ain is one of the more complex sensory perceptions. Sensory neurons in the eye or the ear need detect only a single type of stimulus, such as light or sound. But pain-sensing neurons must detect more diverse stimuli, including heat, cold, acid, and mechanical pressure. To do this, they send out a complex web of sensory fibers, ending in specialized nerve endings known as nociceptors. Although nociceptors have been known about for more than a century, only in the last decade have researchers identified the specific ion channels that respond to noxious, potentially tissue-damaging stimuli. This is our "first molecular insight into the detection of stimuli under normal, acute pain conditions," says David Julius, of the University of California, San Francisco, whose group discovered transient receptor potential V1 (TRPV1), one of the first channels implicated in nociception.

Nociceptors reach into every tissue of the body, from the skin to the gastrointestinal tract. They arise from clusters of sensory neuron cell bodies, known as dorsal root ganglia, located at the junction between the spinal cord and the peripheral nervous system. When nociceptors are activated by painful stimuli, these neurons generate an action potential, resulting in synaptic transmission to "second-order" neurons buried in the spinal cord's grey matter. These second-order neurons in turn transmit the sensation of pain to the brain for processing and interpretation at a conscious level.

According to John Wood, who leads the molecular nociception group at University College London, there are two classes of channels involved in nociception. One class is responsible for detecting noxious stimuli and the products of tissue damage. Of the 20 to 30 TRP channels identified, at least nine are involved in these functions, in addition to members of the acid-sensing channel, or ASIC, family, and the adenosine triphosphate (ATP)-gated P2X receptor family.

© 2002 Elsevier

MEASURED RESPONSE: A sensory neuron in tissue culture exhibiting robust growth of sensory neuron processes. A microelectrode at the top touches the neuron to measure signals. A second electrode on the right imbeds in a group of keratinocytes. (from S.P. Cook, E.W. McCleskey, Pain, 95:41-7,2002.)

A second class of channels is responsible for setting the threshold at which nociceptors can generate an action potential. Under normal conditions, nociceptors have very high thresholds. They are not activated by mild sensations such as warmth or light touch, but only by harsh, potentially tissue-damaging sensations. Under pathological conditions, however, such as inflammatory pain, their thresholds become lower and they respond to much weaker stimuli. Certain newly discovered members of the voltage-dependent sodium channel family, distinguished by their resistance to the channel blocker tetrodotoxin (TTX), seem to be involved in setting this threshold.

But the complexity of the system does not lie in the number of channels alone. "Redundant molecules are involved in handling pain of any kind," says Edwin McCleskey of the Vollum Institute, Portland, Ore. These channels have overlapping sensitivities. For example, four different TRP channels respond to heat. Individual TRP channels can also respond to more than one stimulus, such as heat and acid or heat and pressure. Moreover, individual nociceptors express different channels in different combinations. Thus, channel identification is only the first step to unraveling this complex system. Researchers still have much to do to determine which channels are involved in which types of pain and where in the body, and to understand how these channels work together to produce chronic pain states.

TRP channels have emerged as the dominant class of channels in nociception, just as they have in other forms of sensory perception.¹ Julius' group cloned TRPV1 in 1997 by virtue of its sensitivity to capsaicin, the vanilloid that gives hot peppers their spice.² TRPV1 is also activated by heat and low pH. Since its discovery, three other heat-responsive channels have been identified, TRPs V2-V4. TRPM8, the receptor for cold and cooling compounds like menthol, was also cloned by Julius in 2002.³

© 2002 Elsevier

DAMAGE CONTROL: The primary afferent nociceptor responds to inflammatory mediators released at the site of tissue injury. Components include peptides such as bradykinin, lipids such as the prostaglandins, neurotransmitters such as serotonin and ATP and neurotrophins in a slightly acidic inflammatory soup. These factors can sensitize or excite nociceptor termini by interacting with cell-surface receptors expressed by these neurons. Beyond transmission of the signal, activation of these nociceptors initiates the process of neurogenic inflammation. Release of neurotransmitters such as substance P and calcitonin gene related peptide (CGRP) activates mast cells and neutrophils and induces vasodilation and leakage of capillaries. (Adapted from D. Julius, A.I. Basbaum, Nature, 413:203-10, 2001.) View enlarged diagram

Michael Caterina, who carried out the TRPV1 cloning in Julius' lab, now studies knockout mice for TRPs V1-V4 in his own lab at Johns Hopkins University. He is attempting to unravel these channels' overlapping temperature sensitivities, studying their roles in discriminating between painful heat and pleasurable warmth. So far, he says, "the degree to which these mechanisms overlap is unclear." TRPV1 is clearly associated with pain, and TRPV2 is specialized to respond only to higher temperatures. "But for V3 and V4, we don't have an answer yet," he says.

Caterina points out that all TRP channels can be activated in more than one way, suggesting that the same channel may play different roles in different tissues. TRPV2, for example, opens at temperatures greater than 52°C. Nevertheless, it is highly expressed in the lung, where such temperatures are unlikely, suggesting that it responds to other, unknown, endogenous activators. The same is likely to be true for the other TRP channels. "What we know so far is probably just the tip of the iceberg," he says.

Besides teasing out the roles of different TRP channels in acute pain sensation, researchers would like to understand how TRP channels are regulated and thus change nociceptor sensitivity. Most sensory systems adapt to stimuli, becoming less sensitive over time. "Pain is different," says Peter MacNaughton of Cambridge University. "It gets worse over time." Julius, MacNaughton, and others are studying how TRP channels are regulated in inflammatory pain. Not surprisingly, they have uncovered "a whole raft of mechanisms," MacNaughton says.

The root cause of inflammatory pain is tissue damage. Broken cells discharge their contents, releasing multiple bioactive molecules, including protons, neurotransmitters such as serotonin and ATP, peptides such as bradykinin, and neurotrophins such as nerve growth factor (NGF). These molecules bind to receptors and alter sensory neurons' sensitivity thresholds, so that previously innocuous stimuli become painful. "It's an adaptive function," says Clifford Woolf of Harvard University, "that promotes repair and healing by avoiding contact with anything."

MacNaughton is studying the signaling pathways activated by the binding of bradykinin and NGF to their receptors. He finds that TRP channel regulation involves multiple kinases, including protein kinase C and calcium-calmodulin-dependent kinase II.⁴ The exact targets of these kinases are still unknown. But, at the same time, Julius has shown that these peptides can act by a completely different pathway, stimulating phospholipase C to break down phosphatidylinositol-4,5-bisphosphate (PIP₂), releasing PIP₂ inhibition of the channel.⁵ And Lorne Mendell's group, at the State University of New York at Stony Brook, has found evidence for protein kinase A involvement.⁶ "These different views of how TRP channels are activated will need to be sorted out," says MacNaughton. It is not yet clear under what circumstances each pathway may be used.

TRP channels are generalists, sensing multiple types of noxious stimuli. The ASICs, by contrast, are specialists, responding almost

exclusively to low pH. According to McCleskey, low pH is of interest in three pain scenarios. One is bone cancer, in which high levels of acid can leak into surrounding tissues. A second is inflammation, in which the pH can drop to as low as 5.5 due to release of cell contents. A third is ischemia, in which an organ gets insufficient oxygen--for example, during a heart attack. In this case, pH drops due to the metabolites produced by anaerobic respiration, particularly lactic acid.

McCleskey has targeted ASIC3 for its role in heart attack pain. Acid was initially discounted as a painful stimulus in heart attack, in part because the pH drop is very modest, from 7.4 to 7.0. But McCleskey observed that ASIC3 is highly expressed in sensory neurons innervating the heart. He has since found that "this channel may be the most sensitive detector on the planet to detect the pH drop in heart attack." Not only does it respond to a very small pH decrease, but its activity is potentiated by other chemicals released during ischemia, including lactate and ATP.⁷

Heat, cold, and chemical sources of pain thus have multiple candidate receptors. But one major source of pain, that caused by mechanical pressure, still lacks a defined receptor. "This is the big puzzle of the moment," says Wood. There are many candidate mechanosensitive channels, including TRP channels, the DEG/ENaC sodium channels, and the two pore domain potassium channels. "Probably there will be a lot of movement in the mechanosensation area in next few years," he says.

ASICs and TRP channels are direct sensors of noxious stimuli. But other channels can also stimulate sensory neurons to signal that something is wrong. Extracellular ATP is a major product of inflammation, prompting interest in the ATP-gated P2X receptors as pain mediators. James Galligan, Michigan State University, who studies gastrointestinal innervation, suggests that P2X receptors may be especially important in visceral pain hypersensitivity, such as in inflammatory bowel syndrome. One particularly "trendy" theory, he says, is that visceral hypersensitivity often follows an acute episode of gastroenteritis. The resulting inflammation may promote changes in sensory nerves that persist for many years, causing chronic pain.

While basic researchers are trying to unravel the relative contributions of different sensory channels, those interested primarily in pain relief would like to identify "points of convergence," says Michael Gold, of the University of Maryland. He and many others are focusing on TTX-resistant sodium channels. Certain subtypes are expressed almost exclusively in peripheral sensory neurons, potentially providing a selective drug target.

Gold is studying specific pain syndromes, including temporomandibular joint disorder and migraine, to identify channels that might increase sensory neuron excitability. He and others have found that TTX-resistant sodium channels are often redistributed after tissue injury. By studying their trafficking, they may be able to develop drugs that prevent chronic pain by disrupting this redistribution. This would avoid the necessity of finding blockers selective for these channel subtypes alone.

Interest in nociceptive channels is thus likely to remain high and not just among basic researchers. "At this stage," says Julius, "any new targets are interesting for pain." This is because the most commonly used pain medications, such as opioids and non-steroidal inflammatory agents, also act on receptors outside the pain pathway, causing unwanted side effects. According to Jesus Gonzalez, of Vertex Pharmaceuticals, his company and others are placing "significant emphasis on targeting peripheral mechanisms for pain," hoping to come up with new, side-effect-free methods for controlling pain.