The detection and processing of painful stimuli in afferent sensory neurons is critically dependent on a wide range of different types of voltage- and ligand-gated ion channels, including sodium, calcium, and TRP channels, to name a few. The functions of these channels include the detection of mechanical and chemical insults, the generation of action potentials and regulation of neuronal firing patterns, the initiation of neurotransmitter release at dorsal horn synapses, and the ensuing activation of spinal cord neurons that project to pain centers in the brain. Long-term changes in ion channel expression and function are thought to contribute to chronic pain states. Many of the channels involved in the afferent pain pathway are permeable to calcium ions, suggesting a role in cell signaling beyond the mere generation of electrical activity. In this article, we provide a broad overview of different calcium-permeable ion channels in the afferent pain pathway and their role in pain pathophysiology.
Acute pain is an essential sensory input that protects individuals from harmful environmental stimuli such as heat, extreme cold, chemical irritants, and mechanical tissue damage (803, 956). Nociception alerts us to internal threats such as infections, broken bones, and torn tendons. Without the ability to feel acute pain, there would be little stopping an individual from continuing to engage in harmful behavior. This is exemplified in patients with congenital insensitivity to pain (CIP), a condition that has been linked to several different genes (214) and which has been featured in popular literature, perhaps most notably in the form of villain Ronald Niedermann in Stieg Larsson’s novel The Girl Who Played with Fire. Just like the Niedermann character, real life CIP patients are unable to feel acute pain, while maintaining a normal sensation of touch (214). Children with CIP are at risk of self-mutilation without realizing the associated tissue damage (124). An intriguing report on a group of six Pakistani children (aged 6 to 14 yr) further highlights the dangers associated with CIP. These children were completely insensitive to pain associated with physical injury (204) and, as a result, had endured a host of physical injuries such as burns and fractures. All six Pakistani children shared a null mutation in the Nav1.7 sodium channel, thus losing all ability to sense thermal and mechanical pain (204). CIP patients that survive childhood can lead productive lives, but constant vigilance is necessary for protecting against injury.
In contrast to acute, nociceptive pain, there are chronic pain conditions that do not appear to fulfill a useful physiological function, such as inflammatory and neuropathic pain (912). These painful conditions are often difficult to manage and negatively impact not only the patient’s quality of life (704), but the associated reduced ability to work also results in an economic burden that is conservatively estimated to be $600 billion in the United States alone (419). It thus remains a high priority to identify novel analgesics that target chronic (undesired) pain, while sparing an individual’s ability to detect noxious stimuli. Chronic pain involves changes in expression and/or function of a number of different types of ion channels in peripheral pain-sensing neurons and the central nervous system (CNS) (912) including upregulation of N-methyl-Daspartate receptors (NMDARs) and voltage-gated calcium channels among many others (954). Numerous ion channels contribute to the detection and processing of pain signals. A subset of these channels are permeable to calcium ions (266), which in turn mediate a host of cell signaling functions such as the release of neurotransmitters (644), the activation of calcium-dependent enzymes (330), and calcium-dependent changes in gene expression (249, 640, 946).Thus aberrant calcium signalingis a key step that alters activity of neural networks engaged in the modulation of pain; changes in these networks form the cellular underpinnings of chronic pain. Here, we review the role of calcium-permeable ion channels in the detection, transmission, and processing of pain signaling in the primary afferent pain pathway.
II. ANATOMY OF THE AFFERENT PAIN PATHWAY
Pain signaling is initiated by the detection of noxious stimuli through specialized primary nociceptors located in peripheral endings within the skin and in internal organs. The cell bodies of these neurons are contained within the dorsal root ganglia (DRG) or in the trigeminal ganglia (for cephalic sensory innervation), whereas their nerve terminals are localized in the superficial layers (laminae I and II) of the dorsal horn of the spinal cord (for DRG) or in the brain stem (for trigeminal ganglia) (67). In humans, 29 pairs of DRG (at each vertebral level) and 1 pair of trigeminal ganglia contain sensory neurons. These neurons have a peculiar morphology with a pseudo unipolar axonal projection arising from the cell body and bifurcating in two branches: one very long projection targets the peripheral receptive fields, and a second projection connects to the CNS in the spinal cord or brain stem (835) (FIGURE 1). Therefore, the vast majority of the afferent neuron is comprised of axonal structures (more than 99% of the cell membrane; Ref. 236). The distal parts detect external stimuli that give rise to action potentials propagating along the axonal fibers up to central synapses in the CNS. The role of the sensory neuron cell body in coding sensory information is less defined (236). More globally, peripheral sensory neurons convey a diversity of sensory modalities including pain and itch, discriminative touch, and perception of body muscle tension (proprioception). The classification of sensory fiber subtypes depends both on their function [i.e., conduction velocity (CV)] and on their anatomical features (such as axonal fiber size and myelination; Refs. 274, 275, 697, 1024). Fast-conducting A and A fibers (CV: A 70 –120 m/s, A 70 –30 m/s) have large calibers (5–20 m) and a large cell body (40 m). They are heavily myelinated and correspond to proprioceptive neurons (A) and proprioceptive and tactile neurons (A). Some subclasses of Afibers also support nociceptive signals (246, 281). Lightly myelinated A fibers, with a slower CV (12–30 m/s) and a smaller diameter (2–5 m) and cell body size (30 –40 m), convey tactile and nociceptive information. Finally, slow-conducting C fibers (CV 0.5–2 m/s) with unmyelinated thin axons (0.4 –1.2 m) and small soma size (25 m) are mainly involved in detecting pain and itch signals, but also participate in light touch related to tickling (447, 1024). Studies using skin-nerve preparations (727, 1023) or in vivo singleunit recording of peripheral nerve axons with microneurography (727, 771, 894, 1023) further highlight their diversity. To mirror these functional data gathered over the years, detailed description of the anatomy of the distal and central nerve endings required novel technical approaches. The use of genetically modified mice for specific labeling of fiber subtypes allowed researchers to address this issue, revealing that the structural organization of distal fibers in the skin (958) and the central fibers in the dorsal horn of the spinal cord (532) is extremely complex. Thus, based on these criteria, nociceptive neurons encompass a highly heterogeneous population of neurons with respect to their morphological, anatomical, and electrophysiological properties (507, 580, 726).
Over the past few years the molecular characterization of nociceptive neurons has been intensively explored, revealing that a number of factors/markers define specific subsets of neurons. For example, nociceptive neurons in the adult animal have been classified into two major categories according to their expression of neurotrophin receptors: 1) neurons dependent on the neurotrophin nerve growth factor (NGF) that express TrkA receptors and 2) neurons responsive to members of the glial-derived neurotrophic factor (GDNF) family that express Ret receptors (613, 614). These populations are even more diverse than just two ensembles, since they originate from distinct lineages during development with early and late TrkA or Ret neurons (50, 562). Therefore, subcategories can be separated by distinct molecular markers. Among these markers the TrkA-positive neurons express calcitonin gene-related peptide (CGRP) and substance P (SP) and are thus referred to as peptidergic nociceptors. In contrast, nonpeptidergic nociceptors are mainly comprised of Ret positive neurons (614). These two classes of neurons anatomically project to distinct laminae in the dorsal horn with TrkA fibers innervating the outermost region (lamina I) and the Ret fibers innervating distinct layers of lamina II (507, 532, 613). In addition, these two populations are not homogeneous and contain distinct cytological markers that reflect their specific roles in detecting sensory information. For example, the Ret-positive cells contain a population of neurons that express cell surface glycol conjugates that are specifically recognized by isolectin B4 (IB4) from Griffonia simplicifolia (613). A specific subgroup of Retpositive and IB4-negative cells corresponds to low-threshold mechanoreceptive C fibers that specifically express tyrosine hydroxylase and the vesicular glutamate transporter VGlut3 (532, 767). A number of studies have revealed that small nociceptive IB4 positive and negative neurons play distinct roles in pain (245, 809, 964). Going forward, it will be important albeit challenging to identify nonoverlapping molecular markers of the different subpopulations of sensory neurons and link these to specific pain responses or tactile sensations.
Some of the markers associated with specific afferent fiber populations include calcium-permeable ion channels reviewed here. The cold/menthol receptor TRPM8 (689) and the heat/ vanilloid receptor TRPV1 (138) segregate into nonoverlapping classes of nociceptors, although TRPV1 and TRPM8 coexpression has been observed in cultured neurons (237). The mustard oil receptor TRPA1 and the purinergic receptor P2X3 are predominantly expressed in IB4-positive neurons (55, 109). Neurons that express low voltage-gated calcium channels appear to be negative for -opioid receptor expression (963). Altogether, this illustrates that calcium-permeable channels can be useful markers of specific primary afferent fiber types.
Multiple other signaling proteins such as Mrgpr/SNSR class G protein-coupled receptors (GPCRs) are largely expressed in a mutually exclusive fashion (553). As another molecular twist of diversity, distinct splice variants of a single gene can be specifically expressed within a subpopulation of sensory neurons. It has been demonstrated for example that for the N-type calcium channels encoded by the Cav2.2 subunit, the expression of the exon 37a variant is restricted to nociceptive neurons and acts as a molecular switch that tailors the channel toward specific roles in pain perception and modulation by GPCRs (25, 36, 82). Deciphering how these subpopulations of nociceptive neurons are molecularly specified and functionally diversified will greatly expand the understanding of pain biology, but this also represents a challenge in many laboratories working on molecular pain physiology.
The spinal cord dorsal horn is the essential CNS sensory processing hub connecting the periphery to the brain. In this nociceptive pathway, dorsal horn neurons integrate inputs from peripheral nociceptors, local interneurons, and descending projections and transmit processed signals to the brain pain network (851) (FIGURE 1). Neurons in the superficial layers of the dorsal horn (laminae I and II) primarily receive nociceptive-specific inputs through high-threshold A- and C-fiber primary afferents. Lamina I and II neurons display considerable heterogeneity in molecular, functional, and morphological properties and can be divided into subpopulations based on their morphological, biochemical, and electrophysiological profiles (338). Excitatory and inhibitory interneurons predominate in lamina I and II, while a subset of lamina I neurons project directly to brain pain centers which include the lateral parabrachial area, the periaquedecutal grey matter, and the thalamus. Within deeper laminae of the dorsal horn (lamina V), wide dynamic range neurons respond to both innocuous and noxious inputs and project to brain pain networks through the spinothalamic tract. Combining recently developed optogenetic approaches with spinal cord imaging and recording techniques has the potential to unlock remaining mysteries regarding how innocuous and noxious sensory information is processed within the complex synaptic circuitry of the spinal cord dorsal horn during normal and pathological pain conditions.
III. MAJOR TYPES AND KEY ANIMAL MODELS OF PAIN
As stated above, nociceptive pain is primarily a protective mechanism. Acute pain is therefore a physiological phenome non that does not involve abnormal expression of ion channels and receptors. It has been investigated in clinical studies involving human patients, and in animal models with the use of pharmacological strategies, and gene knockout or overexpression experiments in rodents or simpler organisms, such as Drosophila or zebrafish (518, 519, 555, 864). Testing of acute pain responses can be achieved through application of a range of stimuli (electrical, thermal, mechanical, or chemical) that can be more or less controlled in time and intensity. Cutaneous somatic or cephalic nociception (stimulation of skin nerve endings) is by far the most widely used approach to investigate acute pain in animals, but visceral pain has also been explored through stimulation of nerve endings in hollow organs (gastrointestinal tract, bladder) (174, 325). Acute reactions due to excessive nociceptive pain can be induced by thermal and mechanical stimuli, or via chemical stimuli such as subcutaneous Formalin injection (559, 632) and by application of chemical agonists of ion channels that are involved in the detection of nociceptive signals (for example, capsaicin, mustard oil, acid). In the case of the Formalin test, the behavioral consequence of this strong tonic nociceptive stimulation is a biphasic response reflecting the initial stimulation of peripheral nociceptive sensory neurons, and a delayed second phase linked to a facilitation of dorsal horn responses (central sensitization) produced by a marked inflammatory reaction (405).