sci U S A 84:8735-9 Hodgkin AL, Katz B. 1949. The effect of temperature on the electrical activity of the giant axon of the squid. J Physiol 109:240-9 Hoenderop JG, Voets T, Hoefs S, Weidema F, Prenen J, et al. 2003. Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6. EMBO J 22:776-85 Hu HZ, Gu Q, Wang C, Colton CK, Tang J, et al. 2004. 2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3. J Biol Chem 279:35741-8 Hui K, Guo Y, Feng ZP. 2005. Biophysical properties of menthol-activated cold receptor TRPM8 channels. Biochem Biophys Res Commun 333:374-82 Jordt SE, Bautista DM, Chuang HH, McKemy DD, Zygmunt PM, et al. 2004. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427:260-5 Julius D, Basbaum AI. 2001. Molecular mechanisms of nociception. Nature 413:203-10 Jung J, Shin JS, Lee SY, Hwang SW, Koo J, et al. 2004. Phosphorylation of vanilloid receptor 1 by Ca2+/calmodulin-dependent kinase II regulates its vanilloid binding. J Biol Chem 279:7048-54 Karashima Y, Prenen J, Meseguer V, Owsianik G, Voets T, Nilius B. 2008. Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators. Pflugers Arch 457:77-89 Karashima Y, Talavera K, Everaerts W, Janssens A, Kwan KY, et al. 2009. TRPA1 acts as a cold sensor in vitro and in vivo. Proc Natl Acad Sci U S A 106:1273-8 Knowlton WM, McKemy DD. 2010. TRPM8: From Cold to Cancer, Peppermint to Pain. Curr Pharm Biotechnol 12(1):68-77

148

Sarria, I

Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, et al. 2005. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c- fibers and colocalization with trk receptors. J Comp Neurol 493:596-606 Lai J, Gold MS, Kim CS, Bian D, Ossipov MH, et al. 2002. Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 95:143-52 Lambers TT, Weidema AF, Nilius B, Hoenderop JG, Bindels RJ. 2004. Regulation of the mouse epithelial Ca2(+) channel TRPV6 by the Ca(2+)-sensor calmodulin. J Biol Chem 279:28855-61 Lattanzio FA, Jr. 1990. The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems. Biochem Biophys Res Commun 171:102-8 Lee SY, Lee JH, Kang KK, Hwang SY, Choi KD, Oh U. 2005. Sensitization of vanilloid receptor involves an increase in the phosphorylated form of the channel. Arch Pharm Res 28:405-12 Leung PC, Taylor WA, Wang JH, Tipton CL. 1985. Role of calmodulin inhibition in the mode of action of ophiobolin a. Plant Physiol 77:303-8 Levine JD, Alessandri-Haber N. 2007. TRP channels: targets for the relief of pain. Biochim Biophys Acta 1772:989-1003 Li HS, Zhao ZQ. 1998. Small sensory neurons in the rat dorsal root ganglia express functional NK-1 tachykinin receptor. Eur J Neurosci 10:1292-9 Li Q, Wang X, Yang Z, Wang B, Li S. 2009. Menthol induces cell death via the TRPM8 channel in the human bladder cancer cell line T24. Oncology 77:335-41 Lin CR, Amaya F, Barrett L, Wang H, Takada J, et al. 2006. Prostaglandin E2 receptor EP4 contributes to inflammatory pain hypersensitivity. J Pharmacol Exp Ther 319:1096-103 Linte RM, Ciobanu C, Reid G, Babes A. 2007. Desensitization of cold- and menthol-sensitive rat dorsal root ganglion neurones by inflammatory mediators. Exp Brain Res 178:89-98 Lishko PV, Procko E, Jin X, Phelps CB, Gaudet R. 2007. The ankyrin repeats of TRPV1 bind multiple ligands and modulate channel sensitivity. Neuron 54:905-18 Liu B, Qin F. 2005. Functional control of cold- and menthol-sensitive TRPM8 ion channels by phosphatidylinositol 4,5-bisphosphate. J Neurosci 25:1674-81 Liu D, Liman ER. 2003. Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5. Proc Natl Acad Sci U S A 100:15160-5 Lukacs V, Thyagarajan B, Varnai P, Balla A, Balla T, Rohacs T. 2007. Dual regulation of TRPV1 by phosphoinositides. J Neurosci 27:7070-80 Macpherson LJ, Hwang SW, Miyamoto T, Dubin AE, Patapoutian A, Story GM. 2006. More than cool: promiscuous relationships of menthol and other sensory compounds. Mol Cell Neurosci 32:335-43 Madrid R, de la Pena E, Donovan-Rodriguez T, Belmonte C, Viana F. 2009. Variable threshold of trigeminal cold-thermosensitive neurons is determined by a balance between TRPM8 and Kv1 potassium channels. J Neurosci 29:3120-31 Malkia A, Pertusa M, Fernandez-Ballester G, Ferrer-Montiel A, Viana F. 2009. Differential role of the menthol-binding residue Y745 in the antagonism of thermally gated TRPM8 channels. Mol Pain 5:62

149

Sarria, I

McKemy DD. 2005. How cold is it? TRPM8 and TRPA1 in the molecular logic of cold sensation. Mol Pain 1:16 McKemy DD, Neuhausser WM, Julius D. 2002. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416:52-8 McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, et al. 2007. TRPA1 mediates formalin-induced pain. Proc Natl Acad Sci U S A 104:13525-30 McNeill DL, Coggeshall RE, Carlton SM. 1988. A light and electron microscopic study of calcitonin gene-related peptide in the spinal cord of the rat. Exp Neurol 99:699-708 Meeusen R, Lievens P. 1986. The use of cryotherapy in sports injuries. Sports Med 3:398-414 Mishra R, Rao V, Ta R, Shobeiri N, Hill CE. 2009. Mg2+- and MgATP-inhibited and Ca2+/calmodulin-sensitive TRPM7-like current in hepatoma and hepatocytes. Am J Physiol Gastrointest Liver Physiol 297:G687-94 Mohapatra DP, Nau C. 2003. Desensitization of capsaicin-activated currents in the vanilloid receptor TRPV1 is decreased by the cyclic AMP-dependent protein kinase pathway. J Biol Chem 278:50080-90 Morin C, Bushnell MC. 1998. Temporal and qualitative properties of cold pain and heat pain: a psychophysical study. Pain 74:67-73 Mukerji G, Yiangou Y, Corcoran SL, Selmer IS, Smith GD, et al. 2006. Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations. BMC Urol 6:6 Munns C, AlQatari M, Koltzenburg M. 2007. Many cold sensitive peripheral neurons of the mouse do not express TRPM8 or TRPA1. Cell Calcium 41:331-42 Nealen ML, Gold MS, Thut PD, Caterina MJ. 2003. TRPM8 mRNA is expressed in a subset of cold-responsive trigeminal neurons from rat. J Neurophysiol 90:515-20 Niemeyer BA, Bergs C, Wissenbach U, Flockerzi V, Trost C. 2001. Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin. P Natl Acad Sci USA 98:3600-5 Nilius B, Prenen J, Janssens A, Owsianik G, Wang C, et al. 2005a. The selectivity filter of the cation channel TRPM4. J Biol Chem 280:22899-906 Nilius B, Prenen J, Tang J, Wang C, Owsianik G, et al. 2005b. Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4. J Biol Chem 280:6423-33 Noel J, Zimmermann K, Busserolles J, Deval E, Alloui A, et al. 2009. The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception. EMBO J 28:1308- 18 Numazaki M, Tominaga T, Takeuchi K, Murayama N, Toyooka H, Tominaga M. 2003. Structural determinant of TRPV1 desensitization interacts with calmodulin. Proc Natl Acad Sci U S A 100:8002-6 Okazawa M, Inoue W, Hori A, Hosokawa H, Matsumura K, Kobayashi S. 2004. Noxious heat receptors present in cold-sensory cells in rats. Neurosci Lett 359:33-6 Okazawa M, Takao K, Hori A, Shiraki T, Matsumura K, Kobayashi S. 2002. Ionic basis of cold receptors acting as thermostats. J Neurosci 22:3994-4001 Oosterveld FG, Rasker JJ. 1994. Treating arthritis with locally applied heat or cold. Semin Arthritis Rheum 24:82-90

150

Sarria, I

Ordaz B, Tang J, Xiao R, Salgado A, Sampieri A, et al. 2005. Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation. J Biol Chem 280:30788-96 Owsianik G, Talavera K, Voets T, Nilius B. 2006. Permeation and selectivity of TRP channels. Annu Rev Physiol 68:685-717 Patapoutian A, Peier AM, Story GM, Viswanath V. 2003. ThermoTRP channels and beyond: mechanisms of temperature sensation. Nat Rev Neurosci 4:529-39 Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, et al. 2002. A TRP channel that senses cold stimuli and menthol. Cell 108:705-15 Phillips JR, Matthews PB. 1993. Texture perception and afferent coding distorted by cooling the human ulnar nerve. J Neurosci 13:2332-41 Pierau FK, Torrey P, Carpenter DO. 1974. Mammalian cold receptor afferents: role of an electrogenic sodium pump in sensory transduction. Brain Res 73:156-60 Premkumar LS, Raisinghani M, Pingle SC, Long C, Pimentel F. 2005. Downregulation of transient receptor potential melastatin 8 by protein kinase C-mediated dephosphorylation. J Neurosci 25:11322-9 Proudfoot CJ, Garry EM, Cottrell DF, Rosie R, Anderson H, et al. 2006. Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain. Curr Biol 16:1591-605 Qin N, Flores CM. 2007. Polypeptide complex of TRPM8 and calmodulin and its uses thereof. U.S. Patent 2007/0105155 A1 May 10, 2007 Rasband MN, Park EW, Vanderah TW, Lai J, Porreca F, Trimmer JS. 2001. Distinct potassium channels on pain-sensing neurons. Proc Natl Acad Sci U S A 98:13373-8 Reid G, Amuzescu B, Zech E, Flonta ML. 2001. A system for applying rapid warming or cooling stimuli to cells during patch clamp recording or ion imaging. J Neurosci Methods 111:1- 8 Reid G, Babes A, Pluteanu F. 2002. A cold- and menthol-activated current in rat dorsal root ganglion neurones: properties and role in cold transduction. J Physiol 545:595-614 Reid G, Flonta M. 2001a. Cold transduction by inhibition of a background potassium conductance in rat primary sensory neurones. Neurosci Lett 297:171-4 Reid G, Flonta ML. 2001b. Physiology. Cold current in thermoreceptive neurons. Nature 413:480 Reid G, Flonta ML. 2002. Ion channels activated by cold and menthol in cultured rat dorsal root ganglion neurones. Neurosci Lett 324:164-8 Roe MW, Lemasters JJ, Herman B. 1990. Assessment of Fura-2 for measurements of cytosolic free calcium. Cell Calcium 11:63-73 Rohacs T, Lopes CM, Michailidis I, Logothetis DE. 2005a. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nat Neurosci 8:626-34 Rohacs T, Lopes CM, Michailidis I, Logothetis DE. 2005b. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain. Nat Neurosci 8:626-34 Rosenbaum T, Gordon-Shaag A, Munari M, Gordon SE. 2004. Ca2+/calmodulin modulates TRPV1 activation by capsaicin. J Gen Physiol 123:53-62 Rush AM, Brau ME, Elliott AA, Elliott JR. 1998. Electrophysiological properties of sodium current subtypes in small cells from adult rat dorsal root ganglia. J Physiol 511 ( Pt 3):771-89

151

Sarria, I

Sabnis AS, Reilly CA, Veranth JM, Yost GS. 2008a. Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures. Am J Physiol Lung Cell Mol Physiol 295:L194-200 Sabnis AS, Shadid M, Yost GS, Reilly CA. 2008b. Human lung epithelial cells express a functional cold-sensing TRPM8 variant. Am J Respir Cell Mol Biol 39:466-74 Sarria I, Gu J. 2010. Menthol response and adaptation in nociceptive-like and nonnociceptive- like neurons: role of protein kinases. Mol Pain 6:47 Sarria I, Ling J, Zhu MX, Gu JG. 2011. Trpm8 Acute Desensitization Is Mediated by Calmodulin and Requires Pip2: Distinction from Tachyphylaxis. J Neurophysiol 106:(6) 3056-3066 Schmidt M, Dubin AE, Petrus MJ, Earley TJ, Patapoutian A. 2009. Nociceptive signals induce trafficking of TRPA1 to the plasma membrane. Neuron 64:498-509 Schnizler K, Shutov LP, Van Kanegan MJ, Merrill MA, Nichols B, et al. 2008. Protein kinase A anchoring via AKAP150 is essential for TRPV1 modulation by forskolin and prostaglandin E2 in mouse sensory neurons. J Neurosci 28:4904-17 Schonthal AH. 1998. Role of PP2A in intracellular signal transduction pathways. Front Biosci 3:D1262-73 Sculptoreanu A, Yoshimura N, de Groat WC. 2004. KW-7158 [(2S)-(+)-3,3,3-trifluoro-2- hydroxy-2-methyl-N-(5,5,10-trioxo-4,10-dihydro thieno[3,2-c][1]benzothiepin-9- yl)propanamide] enhances A-type K+ currents in neurons of the dorsal root ganglion of the adult rat. J Pharmacol Exp Ther 310:159-68 Shi J, Mori E, Mori Y, Mori M, Li J, et al. 2004. Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells. J Physiol 561:415-32 Spray DC. 1986. Cutaneous temperature receptors. Annu Rev Physiol 48:625-38 Stein RJ, Santos S, Nagatomi J, Hayashi Y, Minnery BS, et al. 2004. Cool (TRPM8) and hot (TRPV1) receptors in the bladder and male genital tract. J Urol 172:1175-8 Stewart AP, Egressy K, Lim A, Edwardson JM. 2010. AFM imaging reveals the tetrameric structure of the TRPM8 channel. Biochem Biophys Res Commun 394:383-6 Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, et al. 2003. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112:819-29 Strotmann R, Schultz G, Plant TD. 2003. Ca2+-dependent potentiation of the nonselective cation channel TRPV4 is mediated by a C-terminal calmodulin binding site. J Biol Chem 278:26541-9 Strotmann R, Semtner M, Kepura F, Plant TD, Schoneberg T. 2010. Interdomain interactions control Ca2+-dependent potentiation in the cation channel TRPV4. PLoS One 5:e10580 Stucky CL, Dubin AE, Jeske NA, Malin SA, McKemy DD, Story GM. 2009. Roles of transient receptor potential channels in pain. Brain Res Rev 60:2-23 Tahmoush AJ, Schwartzman RJ, Hopp JL, Grothusen JR. 2000. Quantitative sensory studies in complex regional pain syndrome type 1/RSD. Clin J Pain 16:340-4 Takashima Y, Daniels RL, Knowlton W, Teng J, Liman ER, McKemy DD. 2007. Diversity in the neural circuitry of cold sensing revealed by genetic axonal labeling of transient receptor potential melastatin 8 neurons. J Neurosci 27:14147-57

152

Sarria, I

Tan ZY, Donnelly DF, LaMotte RH. 2006. Effects of a chronic compression of the dorsal root ganglion on voltage-gated Na+ and K+ currents in cutaneous afferent neurons. J Neurophysiol 95:1115-23 Thut PD, Wrigley D, Gold MS. 2003. Cold transduction in rat trigeminal ganglia neurons in vitro. Neuroscience 119:1071-83 Tominaga M, Caterina MJ. 2004. Thermosensation and pain. J Neurobiol 61:3-12 Tsavaler L, Shapero MH, Morkowski S, Laus R. 2001. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer Res 61:3760-9 Tsuzuki K, Xing H, Ling J, Gu JG. 2004. Menthol-induced Ca2+ release from presynaptic Ca2+ stores potentiates sensory synaptic transmission. J Neurosci 24:762-71 Ullrich ND, Voets T, Prenen J, Vennekens R, Talavera K, et al. 2005. Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice. Cell Calcium 37:267-78 Vellani V, Zachrisson O, McNaughton PA. 2004. Functional bradykinin B1 receptors are expressed in nociceptive neurones and are upregulated by the neurotrophin GDNF. J Physiol 560:391-401 Viana F, de la Pena E, Belmonte C. 2002a. Specificity of cold thermotransduction is determined by differential ionic channel expression. Nat Neurosci 5:254-60 Viana F, de la Pena E, Belmonte C. 2002b. Specificity of cold thermotransduction is determined by differential ionic channel expression. Nat Neurosci 5:254-60 Voets T, Droogmans G, Wissenbach U, Janssens A, Flockerzi V, Nilius B. 2004a. The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430:748-54 Voets T, Janssens A, Droogmans G, Nilius B. 2004b. Outer pore architecture of a Ca2+- selective TRP channel. J Biol Chem 279:15223-30 Voets T, Nilius B. 2003. TRPs make sense. J Membr Biol 192:1-8 Voets T, Owsianik G, Janssens A, Talavera K, Nilius B. 2007. TRPM8 voltage sensor mutants reveal a mechanism for integrating thermal and chemical stimuli. Nat Chem Biol 3:174-82 von Mackensen S, Hoeppe P, Maarouf A, Tourigny P, Nowak D. 2005. Prevalence of weather sensitivity in Germany and Canada. Int J Biometeorol 49:156-66 Vydyanathan A, Wu ZZ, Chen SR, Pan HL. 2005. A-type voltage-gated K+ currents influence firing properties of isolectin B4-positive but not isolectin B4-negative primary sensory neurons. J Neurophysiol 93:3401-9 Wang W, Gu J, Li YQ, Tao YX. 2011. Are voltage-gated sodium channels on the dorsal root ganglion involved in the development of neuropathic pain? Mol Pain 7:16 Wei ET, Seid DA. 1983. AG-3-5: a chemical producing sensations of cold. J Pharm Pharmacol 35:110-2 Wiesner TF, Berk BC, Nerem RM. 1996. A mathematical model of cytosolic calcium dynamics in human umbilical vein endothelial cells. Am J Physiol 270:C1556-69 Xiao B, Dubin AE, Bursulaya B, Viswanath V, Jegla TJ, Patapoutian A. 2008a. Identification of transmembrane domain 5 as a critical molecular determinant of menthol sensitivity in mammalian TRPA1 channels. J Neurosci 28:9640-51

153

Sarria, I

Xiao R, Tang J, Wang C, Colton CK, Tian J, Zhu MX. 2008b. Calcium plays a central role in the sensitization of TRPV3 channel to repetitive stimulations. J Biol Chem 283:6162-74 Xing H, Chen M, Ling J, Tan W, Gu JG. 2007a. TRPM8 mechanism of cold allodynia after chronic nerve injury. J Neurosci 27:13680-90 Xing H, Chen M, Ling J, Tan WH, Gu JGG. 2007b. TRPM8 mechanism of cold allodynia after chronic nerve injury. Journal of Neuroscience 27:13680-90 Xing H, Ling J, Chen M, Gu JG. 2006. Chemical and cold sensitivity of two distinct populations of TRPM8-expressing somatosensory neurons. J Neurophysiol 95:1221-30 Xing H, Ling JX, Chen M, Johnson RD, Tominaga M, et al. 2008. TRPM8 mechanism of autonomic nerve response to cold in respiratory airway. Mol Pain 4:22 Yamamura H, Ugawa S, Ueda T, Morita A, Shimada S. 2008. TRPM8 activation suppresses cellular viability in human melanoma. Am J Physiol Cell Physiol 295:C296-301 Yang XR, Lin MJ, McIntosh LS, Sham JS. 2006. Functional expression of transient receptor potential melastatin- and vanilloid-related channels in pulmonary arterial and aortic smooth muscle. Am J Physiol Lung Cell Mol Physiol 290:L1267-76 Yoshimura N, White G, Weight FF, de Groat WC. 1996. Different types of Na+ and A-type K+ currents in dorsal root ganglion neurones innervating the rat urinary bladder. J Physiol 494 ( Pt 1):1-16 Zakharian E, Cao C, Rohacs T. 2010. Gating of transient receptor potential melastatin 8 (TRPM8) channels activated by cold and chemical agonists in planar lipid bilayers. J Neurosci 30:12526-34 Zakharian E, Thyagarajan B, French RJ, Pavlov E, Rohacs T. 2009. Inorganic polyphosphate modulates TRPM8 channels. PLoS One 4:e5404 Zhang L, Barritt GJ. 2004. Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival of prostate cancer cells. Cancer Res 64:8365-73 Zhang X, Huang J, McNaughton PA. 2005a. NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels. EMBO J 24:4211-23 Zhang Z, Okawa H, Wang Y, Liman ER. 2005b. Phosphatidylinositol 4,5-bisphosphate rescues TRPM4 channels from desensitization. J Biol Chem 280:39185-92 Zhang Z, Zhao Z, Margolskee R, Liman E. 2007. The transduction channel TRPM5 is gated by intracellular calcium in taste cells. Journal of Neuroscience 27:5777-86 Zhu MX. 2005. Multiple roles of calmodulin and other Ca(2+)-binding proteins in the functional regulation of TRP channels. Pflugers Arch 451:105-15 Zimmermann K, Leffler A, Babes A, Cendan CM, Carr RW, et al. 2007. Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures. Nature 447:855-8 Zurborg S, Yurgionas B, Jira JA, Caspani O, Heppenstall PA. 2007. Direct activation of the ion channel TRPA1 by Ca2+. Nature Neuroscience 10:277-9

154

Sarria, I

155