The Role of Transient Receptor Potential Channels in Pain

Index of Abbreviations

TRP, transient receptor potential

TRPA, transient receptor potential ankyrin

TRPM, transient receptor potential melastatin

TRPV, transient receptor potential vanilloid

CNS, central nervous system

ATP, adenosine tri-phosphate

PCR, polymerase chain reaction

CHO, Chinese hamster ovary

HEK, human embryonic kidney

CMR; cold and menthol sensitive receptor

VR, vanilloid receptor

VRL, vanilloid-like receptor

DRG, dorsal root ganglia

Introduction

Pain is one of the most common reasons for a person to visit a healthcare provider. Many ailments usually have at least one associated symptom that is painful. Understanding the complex circuitry allowing one to detect pain is essential in understanding the cause of that pain and how to effectively treat it. To date, pain is mainly able to be treated by suppressing inflammation, or by blocking the signal of pain entirely. A special family of channels, transient receptor potential (TRP) channels, has been identified as being primarily responsible for the initial detection and transmission of an electrical signal initiated by a painful stimulus. This review will discuss the role these channels play in detecting different types of painful stimuli, possible ways to antagonize these channels to alleviate pain, and future endeavors that could be of importance in coming to a better understanding of these channels and how they can possibly be manipulated to treat both acute and chronic pain.

While the initial thought of pain is undesirable, one must understand the importance and significance such an undesirable sensation has on injury prevention. Without the sensation of pain, one would not be able recognize a situation or stimulus as being painful, and could therefore lack the ability to prevent further bodily harm or tissue damage. Pain, itself, must first be understood before going any further.

Definitions and Divisions of Pain

Pain can be defined as the sensation perceived at the level of the brain derived from signals carried via nociceptors after being stimulated by injurious stimuli. Nociceptors are the peripheral afferent neurons which carry pain signals from affected tissue to the central nervous system. Pain can be divided into two different categories: nociceptive pain and neuropathic pain. Nociceptive pain originates due to some sort of injury induced stimulation. Neuropathic pain results from an injury of the actual nervous system, either central or peripheral. Neuropathic pain is initiated or caused by abnormal firing of the nociceptor or a neuron further upstream due to a primary lesion or dysfunction in the nervous system (Closs et al., 2009). These lesions or states of dysfunction can be due to a number of illnesses (diabetes, multiple sclerosis, stroke, etc) or inflammation, however, a common example of this subset of pain is phantom limb syndrome. Phantom limb syndrome is the sensation of pain resulting from the amputation of a limb. The resulting pain is thought to be at least partially due to the lesion on the nociceptor which now will function abnormally. Making this distinction between nociceptive and neuropathic pain is important clinically and pharmacologically. Methods used to treat the more well known and understood nociceptive pain are generally different than those used to treat neuropathic pain, which often involves several pathways and mechanisms. Pharmacological agents commonly used to treat pain, like non-steroidal anti-inflammatory drugs (NSAIDs), are beneficial against nociceptive pain generated by external stimuli. However, they are therapeutically not as useful against neuropathic pain generated by internal dysfunction or lesion of neural tissue. Besides being stimulated by an original stimulus, nociceptive pain is also enhanced by modulation of the nociceptor due to other factors released at the site of injury. Inhibiting the production of these modulators by, for example by NSAIDs, has the potential to attenuate pain. Conversely, neuropathic pain usually does not involve these modulators and, therefore, blocking their production would not serve a purpose. A more widespread and centrally targeted approach, in terms of the nervous system, is sometimes more beneficial in treating neuropathic pain. Nevertheless, targeting certain peripheral receptors or channels (most importantly, TRP channels) on the nociceptors might have beneficial therapeutic effects for neuropathic pain. These actions, to be discussed later, work in part by dampening any reaction to a stimulus that could result in premature firing at a neuropathy occurring more centrally in the nervous system (upstream).

Nociception and Nociceptors

Nociception is the process of generating an electrical signal in the primary nociceptors due to some sort of painful stimulus. This signal is then sent to the central nervous system where it can be processed and, ultimately, perceived as pain. Nociceptors are free nerve endings that distribute throughout the periphery, varying in densities of distribution based on the relative sensitivity of that particular area. Nociceptors, being the primary afferent neurons involved in sending the signal upstream that was generated from a painful stimulus, can be divided and defined more acutely. One subset of nociceptors is responsible for the reaction of pulling ones hand away from the oven, preventing any further injury, and the perception of pain afterwards that comes slower and is a more dull and aching sensation is due to another subset of nociceptors (non-myelinated, discussed later). Nociceptors are found all over the body, including the skin, internal organs, bone and teeth, and the heart. Most nociceptors are polymodal in the sense that they can respond to an array of stimuli. However, there are nociceptors responsible for only certain types of stimuli. A general allotment can be made by separating the nociceptors into what they primarily detect; namely, that of chemical, thermal, and mechanical stimuli. Nociceptors are divided based on their development and mature functionality into two categories, peptidergic and nonpeptidergic. This distinction is not only important in classification, but also has value in showing the different types of receptors and channels expressed in each class, as well as how they project to the brain. Peptidergic nociceptors express substance P and calcitonin gene-related peptide (CGRP), whereas nonpeptidergic nociceptors lack these peptides and have receptors for the lectin IB4 (Braz et al., 2005). Peptidergic and nonpeptidergic nociceptors take different routes on their course to higher levels of the nervous system. The peptidergic C-fibers transmit signals to the brainstem or thalamus. However, the nonpeptidergic C-fibers transmit signals to limbic parts of the brain including the globus pallidus, located in the sub cortex. Using wheat germ agglutinin (WGA), Braz et al (2005) traced the routes of these two subsets of C-fibers. What they concluded was that these two classes of primary afferent nociceptors are at the origin of parallel pathways that process nociceptive information. They also concluded that although they can not definitely prove the two routes do not cross or converge in the higher levels of the CNS, “the circuits engaged at the level of the spinal cord appear to be remarkably segregated” (Braz et al., 2005). Their findings also suggest that the information relayed on nonpeptidergic nociceptors contributes more to the affective component of the pain experience rather than to the sensory discriminative component (Braz et al., 2005). Evidence supporting this claim is based on the localization of these fibers in the hypothalamus, the amygdala, nuclei in the stria terminals, and the lack of localization topographically in the thalamus (Braz et al., 2005).

The primary afferent neurons in the pain pathway, nociceptors, can also be divided based on their axonal composition into two families: A-fibers and C-fibers. The division is important in the types of pain either one contributes to. A-fibers constitute 30% of all nociceptors. These A-fibers mediate those pains that are fast and prickly. These fibers are lightly myelinated, have medium or large axons, and conduct rather fast action potentials as compared to the unmyelinated, small diameter axons of the C-fibers. These C-fibers compose about 70% of all nociceptors. C-fiber nociceptors mediate the slower, throbbing, burning sensations of pain. Nociceptors, in general, contain a variety of channels and receptors needed to initiate the pain signal (start an action potential) to be sent to higher regions for processing (Figure 1). Nociception begins with the channels and receptors that are contained in or on the membranes of nociceptors (Figure 1). For example, stimuli that affect this membrane by physical means such as bending or stretching can start an action potential by activating the ion-gated channels and/or receptors which are stretch-activated. Furthermore, stimulation of the tissue can release certain chemicals including ATP, hydrogen ions, and proteases. ATP and hydrogen ions can contribute to an action potential by binding directly to the ATP-receptors or hydrogen receptors on the nociceptors and sensitizing the terminals to further stimulation. Proteases contribute to producing an action potential by breaking down extracellular proteins converting them to bradykinin which can also bind to some other specific receptors on nociceptors and lead to a sensitization or modulation of the nerve. Nociception can also involve temperature sensitive ion channels or chemical sensitive ion channels. An example of how chemical sensitive ion channels work would be the common muscle burn due to excessive lactic acid build up which leads to nociceptive action potentials. This most likely happens due to lactic acid stimulating acid sensing channels and receptors located on the peripheral nerve terminal. Upon stimulation, an action potential is generated and sent to the brain for processing, and is later perceived as a burning pain.

In order to initiate the nerve signal, the nociceptor must be activated. There are many different gates and channels on the most peripheral portions of the nociceptor that are responsible for determining whether or not an action potential is triggered. This rich mixture of membrane proteins, both channels and receptors, allows the nerve ending to integrate information from the outside world in order to sense whether or not something is noxious or injurious. These channels and receptors are made to be able to sense different aspects of the world, like thermal, mechanical, or chemical stimuli each with differing degrees of sensitivity. Intrinsic high thresholds for activation in these proteins allow them to operate when triggered by stimuli different from those that result in normal sensory signaling. The peripheral end of nociceptors are loaded with a variety of these transducers, including the acid sensing ion channel (ASIC) family, prostaglandin E2 receptors, P2Y receptors, Neurotrophic tyrosine kinase (TrkA) receptors, Bradykinin receptors (B1/B2), Interleukin receptor (ILR) 1, some ligand-gated channels, and transient receptor potential (TRP) channels (Figure 2). All of these receptors and channels work synchronously with one another to either sensitize or desensitize the nociceptor and, ultimately, start an action potential or not. Although all of these components are quite important in order to have a system working in a physiological non-disease state, one class in particular is of central importance. These are the family of transient receptor potential (TRP) channels.

The Transient Receptor Potential (TRP) Channels

Found mainly on the peripheral terminal of the nociceptor, TRP channels act to detect certain thresholds of stimulation. Whether the stimulation is in the form of some sort of noxious temperature, pressure, or chemical, TRP channels activate and initiate the beginning of the action potential which will eventually be interpreted by the CNS as pain. Attempting to understand pain where it actually first causes neural activity could lead to novel techniques of treating pain at the source of detection, rather than using the methods currently adopted such as anti-inflammatory drugs or central blockers like opiates. Since the initial detection of a painful stimulus has been strongly correlated with the activation of TRP channels, understanding their discovery, how they work, and what both activates and inhibits these channels will help to shed more light on how to utilize these channels in the control of pain.

Discovery of TRP Channels

TRP channels were first discovered and identified in Drosophila melanogaster (the common fruit fly). Normal Drosophila respond to light by activation of a rhodopsin GTP (guanosine triphosphate) coupled protein which absorbs photons and activates phopholipase C (PLC), inositol triphosphate (IP3) and diacylglycerol (DAG), and, in turn, leads to a depolarization of the membrane (Pedersen et al., 2005). Whereas normal Drosophilia respond to light with a sustained response, a mutant Drosophilia was found that exhibited photoreceptors which showed a transient response in voltage when under a stimulus of continuous light (Clapham, 2003). The normal Drosophilia which exhibited a sustained flat receptor potential was very different from the mutant which demonstrated a transient receptor potential, hence the term transient receptor potential channel (Cosens & Manning, 1969; Pedersen et al., 2005). Most ion channels or receptors are named so based on what ion they are most selective for or which ligand has the highest selectivity or rate of binding, as is the case with sodium channels, potassium channels, calcium channels, or NMDA (N-methyl-D-aspartic acid) receptors. TRP channels were identified based on their homology rather than by what ligand activated them or by what type of ion selectivity they had (Clapham, 2003). TRP channels were then cloned and purified to determine the correct amino acid sequences. From here, TRP channels were identified in mammals based on similarities among genetic sequencing which was determined based on a search of the Expressed Sequence Tag database in conjunction with PCR (polymerase chain reaction) techniques (Nishida et al., 2006). PCR is a commonly used method where a very small sample of DNA sequence is copied and amplified so as to produce many, many copies of the sequence to be studied or manipulated.

Division of the TRP Superfamily

After being discovered and cloned, the family of transient receptor potential channels has been divided into a number of subgroups. In total, TRP channels can been separated into 7 subgroups; TRP Ankyrin, TRP Canonical, TRP Melastatin, TRP Mucolipin, TRP NOMPC (No Mechanoreceptor Potential C), TRP Polycystin, and TRP Vanilloid. TRP NOMPC is the only one out of the seven not found in mammals, so it will not be discussed in this review. Of the remaining six, only TRPA, TRPM, and TRPV are most involved in pain, and will remain the focus throughout.

Mechanism of TRP Channel Activation

TRP channels have been identified as being crucial for the detection of noxious stimuli, ranging from those of heat and chemical, to those of mechanical stretch and pressure. TRP channels can be activated by a wide range of physiochemical stimuli. However, the end result of TRP channel activation is an influx of cations, mainly sodium and calcium, into the cytosol of the cell through the channel formed from TRP channel subunits (homo- or heterotetramers, discussed later). There have been many theories put forth about how these physiochemical stimuli are translated into activation of TRP channels (figure 3). One generally accepted thought is that TRP channels are activated by G-protein coupled receptors that initially respond to the stimuli and activate the TRP channels by way of intracellular mediators (Clapham et al., 2003). Once activated, TRP channels allow an influx of mainly calcium, which once inside the cell, can have a wide variety of effects. TRP channels can also be activated by ligands which work directly with the channel to modulate its function and lead to an increase in cation influx. Some activators are lipophilic and actually work by flowing through the plasma membrane and modulating the intracellular domains of the TRP channels (Jara-Oseguera et al., 2008). An activation of TRP channels is also thought to be achieved by simple mechanical stimuli like touch and pressure which cause stretching of the membrane and activation in this sense due to a conformational change in the channel. Changes in extracellular osmolarity have been shown to be involved with the direct activation of TRP channel subfamilies. Changes in extracellular osmolarity can also lead to changes in intracellular pH. The change can modulate the intracellular domains of the TRP channel in a secondary response to extracellular damage signified by osmotic stress outside of the cell. Thermal stimulation is also known to modulate and activate the channel, and until recently, the mechanism had not been very clear. Some more recent research suggests that the distal portion of the cytosolic C-terminal on TRP channels is responsible for sensing heat changes

An idealized view of the peripheral nociceptor with a TRP channel present. Important to realize all of the ways in which TRP channels can be modulated by ligands working either directly on TRP channels or through another protein. Many signaling cascades can be generated, however, the end result is the generationg of an action potential. (Jara-Oseguera et al., 2003; Vlachova et al., 2003)

Whichever theory holds true for the mode of activation for TRP channels (activation due to a stimulated G-protein coupled receptor, direct or indirect activation by ligands, direct conformational change from pressure or stretch, changes in extracellular osmolarity or pH, or thermal activation), the action potential is generated from the influx of both calcium and sodium through the non-selective cation pore formed by TRP channels. Once an action potential has been initiated via entry of cations through the TRP channel, the signal will propagate upstream via sodium channels until it reaches the higher levels of the central nervous system for processing.

The Pain Pathway

The next step in understanding pain is to understand the pathways by which signals are conducted after an action potential has been generated and the brain regions where they are ultimately processed for determining loci and severity of the pain signal. When discussing nociceptive or neuropathic pain, the initial afferent signal sent to the brain travels along the same path. This path that was initiated in the periphery on nociceptors continues on the dorsal root axons within the zone of Lissauer and finally synapses in an area of the dorsal horn referred to as the substantia gelatinosa. From here, axons travel the spinothalamic tract ascending without synapse until finally making a synapse in the thalamus. Pain can travel along the spinothalamic tract or trigeminothalamic tract depending on the origin of the stimulus (Bear et al., 2007). If the stimuli are traveling the trigeminothalamic tract, it must first synapse with the spinal trigeminal nuclei in the brain stem before going to any higher synapse. In either scenario, both tracts synapse in the intralaminar or ventral posterior nuclei which are located in the thalamus (Bear et al., 2007). The next stop for the signal on the ascending path is to finally synapse in the primary somatosensory cortex, depending on where the initial stimulus originated (figure 4). These nerves then project into the somatosensory cortex and lay out in a sort of topographical map based on where the injury had occured. For example, if someone was to burn their hand, the pain signal generated at the nociceptors in the hand would travel up the spinothalamic tract and finally synapse in the region of the somatosensory cortex which correlates to the region of the hand. The ascending course in which nociception takes is very similar to that of somatosensory nerves which are involved in sensing non-injurious stimuli, even synapsing in similar areas of the thalamus. This shows the dynamic capability of the higher cortical regions when it comes to relaying, differentiating, and processing stimuli originating from the periphery. An ability to distinguish stimuli that cause harmless feelings from those causing pain is essential to life and the avoidance of injury.

Once the nerve impulse resulting from some sort of noxious stimulus has reached the brain, it can be processed, an efferent signal can be returned to the peripheral site, and the proper reaction can be taken. This intricate orchestration of signaling to and from the thalamus is essential in the circuitry of nociception. Without this connectivity, the ability to recognize a stimulus as being painful or not, and then being able to react to the pain would not be possible. By understanding the pain center and both the ascending and descending paths, clinicians can target certain areas with either pharmacological agents or surgical techniques to alleviate pain. When looking at the very beginning of the ascending tract, it can be determined that this “spark”, initiated by some noxious stimuli, starts at the most peripheral segments of nociceptors due to TRP channel (TRPA, TRPM, and TRPV) activation.

General Structure of TRP Channels

Essential to understanding the mechanistic operation and function of these fascinating channels, the structure must be taken into account. Overall, TRP channels are classified as six transmembrane cation channel proteins, with both the amino- and carboxyl- termini located intracellular as well as having a partial membrane spanning region between transmembrane domains 5 and 6 (Gaudet, 2008). The transmembrane segments are homologous to the membrane spanning domains in Shaker potassium channels. To become functional, the channels must assemble as a tetramer of subunits (also known as a multimer) (Gaudet, 2008). These channels are very dynamic in the sense that they can be activated by physical stimuli or ligands which can be affecting the channel either on its intracellular domains or on its extracellular domains. (Figure 5 and Figure 6)

TRP Ankyrin Channel Structure

This subfamily of channels consists of six transmembrane domains with both the amino and carboxyl termini located intracellularly. TRP ankyrin channels were named so based on a repeated domain found on the intracellular amino tails. This ankyrin domain localized to the amino terminal consists of usually about 14 ankyrin repeats (Story et al., 2003). An ankyrin repeat can be defined structurally and functionally as “30-34 amino acid residues and exclusively functions to mediate protein-protein interactions” (Li et al., 2006). Postulation into the ankyrin repeat being involved with the functionality of binding intracellular modulators which can either sensitize or desensitize the channel have been put forth. This being said, it has been theorized that interactions with this domain may alter the activity of the channel by either enhancing or inhibiting the flow of cations through the pore. Ankyrin repeats may also be a target for intracellular regulatory molecules. Similar to other TRP channel families, TRPA assembles in a multimer to be rendered functional, contains 6-transmembrane domains with a pore-loop in between transmembrane segments 5 and 6, and both of the termini are found on the intracellular face of the plasma membrane. (Figure 7)

TRP Ankyrin Channels and their association with Pain

The subfamily of TRP Ankyrin channels consists of one protein, TRPA1. TRPA1 channels have been closely associated with the pain cascade in many different instances. It has been identified that many environmental compounds and endogenous inflammatory factors can activate TRPA1 by a multitude of mechanisms, all causing the sensation of pain. It has also been well documented that TRPA1 may have some involvement in sensing temperatures so cold that they illicit pain (Anand et al., 2008; Bandell et al., 2004; Jordt et al., 2004).

TRPA1 has been found to respond when in the presence of a number of injurious chemicals found in the environment. There has been quite a lot of evidence shown to validate the involvement of TRPA1 in reacting and becoming activated in response to these compounds. Activation in this sense refers to opening of the channels and an influx of cations, mostly that of calcium and sodium. “In addition to noxious cold, pungent natural compounds found in cinnamon oil (cinnamaldehyde-CA), wintergreen oil (methyl salicylate), clove oil (Isoeugenol-IE), mustard oil, and ginger (zingerone), all activate TRPA1, to produce a burning sensation” (Anand et al., 2008). The exact mechanism of action in which these agonists bind and activate this channel is still in the midst of being understood, however, some evidence has been shown that supports a covalent linkage to cysteine residues on the extracellular domains of TRPA1 channel subunits (Macpherson et al., 2007). Through experimentation by Bandell et al (2004), they were able to show conclusive evidence supporting the involvement of TRPA1 in sensing environmental chemical irritants (figure 8). “A variety of pungent compounds—oils of cinnamon, mustard, wintergreen, ginger, and clove—activate TRPA1”. Cinnamaldehyde is the main constituent of cinnamon oil (70%) and is extensively used for flavoring purposes in foods, chewing gums, and toothpastes. Allyl isothyocianate (mustard oil) is one of the active ingredients in horseradish and wasabi. "Methyl Salicylate (wintergreen oil) is used commonly in products such as mouthwash and as a counterirritant in topical analgesic ointments” (Bandell et al., 2004). Bandell et al (2004) used a FLIPR (fluorescent imaging plate reader) to look for any intracellular calcium level increases after application of each of these compounds. FLIPR works by measuring the change in fluorescence given off by certain fluorescent calcium sensitive dyes which are activated in the presence of calcium. They found that cinnamaldehyde, allyl isothiocyanate, and methyl salicylate were specifically activating TRPA1, with cinnamaldehyde and allyl isothiocyanate having the most efficacious effects on TRPA1. Matched experimentation on cell lines expressing TRPV1, TRPV4, or TRPM8 showed no increases in intracellular calcium when exposed to cinnamaldehyde or allyl isothiocyanate (figure 8). Thus, this further showed that these environmental irritants worked specifically via TRPA1.

The experimental results of (A) FLIPR and (B) another calcium imaging technique when cells expressing TRPA1 were exposed to cinnamaldehyde and allyl isothiocyanate. C) Results showing the changing intracellular calcium levels in the presence of RR (ruthenium red - a known TRPA antagonist). D) Results of experimentation where cells expressing TRPV1,4 and TRPM8 were exposed to cinnamaldehyde. No response was elicited. (Bandell et al., 2004)

Patch-clamp methodology gave reassurance to the conclusion that was drawn. Patch-clamping works by isolating a specific channel of interest in a cell and recording the currents evoked under varying circumstances. By conducting patch-clamp analysis on the TRPA1 channels that were exposed to these irritants, it was determined that currents were elicited. Later experiments completed by Bautista et al (2006) supplemented the previous findings pointing towards TRPA1 being essential to initiating the pain cascade stimulated by environmental irritants such as mustard oil and garlic (allicin). Also highlighting the specificity, these compounds proved to work solely by a TRPA1-dependent pathway that is completely TRPV1-independent. These results were documented based on experiments comparing both intracellular calcium changes as well as behavioral changes in mice expressing TRPA1 versus mice lacking any functional TRPA1. Mice that were heterozygous or haploinsuffiecient for a phenotype, in this case for TRPA1, were tested in the same round of experiments. “This haploinsufficiency phenotype suggests that the number of functional TRPA1 channels is limiting such that changes in their expression or sensitivity can alter neuronal excitation in a linear and dynamic fashion” (Bautista et al., 2006). This gave evidence that the amount of TRPA1 channels can change based on the extent of expression or sensitivity and this modifies the amount of excitation (Bautista et al., 2006).

Noxious chemicals are not limited to those that are applied topically to the skin or internally in the gastrointestinal tract. Injurious or painful chemicals are also produced endogenously as a result of normal physiological processes. There have been many experiments performed to give solid evidence of this, and one example is the following. In the experimentation by Macpherson, Xiao et al (2007), they test for the responsiveness of TRPA1 to analdehydes and an endogenously produced chemical. In this case, they hypothesized that formaldehyde, other aldehydes, and 4-hydroxynonenal (4-HNE), an endogenous product made during oxidative stress in cells will activate TRPA1 channels (Macpherson, Xiao et al., 2007). When the hypothesis was tested, they found that both formaldehyde and 4-HNE did stimulate the TRPA1 channel. Using TRPA1 knock-out mice they were able to compare effects of injections of these injurious compounds to mice still expressing the TRPA1 channel. Results conclusively showed in both instances that these chemicals did activate TRPA1 channels. Most notable in this experiment is the discovery that TRPA1 can be activated by an endogenously produced chemical like 4-HNE, which results from lipid peroxidation in the cells. There has also been evidence shown that endogenous inflammatory agents can directly stimulate this channel as well. By expanding on previous studies, Taylor-Clark et al (2008) were able to demonstrate “that electrophilic molecules that are produced downstream of COX activity during inflammation can also directly activate the channel” (Taylor-Clark et al., 2008). COX (cyclooxygenase) is an enzyme involved in inflamed tissue and leads to the production of prostaglandins which leads to increased inflammation and pain. Metabolites of endogenous inflammatory molecule like prostaglandins were shown to activate TRPA1. Using mouse models along with calcium imaging and patch-clamping to show results, it was verified that these compounds do activate TRPA1. Since TRPA1 and TRPV1 are co-expressed on the nociceptor, they needed to prove whether or not the activation of the nerve by these inflammatory molecules was based solely on TRPA1 activation. In order to test this hypothesis, they used a TRPV1 selective antagonist (I-RTX) to show results of nerve activation strictly based on TRPA1 activity. Upon application of I-RTX, the cell line became desensitized to capsaicin, as would be expected due to capsaicin being a primary agonist of TRPV1. They also used HC-030031, a selective TRPA1 antagonist to verify these results by showing that nerve terminals were drastically reduced in activation when exposed to inflammatory metabolites while TRPA1 was antagonized. HC-030031 eliminated calcium influx that was originally seen when the cells were exposed to prostaglandins simultaneously with I-RTX, the TRPV1 antagonist. Lastly, to make sure that there was no other “third-party” channel of the TRP family involved, they administered the same test while subjecting the cell lines to ruthenium red, a non-selective TRP channel blocker (Taylor-Clark et al., 2008). Under administration of ruthenium red, responses to any of the agonists (prostaglandin PGJ2, TRPA1 agonist AITC, and TRPV1 agonist capsaicin) were reduced. Results of these tests gave evidence supporting the sole involvement of TRPA1 in sensing inflammatory endogenous metabolites, resulting in increased pain perception or sensitization of the inflamed area. Extra proof was provided in this same experiment by expressing the HEK293 solely with TRPA1 and introducing the known agonists. Compared to HEK293 cells lacking TRPA1, the conclusion could be drawn that renders TRPA1 as a candidate for being responsible in sensing endogenous inflammatory pain causing molecules.

As for TRPA1 and its involvement in thermosensation, it has been found to sense temperatures that are noxiously cold. There has, however, been some dispute as to whether TRPA1 is actually involved in thermosensing. The following experiments have been employed in order to test hypotheses on whether or not TRPA1 is involved directly in thermosensing of noxious cold.

In one study by Jordt et al (2004), they attempted to determine if TRPA1 senses noxious cold temperatures. In order to test this hypothesis, they first identified TRPA1 channels by exposing them to mustard oil. Calcium imaging techniques were used to show whether or not the channels were activated by the mustard oils, which in fact they were. Calcium imagery allowed scientists to visualize the large increases of intracellular calcium which is assumed to be due to opening of the TRPA1 channel from some sort of extracellular binding of the activators (Jordt et al., 2004). Once the channels were identified by this manner, the channels were subjected to noxious temperatures to see if the same response was generated. Since it had been identified that temperatures below 20°C were considered noxious, the channels were subjected to 5°C temperatures. Using similar calcium imaging methods, it was determined that 96% of the channels experienced no calcium influx, therefore demonstrating the channels inability to detect and respond to noxious temperature. This led to the conclusion that TRPA1 was, in fact, only sensitive to chemical compounds like mustard oil and menthol, while being insensitive and unresponsive to noxious cold temperature.

In contrast, Karashima et al (2009) showed evidence that TRPA1, actually, is a major sensor for cold temperatures that plunge into noxious ranges. In fact, most other reports also agree with the hypothesis that TRPA1 is activated by noxious cold temperatures. They hypothesized that TRPA1 does play a role in cold sensation via a calcium independent method. To clarify, when they tested for the activation via a calcium independent mechanism, they were trying to determine whether channels are activated secondarily from cold activation releasing intracellular stores of calcium as opposed to some sort of activation or conformational change induced directly on the channel from the decreased temperature. To test this hypothesis, they used a patch-clamp method to see whether or not cold temperatures induced activation of inward currents in murine CHO (Chinese hamster ovary) cell lines transfected with TRPA1 channels. They compared the results found to CHO cell lines lacking TRPA1 channels. When subjected to temperatures of 26°C, the TRPA1 positive cells showed strong activation currents. When chilled to 10°C, the TRPA1 cells were activated even more vigorously. When the same temperatures were induced on TRPA1 negative cell lines, patch-clamping showed a flat line response to both 26°C and 10°C temperatures. Since it had been hypothesized that TRPA1 activation could be directly linked to intracellular calcium stores, they needed to make evident that the increase in calcium was not calcium from intracellular stores released upon being stimulated by cold temperature. In order to verify that this activation was independent of secondarily released intracellular calcium stores, they cleared the cell lines of any intracellular calcium. Extracellular calcium was removed by bathing the cell lines in calcium free solution, while also being treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), a calcium chelator, to rid them of any intracellular calcium. Upon stimulation of the TRPA1 channels with cold temperatures, a strong current of activation was still detected, presumably due to the inward current from other cations, mainly sodium. Thus giving verification that TRPA1 was activated by the cold temperatures alone, independent of intracellular calcium stores. Subsequent calcium release is needed, however, for full activation of the TRPA1 channels. To support these claims, Karashima et al (2009) then tested TRPA1 channels for cold induced activation after “TRPA1 was fully preserved in cells pretreated for 30 min in Ca2+-free medium supplemented with the SERCA pump inhibitor cyclopiazonic acid (CPA) to deplete intracellular Ca2+ stores before cooling” (Karashima et al., 2009). Results supported the findings that cold alone caused the primary activation of TRPA1, independent of intracellular or extracellular calcium stores; however, calcium flux from the intracellular stores upon cold stimulation leads to an additive effect resulting in activation of the channel with higher amplitude.

A) Results showing the delayed reaction in mice lacking the TRPA1 channels to rub paws that have been on a cold plate. B) Another analysis in the same experiment, this time recording the amount of jumps off of the plate to relieve pain from the cold plate. C) The relationship of probability of jumping in response to time on cold plate. D) Results of immersing the tail of a mouse in a mixture of water and methanol. Mice lacking TRPA1 showed a delay in flicking of the tail. (Karashima et al., 2009)

To further support their hypothesis, Karashima et al then tested the effects of cold induced pain perception via TRPA1 channels in mice models (figure 9). They designated two groups of mice, wild-type and TRPA1-/- null mice. The mice were put on a metal plate measuring 0°C. Of the wild-type mice, 19 of the 25 exhibited jumping behavior, implying the plate caused pain due to the TRPA1 channels being localized in the skin and paws of the mice. As for the TRPA1-deficient mice, only 3 of the 25 exhibited this behavior. Thus, this further exemplifies the importance of TRPA1 in detecting noxious temperatures. Most other recent reports indicate that TRPA1 is involved in noxious cold thermosensation, and the results found earlier suggesting that TRPA1 does not sense temperatures in this range have not been explained.

TRP Melastatin Channel Structure

The second subfamily of TRP channels to be discussed in this review is that of TRP Melastatin. Although this subfamily is one of the largest in terms of number of amino acids, its overall structure is comparable to that of any other TRP channel family member. TRPM channels follow the general structure as defined by any other TRP channel, being that they consist of 6 transmembrane domains, with both termini located on the cytosolic side of the plasma membrane. Unlike some of the other TRP channels, the Melastatin division does not have any ankyrin repeats on the terminal amino tail. This is functionally important in that now a division can be made between TRPM and the other pain TRP channels,TRPA and TRPV. This distinction is not only structurally significant, but also shows significance in what can interact and modulate the channel. This can prove important down the road when discussing probable pharmaceutical agents that would need to have specificity among TRP channel subfamilies. When it comes to variations in channel structure that can differentiate the Melastatin family from other TRP channel subfamilies, the most unique characteristic is a coiled-coil domain present on the cytosolic carboxy terminal tail (Fujiwara & Minor, 2008). In certain studies performed on the TRPM channels, they have been able to determine a couple of important roles involving these coiled-coil structures. This coiled-coil domain, which is a motif present on a number of other proteins, is suggested to assist in the assembly of TRPM channels in multimers (Tsuruda et al., 2006). The coiled-coil domain also has been suggested to be involved in modulating both the assembly and activation of the channels (Tsuruda et al., 2006). Modulation of the assembly of these channels is most likely due to the interaction between the coiled-coil domains of each subunit with one another (Tsuruda et al., 2006). An additional level of structural importance (for the TRPM family as a whole) is the presence of enzyme activity linked to these channels, allowing them to be classified as “chanzymes”. Chanzymes can be envisioned as a channel or pore protein which also houses some sort of enzymatic activity. Although the channels involved are not involved in pain transmission, it is still very remarkable and should be noted that out of the entire TRP superfamily, only the TRPM channel subfamily has members considered chanzymes (TRPM2, TRPM6, and TRPM7). (See Figure 10)

Figure 10: Structural characteristic of TRPM channels. Notice the absence of an ankyrin repeat domain but the presence of enzymes. These enzymes can catalyze an array of messaging cascades upon activation of the channel.

(Pedersen et al., 2005)

TRP Melastatin Channels and their association with Pain

This subgroup of channels is expressed as eight different varieties (TRPM1-8) in Homo sapiens; this is the largest subdivision of the TRP channels expressed. However, not all of these subtypes contribute to sensing pain or possible something that could be painful if prolonged. TRPM8 is the only member of this subfamily which, in terms of nociception, has any functionality. M8s main function deals with thermosensitivity. TRPA1 was discussed above as being responsible for responding to extremely low temperatures. In order to respond to all ranges of the temperature scale, channels have adapted a sort of gradient in which different subclasses recognize and respond to different temperature ranges. TRPM8 is activated by temperatures above those which activate TRPA1.

It was determined that this protein, TRPM8, responds to temperatures specifically in the range of 8°C - 28°C. These channels were originally found in the rat and were named cold and menthol-sensitive receptors (CMR1). These channels were subjected to cold temperatures and, based on patch-clamp analysis, it was found that cold induced a fairly robust inward membrane current. Although specific actions of how thermal stimuli mechanistically induce an inward current and depolarization have yet to be completely elucidated, it has been assumed that temperature has some effect on the channels resulting in an inward flow of cations. Applying temperatures ranging from room temperature to 30°C on this channel evoked no response and applying temperatures in this range actually antagonized the channels responsiveness or ability to react to stimuli that normally evoke a response (McKemy et al., 2002). CMR1 was then cloned and expressed in HEK293 human cells. Here the same experimentation was performed and similar results were collected. These human cells that were transfected with cloned rat CMR1 were then tested for responsiveness to cold temperatures. Indeed, when exposed to temperatures decreasing from 35°C to 5°C, the channels were activated and caused increasing inward currents complementary to that of the decreasing temperature. Later comparison of CMR1 to human TRP channels found CMR1 to be 92% identical to human TRPM8. For that reason, the conclusion was drawn stating that the menthol receptor found in humans was also sensitive to cold temperatures, and was in fact identifiable as TRPM8. To determine localization of this channel in the human nervous system and to provide evidence for it being localized on nociceptors, northern blot and in situ hybridization experiments were carried out. Northern blot is a laboratory technique which makes it possible to observe RNA that codes for a certain protein. During certain situations, RNA levels will fluctuate to adapt for making the proper amount of protein required. It was shown that CMR1, and thus the comparable human TRPM8, was located on trigeminal nerves and dorsal root ganglia axons with an overlap of expression being where TRP Vanilloid receptors were expressed (McKemy et al., 2002). Since there was an overlap in expression with TRPM8 and TRPV, it can be assumed that TRPM8 is also located on peripheral nociceptors and plays a vital role in sensing a partial cascade of temperature, specifically ranging from 8°C-28°C. It has also been documented that TRPM8 is an involved temperature sensing protein in the genitourinary track, therefore giving evidence to the widespread distribution of this channel and the need for further research into possible drug treatment of certain urological disorders (Stein et al., 2004).

It is known that menthol causes a cooling sensation by working directly on cold sensing proteins on nerves that typically sense temperature. In earlier experimentation (2002), Chinese hamster ovary (CHO) cells that were transfected with TRPM8 and incubated at 25°C were tested for reaction to menthol. When treated with menthol, fluorescence testing confirmed a significant increase in intracellular calcium compared to those CHO cells lacking TRPM8. Furthermore, when experimenters took away extracellular calcium and repeated the experiment, they showed that intracellular fluorescence did not increase (Peier et al., 2002). Based on this finding, it can be concluded that TRPM8 channels must have significant calcium permeability. Menthol was also tested by Peier et al (2002) for its effectiveness at varying temperatures. Similar techniques showed that when TRPM8 CHO cells were incubated at 33°C, menthol had no effect on intracellular calcium levels; however, when the incubation temperature was decreased to 30°C, intracellular calcium levels rose substantially. These results give evidence towards the possibility that menthol can act to actually “mimic the effect of lowering temperature on TRPM8-expressing cells” (Peier et al., 2002). Studies performed in 2004 show a range of other agonists that TRPM8 is sensitive to including the following: linalool, geraniol, hydroxycitronellal, eucalyptol, icilin, WS-3, WS-23, Frescolat MGA, Frescolat ML, PMD 38, Collact P, and Cooling agent 10; linalool, geraniol, and hydroxycitronellal actually being odorants (Behrendt et al., 2004). When each agonist was applied to HEK293 cells which had been transfected with TRPM8, a calcium increase could be elicited (figure 11). However, changes in pH can lessen the effect on these agonists on when in the presence of TRPM8 channels. Acidic environments can actually inhibit the response of this channel when in the presence of agonists (Behrendt et al., 2004). This is important in the sense that tissue which has been injured often has an acidic pH, and therefore, a cooling sensation normally caused by these agonists is not felt at the site of injury. TRPV1 channel activity is amplified in acidic pH, giving a possible reason for the sensation of warmth sometimes felt at the site of injury (Behrendt et al., 2004).

After conducting experimentation using FLIPR, the relative responsiveness of TRPM8 to the following substances has been graphed. Since icilin evoked the greatest response, all substances are measured in a “% icilin response” method.

(Behrendt et al., 2004

It is important to note that, besides detecting any possibility of injury occurring from exposure to these compounds, these channels also have a quite disparate function in pain. More specifically, most of the agonists listed above activate TRPM8 in a fashion to create a cooling sensation, which clinically could be important for relieving pain (Behrendt et al., 2004). A large target for the possible analgesic effects from TRPM8 channel agonists has been pointed towards neuropathic pain. Proudfoot et al (2006) has defined neuropathic pain as such that “arises from peripheral nerve damage” or abnormal nervous tissue activity at any place in the nervous system that is in the absence of actual injury. It has been documented that TRPM8 is highly expressed in the periphery after a nerve has been damaged. This heightened expression is crucial for the analgesic property of TRPM8 and is found mainly ipsilateral of the injury versus contralateral (Proudfoot et al., 2006). This means that the elevated expression of TRPM8 channels on the dorsal root ganglia and spinal cord nerves were found on the same side of the body (ipsilateral) as the location of the injury. Heightened expressiveness of TRPM8 is thought to be analgesic based on the fact that upon TRPM8 channel stimulation, a cooling sensation is experienced, and this sensation is thought to have possible therapeutic effects. Prior to nerve injury, the majority of TRPM8 is located on unmyelinated afferent dorsal root ganglia. After the injury, increases in TRPM8 expression was increased in both the population of unmyelinated nerves where it is normally found as well as myelinated afferents (Proudfoot et al., 2006). These results can help determine how TRPM8 mediates an analgesic state when exposed to activators. Using an intrathecal (under the lining of the spinal cord) approach of administering TRPM8 activators to centrally located channels, it had been noted that analgesia can be achieved (Proudfoot et al., 2006). By giving these TRPM8 activators, the reflex sensitization of rats with neuropathic pain was drastically reduced or even abolished when they were standing with their paws immersed in solutions of noxious temperatures (Proudfoot et al., 2006). This gives conclusive evidence that TRPM8 can be modulated either via peripheral channels or via central channels.

TRP Vanilloid Channel Structure

The structure of TRP Vanilloid channels has recently been elucidated by electron cryomicroscopy. Like other TRP channels, TRPV was also structurally compared to that of voltage gated potassium channels. Unlike the TRPM subfamily, TRPV does contain ankyrin repeats on the intracellular amino terminal. Unlike the regularity of sequence and number of ankyrin repeats present in TRPA, TRPV only has a few ankyrin repeats which are arranged in a more random sequence (Phelps et al., 2007). It has been determined that they contain six repeats on the N-terminal tail (Lishko et al., 2007). Having fewer ankyrin repeats is probably functionally important in determining what types of proteins can interact with the domain and, in the end, modulate the channel. Fewer ankyrin repeats in this domain would also likely contribute to homo- or heteromultimerization. This means that when a multimer of subunits is to form, the amount of ankyrin repeats could determine if the subunits that come together are all the same identical subunits or different. It is also important to note that ankyrin repeats were found to bind small ligands like ATP and calcium calmodulin, each of which give rise to opposing effects (Phelps et al., 2007). ATP results in the prevention of desensitization when the channel is activated, whereas calcium calmodulin is essential for desensitization (Phelps et al., 2007). TRPV channels as a subfamily do have subtle differences in the ankyrin repeats and overall structure, however, it has been theorized that these domains are probable locations for intracellular proteins to dock and/or interact with TRPV channels modulating their overall activity (Moiseenkova-Bell et al., 2008). The ankyrin repeat domain may give a docking site for intracellular messengers to bind and affect the flux of ions through the channel. This conserved feature among all of the TRPV channel family members gives evidence as to how the channel may be modulated or controlled. Understanding how a class of channels is controlled can lend further insight into the development of synthetic agonists or antagonists. TRPV1-4 channels have also been described in forming homo-multimerized pores (Hellwig et al., 2005). Proof of the theory of TRPV channels forming complexes has been shown using differently tagged channels which were immunoprecipitated and found to co-localize on the precipitate band proving their interaction (Kedei et al., 2001). Other prominent bands on the precipitate proved to be dimers, trimers, and tetramers which mean that these channel subunits can form in any of these varieties; however, tetramers are the only functional form (Kedei et al., 2001). The true importance in this finding is that it suggests that TRPV channel subunits may be able to form some sort of heteromeric structure with one, two, or three other subfamily channel subunits giving rise to a possible alteration in function or sensitivity. Kedei et al (2001) has put forth suggestions of agonists like capsaicin inducing the multimerization by binding some extracellular domain on the subunits. By utilizing SDS-PAGE (an experiment that separates proteins based on the length and molecular weight of their polypeptide chain), they determined that when in the presence of an agonist, covalent cross-linking occurred resulting in multimers (Kedei et al., 2001). Either way, complexity is now greatly increased when there is the possibility of these subunits forming multimers with itself or with other subunits. (See Figure 12)

An overview of structural motifs exemplified in the TRPV channel subfamily (Pedersen et al., 2005)

TRP Vanilloid Channels and their association with Pain

Of all of the TRP channel subfamilies, TRP vanilloid channels are some of the most involved in sensory systems and pain sensing. Ranging from mechanosensation to thermosensation, the TRPV subfamily is an intricate component of understanding the big picture of pain. “In mammals there are six TRPV channels that partition into two groups: TRPV1-4, involved in sensory signaling; and the more distantly related TRPV5 and TRPV6, expressed in the intestinal tract and kidneys and important for calcium homeostasis” (Gaudet, 2008). TRPV1-4 will be of focus in this review, as they are involved in sensory signaling and pain, most notably temperature.

TRP Vanilloid Type 1 Channels and Pain

TRPV1, also known as vanilloid, was the first of this subfamily to be discovered. Cloned from a newly found channel originally termed vanilloid receptor (VR1), it was found to have a similar type of amino acid sequence as other TRP channels as well as the prototypical ankyrin repeats. This channel is primarily noted as being activated by noxious heat. Previous reports cite the channel to be activated by heat in a graded response around temperate temperatures. Above temperatures of 43°C, this channel is actively and rapidly opened to calcium influx (Caterina et al., 1999). By increasing the ambient temperature of cells, a prominent increase in intracellular calcium was established in cells transfected with TRPV1 when compared to cells lacking TRPV1 (Caterina et al., 1997). This response is subdued after the cells are not exposed to the noxious heat, but can be evoked again with a subsequent exposure to a channel agonist. This supports the claim that the channel is involved in sensing painful heat because it proves the response to heat was not caused by the disruption of the membranes integrity by the high temperatures. When analyzed by patch-clamping, both heat and vanilloid (an agonist) generated a strong current, giving evidence that the same protein was involved in sensing both thermal and chemical stimuli. All in all, TRPV1 is definitely involved in detecting noxious heat, whereas temperatures below a threshold of about 40°C do not sufficiently activate the channel.

TRPV1 has been indicated quite extensively as being activated specifically by capsaicin. In fact, VR1 was originally cloned from the “capsaicin receptor”. Capsaicin is an active ingredient in many spicy foods including chili peppers. Since capsaicin leads to a psychophysical burning sensation when exposed to TRPV1 channels, it was hypothesized that capsaicin actually mimics the endogenous activators of nociception. After comparing the results of excitation to known endogenous activators in cells transfected with TRPV1, it was found that they did not correlate with the excitation caused from capsaicin (Caterina et al., 1997). Therefore, it can be concluded that capsaicin does not mimic endogenous modulators of pain, and in fact, works via a separate route on the TRPV1 channel. It was also shown that capsaicin works to directly activate the TRPV1 channel, and that the channel does not require activation from soluble components in the cytoplasm (Caterina et al., 1997). Capsaicin works to directly activate the TRPV1 channel extracellularly causing an opening of the pore and an influx of ions. However, later research has suggested quite the opposite in that capsaicin works by crossing the plasma membrane and acts on intracellular binding sites (Jara-Oseguera et al., 2008). Like previously described TRP channels, TRPV1 is also most permeable to calcium. An important observation to note at this juncture is the evidence found supporting an endogenous molecule, N-arachidonoyl-dopamine (NADA), which mimics the action of capsaicin and works in the brain (Huang et al., 2002). Of importance in discovering the reactivity of NADA (N-arachidonoyl-dopamine) on TRPV1 is important because when applied or utilized, it could desensitize TRPV1 and be useful in an analgesic sense (Huang et al., 2002).

Besides being activated by capsaicin, TRPV1 has also been suggested to respond or be activated by varying levels of pH. Injury or inflammation causes a significant increase in molecular mediators found quite prevalently at the site of injury. This increase in molecular proteins can alter the pH of what is found under normal, non-injurious or non-inflammatory states. Using hydrogen ions, tests have been performed to determine the reactiveness of TRPV1 to changes in pH. At first, it was shown that bathing cells in a medium with a pH of 5.5 elicited only a slight response from cells expressing TRPV1. However, when these same cells were exposed to an agonist like capsaicin and then bathed in a slightly acidic environment, a much greater response could be obtained (Caterina et al., 1997). A modest current can be evoked from acidic pH at physiological temperatures and what this suggests is TRPV1 is not especially sensitive to an acidic pH, but more so an acidic environment can potentiate TRPV1 agonists like capsaicin and heat (Tominaga et al., 1998). More recent studies have elaborated in this subset of experimentation including the reaction of TRPV1 to both acidic and basic pH. Dhaka et al (2009) exposed murine cultured models of DRG neurons to ammonium chloride (NH4Cl). They found that the cells responded vigorously, a response similar to that of when they these same cells were exposed to capsaicin, thus proving that the DRG neurons must contain the TRPV1 channel (figure 13). Exposing the same cells to a TRPV1/TRPA1 antagonist (TRPV1 and TRPA1 tend to be co-localized in DRG neurons) like Ruthenium Red (RR), a great decrease in excitation was shown suggesting that TRPV1 is definitely involved in the chemosensation of basic pH (Dhaka et al., 2009). Similarly, when HEK293 cells expressing TRPV1 were exposed with NH4Cl, a similar vigorous response was recorded in comparison to TRPV1 deficient HEK293 cells. Another more specific TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine -1(2H)-carbox-amide (BCTC), was used in correlation with NH4Cl and these inward currents were effectively blocked (figure 13) (Dhaka et al., 2009). Further results proved that the activation of the TRPV1 channels by basic pH happened in an “inside-out” fashion, meaning that an intracellular increase in pH activates TRPV1 (Dhaka et al., 2009). Dhaka et al (2009) then determined a single histidine residue (H378) is responsible for sensing NH4Cl, and a TRPV1 channel lacking H378 is still functionally sensitive to capsaicin. Overall, TRPV1 senses acidic pH by its extracellular domain (Tominaga et al., 1998), where as, it senses basic pH via an intracellular histidine residue (Dhaka et al., 2009). It can be assumed that capsaicin activates TRPV1 by some sort of extracellular binding site.

Figure 13: A) Normal alkaline-sensing cells within the TRPV1 population and their reactivity to NH4Cl and capsaicin. B) The relative responsiveness of DRG neurons when in the presence of RR or BCTC (TRPV1 antagonist). Notice the lack of responsiveness in the TRPV1 knockout model without RR or BCTC. C) Results of responsiveness of cells expressing TRPV1 when exposed to NH4Cl and capsaicin. D) Smaller responsiveness of cells expressing TRPA1 to NH4Cl.

(Dhaka et al., 2009)

This channel, TRPV1, has even shown significance in sensing pain at the level of the viscera. This is quite important seeing as though most of the pain sensing done by TRP channels occurs in the periphery. TRPV1 as stated above is sensitive to changes in pH and it is well established that the gastrointestinal tract is usually in a state outside of physiological pH. When experiments performed on gastrointestinal cells lacking TRPV1 were exposed to acidic media, the response was blunted, thus providing evidence that TRPV1 was an essential protein involved in sensing acidity in the GI tract (Bielefeldt & Davis, 2008). Bielefeldt & Davis (2008) also suggested that TRPV1 may be activated by mechanical stretch in the viscera; however, this finding could be due to other TRP channels co-localized in the gastrointestinal tract.

Type 1 TRPV channels are polymodal in the sense that they can detect a wide variety of stimuli. TRPV1 is such a polymodal channel that it also responds to certain synthetically made products. Of particular importance is the universal TRP blocker, 2-aminoethoxydiphenyl borate (2-APB), which actually activates TRPV1. Originally produced and found to inactivate IP3 (inositol 1,4,5-trisphoshate) receptors, it was later discovered to activate TRPV1 (also TRPV2 and TRPV3) on its extracellular face (Hu et al., 2004). The importance of this finding is directed more so at possible side effects elicited in case an IP3 receptor antagonist is ever used clinically.

TRP Vanilloid Type 2 Channels and Pain

TRPV2 was originally discovered based on a homologous clone of the vanilloid receptor (VR1). This cloned receptor termed, vanilloid-like receptor (VRL-1), was later identified by genetic comparison to be TRPV2. At first, it was determined that unlike TRPV1, TRPV2 is not sensitive to capsaicin or any like activator. However, TRPV2 is heavily involved in thermosensation. The primary feature that has been most observed with TRPV2 is detecting noxious temperature. When exposed to temperatures ranging from physiological to 50°C, no substantial response could be evoked from TRPV2, however, when the channel was exposed to noxious temperatures greater than 53°C, a robust current was induced (Caterina et al., 1999). This was determined to have a higher threshold than its family member TRPV1, giving rise to possible roles in picking up temperature sensing over the threshold limiting temperature of TRPV1. TRPV2 is localized to a different population of afferent nociceptors which have been shown previously to respond to higher thresholds of heat, specifically Ad fiber nociceptors, whereas TRPV1 was localized to C fiber nociceptors (Caterina et al., 1999). More recent experimentation has shown opposite findings of those showing TRPV2 as sensitive to temperatures above 53°C. These reports suggest a more widespread localization of TRPV2 and a difficulty to elicit as strong a heat sensitive response as previously stated (Lawson et al., 2008). However, these findings do not rule out the possibility of TRPV2 being involved in the sensitization of repeated heat exposure or in cases of tissue injury and inflammation. Again, it is not completely understood how temperature causes the opening of TRPV2, but could be related to a portion of the intracellular C-terminal domain (Jara-Oseguera et al., 2008). As for the responsiveness of this channel to fluctuations in pH, it was found to not be as affected by these changes unlike what was found with fluctuating pH and TRPV1 activity (Caterina et al., 1999).

Closer examination of the actual localization of TRPV2 channels to A mechano- and heat-sensitive (AMH) nociceptors suggests that TRPV2 may have some role in sensing mechanotransduction (Caterina et al., 1999). In fact, TRPV2 has been described recently as being a heat sensitive TRP channel that also senses mechanical stresses. More importantly, TRPV2 has been suggested to innervate numerous tissues besides cutaneous epithelia. Experimentation suggested that mechanical sensitivity is the strongest “ligand” for TRPV2 over even that of noxious heat, and that TRPV2 was mainly localized to myelinated mechanosensitive nociceptors (Lawson et al., 2008). Some sort of mechanistic plasticity exists in TRPV2 allowing it to be both sensitive to heat and stretch.

Investigators could not find actual specific agonists for TRPV2 (as capsaicin is for TRPV1) until recently. Previous studies indicated that THC and 2-APB both activate TRPV2, but both of these activators have broad ranges of activation on other receptors and channels (Bang et al., 2007). Bang et al (2007) identified a uricosuric agent, probenecid, which was used and found to activate TRPV2 (figure 13). Further tests found that probenecid activated the same channels that were activated by 2-APB, which non-specifically activates TRPV1-3 (Bang et al., 2007). Based on that information it could be surmised that probenecid was, in fact, activating TRPV2 (figure 14). Bang et al (2007) also showed that probenecid had specific activating affinity for TRPV2 versus other heat sensitive members of the TRP family including the following: TRPV1, TRPV3, TRPV4, TRPM8, and TRPA1 (Bang et al., 2007). Conclusive evidence like this will hopefully lead to further studies of probenecid on TRPV2 and how this channel works at the molecular level, including activation, sensitization, and desensitization.

A) When probenecid (PRO) was applied to a cell expressing TRPV2, a current response was elicited. The cell was also responsive to 2-APB (known TRP activator). B) A comparison of responsiveness in cells expressing TRPV2 when in the presence of PRO, 2-APB, or PRO + RR. C) A measure of the calcium influx in cells with TRPV2 based on the dosage of PRO (x-axis). D) A comparative chart showing the % change in fluorescence due to calcium influx in cells with either TRPA1, TRPV1, TRPV2, TRPV3, TRPV4, or TRPM8. TRPV2 showed the most significant change in intracellular calcium.

(Bang et al., 2007)

TRP Vanilloid Type 3 Channels and Pain

Another member of the TRPV subfamily is TRPV type 3 (TRPV3). After reviewing genomic regions near that of TRPV1 and TRPV4, TRPV3 was identified. Found to be most prominently expressed in keratinocytes, DRG, testis, tongue, and the brain, this channel has a wide variety of functions. However, as for its involvement in pain perception and signaling, this channel is limited to that of thermo detection and some sensitizing agents (Pedersen et al., 2005).

When TRPV1 was first identified and understood, it became apparent that it detected heat in the temperate ranges of around 43°C. However, when TRPV1 knockout cells were exposed to these same temperature ranges, a subtle response was still evoked. This led to the exploration of another member of the TRP channel family, which became known as TRPV3. This channel is expressed in similar locations as TRPV1 and can even form heteromeric channels with TRPV3 in order to modulate responsiveness (Smith et al., 2002). After more extensive study, Smith et al determined that TRPV3 seemed to have a threshold temperature of 39°C, compared to the 43°C threshold of TRPV1, thus showing a subtle difference in temperature sensing. This is very important in fully understanding how temperature is sensed in a graded way. It has been described that TRPV1 sensed temperatures in the range of about 43°C upwards to about 50°C (Caterina et al., 1997), and TRPM8 was responsible for sensing the temperature range of 22°C and lower (McKemy et al., 2002; Peier et al., 2002). Placing TRPV3 in between these two channels was essential in understanding the full range of temperature sensing. TRPV3 channels become sensitized after repeated heat exposure, similar to TRPV1 and TRPV2 (Peier, Reeve et al., 2002). These channels sensing temperatures ranging from warm to hot are found quite numerously in skin keratinocytes, thus yielding a probable gateway for sensing ambient temperatures (figure 15). By elucidating details suggesting that TRPV3 is located in keratinocytes, it must be assumed these keratinocytes must communicate or synapse somehow with the afferent nociceptors in order for the information to reach higher cortical areas. Peier and Reeve at al (2002) suggested that the probable mechanism governing this communication is via chemical mediators like ATP working on P2X3 receptors which are known to lie on the peripheral terminals of nociceptive cell bodies. “Analysis of P2X3 knockout mice show a strong deficit in coding of warm temperatures” and it has also been shown that keratinocytes release ATP, thus strengthening this hypothesis (Peier, Reeve et al., 2002). A similar study proposed that the ATP released from keratinocytes works in both an autocrine fashion on themselves as well as endocrinologically on the nociceptive terminal, stimulating an action potential which can be further propagated to higher levels (Koizumi et al., 2004). Studies performed later have suggested the keratinocytes signal upstream nerves involved in sensory transduction by way of prostaglandin E2 (PGE2) which was shown to be controlled in a calcium and cyclooxygenase-1 (COX-1) dependent manner (Huang et al., 2008).

Figure 15: A) The ramping inward current of cells expressing TRPV3 to increasing temperature. B) An increase of current is apparent when cells in a calcium rich media are exposed to increasing temperature compared to cells lacking an external calcium supply. C) A logarithmic scaled view of increasing current to increasing temperature. D) Current is evident when cells expressing TRPV3 are exposed to 48°C compared to 25°C (room temperature).

(Peier, Reeve et al., 2002)

Unlike its close relative TRPV1, TRPV3 does not respond to pH changes, osmotic stress, or capsaicin (Peier, Reeve et al., 2002; Smith et al., 2002). By studying mice deficient in the TRPV3 gene, it was determined that these mice showed a lack of responsiveness when exposed to moderate and noxious heat. These same mice still responded equally as wild type mice to any other stimulus, thus further suggesting the sole role of TRPV3 in noxious heat detection (Moqrich et al., 2005). Based on the knowledge of an existing non-specific TRPV1-3 activator, 2-aminoethyl diphenylborinate (2-APB), researchers found yet another more specific activator of TRPV3. A compound was found, diphenylboronic anhydride (DPBA), which was structurally similar to 2-APB but had a slightly different profile of activation of the channel (Chung et al., 2005). Chung et al (2005) found that DPBA evoked a sharper activation profile at lower concentrations than 2-APB, however, 2-APB only caused desensitization at its highest concentration whereas use of DPBA caused desensitization at any of the concentrations over 100µM (Chung et al., 2005).

Camphor, a naturally occurring waxy substance found in some trees, is also an effective agonist of TRPV3 channels. The location of action of camphor on TRPV3 was localized to keratinocytes instead of the sensory nerves, supporting its role in pain versus harmless stimulation (Moqrich et al., 2005). A few other common substances have also been indicated in the activation of TRPV3. Oregano, thyme, clove, and savory all are sensed due to activation of TRPV3, which is found quite prevalently in the skin, tongue, and nose (Xu et al., 2006). Xu et al (2006) found that the main ingredients in oregano and thyme (carvacrol and thymol, respectively) instigate a warm feeling along with their bitter smell and taste, thus implying that they are indeed working through TRPV3. Carvacrol and thymol not only cause a warming sensation, they also sensitize the area of application. The lipophilicity of these molecules is the probable mode of action on how TRPV3 becomes activated or sensitized (Xu et al., 2006). Arachidonic acid metabolites have been shown to activate TRPV1, but in the case of TRPV3 arachidonic acid acts through its parent, un-metabolized form to sensitize the channel to heat stimuli (Hu et al., 2006). This is a novel finding due to the fact that it now suggests TRPV3 is activated or affected in a completely separate and distinct method compared to that of TRPV1. This distinction may become useful in the understanding of how this channel is activated or which domain is involved in sensitization.

TRP Vanilloid Type 4 Channels and Pain

The last member of the TRPV subfamily involved in sensory perception and pain is TRPV4. This channel was originally found to be sensitive to osmotic gradient changes, and was later discovered that it may also play a role in mechano-stretch detection and heat. It was first noted that under hypotonic conditions, this channel exhibited a sharp increase in intracellular calcium levels, exemplifying its activation; conversely increasing extracellular osmolarity has no effect (Strotmann et al., 2000). Thus, TRPV4 can be included in the ever increasing polymodal detection of painful stimuli or inflammation states due to injury.

TRPV4 has also been implicated in sensing temperature in the range of normal physiological ranges. It was found that cells transfected with TRPV4 reacted with greater calcium influx much more vigorously at temperatures of 34°C and above, compared to cells that were devoid of the channel (Guler et al., 2002). They used patch-clamp recording to further substantiate this evidence. When dealing with temperature perception by TRPV4, the sensitivity could be modulated by decreasing the osmolarity of the medium in which the transfected cells were treated. In this case, the channels become sensitized to heat and react with a stronger response to the same temperature threshold, which was shown to persist even after the removal from or reversal of the hypoosmotic condition (Guler et al., 2002). Guler et al (2002) suggests that a finding like this shows that the mechanisms for activating the TRPV4 channel by heat and by osmolarity are definitely working via similar modes if not actually interrelated (figure 16).

Figure 16: A) Currnt evoked in cells expressing TRPV4 to continuous heat stimulation. Before the fourth response (II), cells were bathed in a hypertonic solution leading to a decreased current. Filled vertical bars represent untreated cells. The fouth response (II) showed drastic decrease in cell response when exposed to hypertonicity. B, C) An increase in cell responsiveness is seen in cells with TRPV4 when exposed to hypotonic solutions. (Guler et al., 2002)

The involvement of TRPV4 in the detection of osmolarity had given insight into the possibility of the channel being involved in mechanosensation, more specifically, that induced by pressure or stretch of membranes. This insight was due to the fact that changes in osmolarity will undoubtedly result in changes in cell sizes, leading to a stretch or change in pressure. Previous studies of knockout models lacking TRPV1-3 all still responded to pressure, therefore giving rise to the hypothesis that TRPV4 may be the best candidate for taking the title of the pressure sensor. By studying mice models that were deficient in the gene that encodes for TRPV4, experimenters were able to distinguish differences in behavior in comparison to wild type models under varying levels of pressure. In responding to pressures applied to the tail of the mice showed that TRPV4 deficient models showed a greatly decreased response to the painful stimulus (Suzuki et al., 2003). Surprisingly, the TRPV4 negative murine models that responded in a decreased response to pressure also had a diminished response to acid applied to the skin, giving insight into the possible involvement of this channel in certain acid detection (Suzuki et al., 2003). Responsiveness of TRPV4 can be sensitized by the activation of protease activated receptor-2 (PAR-2), which tends to be localized in a similar proximity as that of TRPV4 (Grant et al., 2007). Since proteases are usually found most prominently at areas of injury, it makes sense that when these mediators act on PAR-2, TRPV4 becomes sensitized. The proteases released from inflammatory response or injury can act on PAR-2 by cleaving it and creating secondary messengers and kinases which act to sensitize TRPV4 (Grant et al., 2007). This finding supports the phenomenon of post-injury sensitization to touch and pressure.

Another area of impact that TRPV4 channels have been included is that of visceral pain sensation. Like TRPV1, this channel has been suggested to be involved in visceral pain pathways due to its more frequent localization in these areas. Using TRPV4 deletion techniques and certain TRP antagonists, it has been determined that this channel is highly involved in the initiation of the pain cascade from injury in the viscera (Brierley et al., 2008).

TRP Channel Antagonism

Since the pain cascade has been understood as initiating due to the peripheral activation of TRP channel family members, actively targeting these channels by molecular antagonism could prove to be a new and alternative approach for the treatment or alleviation of pain. Understanding the complexity of TRP channels has now led to the possibility of further attempts to modulate these structures by using endogenous or synthetic agents. When talking about TRP channels and their function in sensing pain and initiating the pain cascade, one must next consider how to possibly alter the functions of these channels with hopes to attenuate pain. This approach could prove invaluable in the clinical setting as well as in the laboratory setting of trying to further understand all of the complexities behind TRP channels and, consequently, how the signal of pain is initially started. It is of interest to try and treat pain at the level of TRP channels because they are some of the most peripheral in terms of activators in the pain cascade, and this could correlate to less of an adverse or toxic effect from pharmaceutical agents. As far as targeting TRP channels for the treatment of neuropathic pain, the idea has been to attempt and reduce neural sensitization from external stimuli or inflammation which would activate the TRP channels and then stimulate the neuropathy. The main thought behind treating neuropathic pain with such a peripheral modulator such as TRP channels is the hope that by inhibiting these channels, nerves will be less active and, therefore, less likely to stimulate a damaged area of the nervous system upstream. To date, some advancement has been made in the direction of finding agents to produce analgesia via direct action upon the TRP channel family members. The next sections outline specific research that has been conducted for each subset of TRP channel family members involved in pain.

TRP Ankyrin Channel Antagonists

In terms of the TRPA subfamily, some specific antagonists have been developed which have so far proved useful in the elimination or attenuation of pain. In experiments testing the responsiveness of HC-030031 to reverse any type of hypersensitivity brought on by activators of TRPA1, it was determined that hypersensitivity was, in fact, reversed (Eid et al., 2008). They determined that oral dosing of the antagonist HC-030031 reduced both agonist induced sensitivity and chronic sensitivity in the rat model. When applied to the clinical setting, it is unknown, but some side effects could arise from oral dosing of such an antagonist. Anytime a drug is administered orally, the range of effects is more generalized. One surprising fact determined from this experiment is that HC-030031 was acting to reverse mechanical hypersensitivity via TRPA1 in both inflammatory and neuropathic pain, but it was unclear how TRPA1 was mechanistically activated and which endogenous mediators were activating the channel (Eid et al., 2008). In this instance, TRPA1 would be functioning properly; however, if there is an upstream sensitized neuropathy, activation of TRPA1 could lead to premature firing at the site of damaged nervouse tissue. If the signals from the periphery could be lessened or totally alleviated, the probability of activating the neuropathy is decreased substantially. The channel TRPA1 has been shown to mostly modulate sensations attributed with exposure to noxiously cold temperatures. Relief from pain induced by noxious cold is still an unmet demand that should be researched further, especially if TRPA1 is such a polymodal channel in that finding a noxious cold antagonist could prove useful in alleviating other painful stimuli which work through this channel.

TRP Melastatin Channel Antagonists

The Melastatin TRP channel subfamily until recently has not had many advances in antagonism as a strategy for analgesia. Recently an antagonist has been developed to work on TRPM8 channels. Although the main TRPM8 channels being targeted by this antagonist are ones located in the bladder, the evidence put forth will help elucidate further directions to be undertaken in subsequent research. The antagonist, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB), was found to successfully block the TRPM8 channel. When cells expressing TRPM8 were exposed to a known activator and compared to similar cells with AMTB, the calcium influx generated by the agonist was reversed in cells exposed to AMTB in a concentration dependent manner (Lashinger et al., 2008). Although AMTB has only a moderate potency on human TRPM8 channels, it does have a high selectivity for this channel compared to any other members of the TRP channel superfamily which could be co-localized to the primary afferent nerves (Lashinger et al., 2008). Further insight or development of more potent and efficacious drugs to act at the level of the human TRPM8 channel has yet to be identified.

TRP Vanilloid Channel Antagonists

The TRP vanilloid subfamily is one of the most well known of all TRP channel families, and thus, most research has been done in relation to this protein. In regards to advances in antagonism of the receptor, TRPV1 has been the most researched. A few examples have been shown to be effective in the alleviation or reduction in neuropathic and inflammatory pain caused from activation of the TRPV1 channel. Most notably, SB-366791 [N-(3-methoxyphenyl)-4-chlorocinnamide] has been described to reduce pain caused from bone cancers, and actually showed effectiveness at levels 10-fold below that of the concentration that was needed by morphine to alleviate the same pain (Niiyama et al., 2009). Another important indication of SB-366791 was that it potentiated the effects of morphine so that with a very small dose of SB-366791 any dose of morphine showed analgesic effects (Niiyama et al., 2009). Another antagonist SB-705498 (N-(2-bromophenyl)-N'-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolidin-3-yl)]urea) was shown to have antinociceptive actions by blocking pain induced from capsaicin, noxious heats, and acid (Gunthorpe et al., 2007). In all cases, SB-705498 worked in alleviating hypersensitivity from any of the previous listed activators of TRPV1. Some more well developed antagonists which have already be inducted into different phases of clinical trials are SB-782443, GRC6211, AZD1386, and NGD8243; all of which have been shown to target some specialized or localized pain (Lambert, 2009). TRPV1 has been a primary target of neuropathic pain treatment in that some people hypothesize that the neuropathy can be attenuated by desensitizing the pain cascade at the very beginning by applying a topical TRPV1 agonist (Dray, 2008). Another approach suggested by Dray (2008) is the application of a TRPV1 antagonist where the same end result is anticipated in that TRPV1 will be less functional and, therefore, less likely to sensitize the nerves.

TRPV2 channels had previously been shown to be inhibited by ruthenium red; however, this action was non-selective and this gave researchers a reason to find an agent more specific for TRPV2. In this process, gadolinium has been identified as a selective antagonist for TRPV2 (Leffler et al., 2007). Gadolinium was shown to block any currents that were previously created by either heat or a specific agonist of TRPV2 (Leffler et al., 2007).

As far as the TRPV3 channel subfamily is concerned, no current research has shown any advances in this field in terms of specific antagonists; however, it has been shown that the TRPV3 channels are up regulated in patients with diabetic neuropathy. A TRPV3 antagonist would therefore prove useful in the alleviation or treatment in neuropathic pain. There has been limited findings on TRPV4 with regards to antagonism, but some research has shown that like TRPV2, TRPV4 has is actively inhibited by gadolinium (Leffler et al., 2007).

Conclusion

Pain has, and always will be, a very prevalent part of life. Although pain is often associated with a negative connotation, the reality is that pain is absolutely essential to survival. Even though this ever important aspect of life is so vital, it is not fully understood. Pain still tends to elude physicians and scientists based on its multiple modes of activation and modulation. However, if the pain cascade is evaluated one section at a time, eventually this great puzzle can be solved and pain can finally be “fully” understood. Starting at the very point of the pain cascade, at the peripheral terminals of the primary afferent nociceptors is the home of transient receptor potential (TRP) channels. These channels have been studied for some time now, and it has become evident that this superfamily is of great importance in both somatosensory detection and, more importantly, nociception.

In particular, TRP channel subfamilies Ankyrin, Melastatin, and Vanilloid have been the most recognized for their participation in the conduction of painful stimuli to higher cortical regions. They are essential for initiating an action potential on the peripheral nerve terminals when exposed to a certain threshold of stimuli deemed painful. Fully understanding how these channels operate and work, from structurally significant aspects to interactions of other proteins, has proven valuable in the course of understanding pain modulation. TRP channels have recently become a hot target in possible pain therapeutics. TRP channels A, M, and V are significant players in the study of pain, including its initiation and possible attenuation, pending the development of specific and efficacious pharmacological agents. The importance of developing agents able to work along the periphery of the nervous system is very ideal in the sense that it could alleviate any type of side effects or toxicity developed from centrally acting pain killers.

In terms of the TRP Vanilloid subfamily, the treatment of pain has been suggested by application of various antagonists and even agonists such as capsaicin. The idea is that after extended exposure to such an agonist, the nerve will eventually become desensitized, pain will subside, and irrelevant signals will not be propogated upstream to activate any neuropathy (Schumacher, M. A., 2010). Schumacher (2010) also summarized the idea of combining known anesthetics with capsaicin to improve the efficacy of the anesthetic. Some other more potent antagonists like I-RTX have shown promise via intrathecal injection in the murine model for modulation of both TRPV1 and TRPA1. Many other agonists are being studied, and the outlook is promising. The TRP Melastatin family should show promise by inducing a cooling sensation after being activated by an agonist. Since TRPM8 senses cool temperatures, administering an agonist should mimic this sensation and could prove useful in some types of pain therapy.

By targeting the most peripheral source of pain at the TRP channels, not only could nociceptive pain be alleviated, but inflammatory pain and possibly neuropathic pain upstream could also be dampened. The reasoning behind this is the fact that a neuropathy (being damaged or sensitized neural tissue) can be excited by downstream currents more easily than when there is not a neuropathy. By dampening the downstream signals, the excitation of the neuropathy could be lessened and this could prove to reduce the pain associated with neuropathic pain.

All in all, pain therapeutics has entered a great new age with the finding and understanding of TRP channels. Some new directions that could prove useful are in synthesizing more specific agonist and antagonists for each channel known to be involved in pain. Besides this, it would be of importance to study the intracellular signaling that TRP channels are involved in. There may be some sort of protein already present that could be targeted to inhibit some TRP channels, and maybe activate other TRP channels. By elucidating the complex signaling involved in nociceptive terminals, it could shed light on some signaling cascade that may be targeted upstream for treating or better understanding neuropathic pain. If further developments in this field prove successful, ground breaking therapy for the treatment of pain will become available. Along with the development of new pain medications, the modulation of pain and the pain cascade will inevitably become better understood at a whole new level. With this, the administration of more addictive pain medications could possibly be alleviated and more direct therapies, with less side effects, can be prescribed.

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The Role of Transient Receptor Potential (TRP) Channels in Pain

Abstract:

Pain is something that is dealt with throughout life. Aside from the obvious feelings of hurt and discomfort that is associated with pain, it also serves as a valuable asset of survival. Without pain, one would not be able to decrease the severity of an injurious situation or learn and remember from a previous injury in order to avoid a similar problem in the future. The origin of pain, the modulation of pain, and the ascending and descending cascades of pain control have long been sought to be fully understood. In this quest, the search for possible ways to alleviate pain at different loci has proved invaluable in the clinical setting. Of recent interest has been the targeting of pain alleviation at the most peripheral activation sites of pain. Here, Transient Receptor Potential (TRP) channels have been identified as playing a critical role in the activation of the pain signal to the brain. This review covers the main subfamilies of the TRP channel superfamily that are involved in the initiation of the pain signal. TRP Ankyrin, Melastatin, and Vanilloid channel subfamilies will be of the most interest in this review due to their prevalence in sensing painful stimuli.