- freely available
Pharmaceuticals 2012, 5(3), 249-278; doi:10.3390/ph5030249
Published: 23 February 2012
Abstract: Recent discoveries indicate that many G-protein coupled receptors (GPCRs) and channels involved in pain modulation are able to form receptor heteromers. Receptor and channel heteromers often display distinct signaling characteristics, pharmacological properties and physiological function in comparison to monomer/homomer receptor or ion channel counterparts. It may be possible to capitalize on such unique properties to augment therapeutic efficacy while minimizing side effects. For example, drugs specifically targeting heteromers may have greater tissue specificity and analgesic efficacy. This review will focus on current progress in our understanding of roles of heteromeric GPCRs and channels in pain pathways as well as strategies for controlling pain pathways via targeting heteromeric receptors and channels. This approach may be instrumental in the discovery of novel classes of drugs and expand our repertoire of targets for pain pharmacotherapy.
1. Introduction: Receptor and Channel Heteromers as Therapeutic Targets for Pain
Receptors and channels are traditional targets for drug development in the battle against a variety of diseases. Current strategies for drug development assume that receptors and channels are monomers or homomers. Conventional models propose that monomeric G-protein coupled receptors (GPCRs) control the sequential activation of cellular signaling cascades and eventually channel-effectors. Similarly, channels acting as monomers (or homomers) possess defined pharmacological, biophysical and functional properties and are regulated in cell-specific ways. Evidence accumulated in recent years suggests that these models are too simplistic to explain the functional flexibility of these receptors and channels. The current view of receptor and channel organization suggests that a majority of receptors and especially channels are present as dimers or heteromers in cells [1,2,3,4,5,6]. Receptor and channel heteromer complexes are formed by direct interaction with either members of the same receptor/channel family or members of structurally and functionally differing families [7,8,9,10,11]. In certain cases, heteromerization is a requirement for the formation of functional receptors/channels. Thus, the NR1 subunit of NMDA receptors does not form glycine-glutamate-responsive channels as it requires the presence of a NR2 subunit to do so . However, a majority of receptors and channels form non-obligatory heteromers which display unique and specific pharmacological, biophysical, regulatory and functional characteristics. Therefore, heteromer complexes may be considered to be novel and distinct entities with the potential for playing unique physiological roles during normal and pathological conditions.
The aim of this review is to present advantages as well as strategies for controlling pain pathways via targeting heteromeric receptors and channels. This approach may be instrumental in the discovery of novel drugs for pain therapy. Although the occurrence of receptor and channel heteromerization is now well recognized (see reviews [1,4,7,8,13]), we are just now beginning to understand the characteristics of different receptor and channel heteromerization. Thus, the proportion of receptor/channel monomers (or homomers) versus heteromers in the plasma membrane of actual physiological cells is still unknown for the majority of receptors and channels. Furthermore, there are functional, regulatory and pharmacological outcomes of heteromerization that are beginning to be acknowledged for a set of receptors and channels. Nevertheless, this area of research is well worth the effort and investment, as targeting receptor/channel heteromers may provide a completely new therapeutic strategy for treatment of different pain conditions.
There are several reasons for engaging in drug discovery which targets receptor and channel heteromer. First, localization of heteromers (i.e., tissue and cell-specificity) may be restricted to fewer cell types than the expression of monomeric and homomeric subunits that comprise the heteromers. More specific expression patterns may provide for a reduction of side-effects in response to heteromeric selective drugs. For example, highly selective TRPV1 receptor antagonists were developed with the intent to block inflammation-induced thermal and mechanical hyperalgesia [14,15,16]. Unfortunately, the TRPV1 receptor is also involved in the control of body temperature  and several critical functions of the CNS [18,19,20]. Thus, TRPV1 receptor antagonists produced unwanted side effects such as fever . Drugs selective for TRPV1 heteromers [22,23] may potentially avoid this side-effect, as TRPV1-heteromers could express selectively in nociceptors and/or skin, but not cells engaged in other body functions . Second, there is increasing evidence that a receptor or channel heteromer is an entity with a distinct pharmacology and/or functionality [1,5,10,24,25]. Heteromers may modulate a given physiological process in a substantially different way compared to monomer/homomer subunits composing these heteromers. As a consequence to unique physiological effects, heteromer-selective drugs may not produce side effects attributed to monomeric or homomeric receptors/channels. Third, the physiological role of a heteromer may include modulation of receptor homomer function [9,26]. For example, heteromerization between TRPV1 and TRPA1 channels can substantially affect the calcium signaling pathways of TRPA1 homomers [26,27]. Thus, it is possible that the signaling pathway activated by the heteromer may be of high importance in some physiological processes, but not others, which are governed by homomers. For example, the D1-D2 dopamine receptor heteromer plays a role in LTP, while the D2–D5 heteromer’s role is restricted to control of motor activity . This underscores the potential for new therapeutic strategies specifically targeting one signaling pathway among the variety of pathways that homomer receptors/channels and their heteromers offer. Altogether, receptor/channel heteromer-selective drugs offer more flexible control of receptor/channel functions, with the potential for a lower incidence of side effects.
2. G-Protein Coupled Receptor Heteromers as Therapeutic Targets for Pain
G protein coupled receptors (GPCRs) are the largest family of cell surface receptors and are the most common targets for therapeutic drugs including drugs used for treatment of pain . Receptor activation in response to ligand binding as well as constitutive receptor activity (i.e., ligand-independent receptor activity) leads to coupling and activation of heterotrimeric G proteins which in turn regulate numerous intracellular signaling events. G proteins are grouped into four subfamilies: Gi/o, Gs, Gq/11 and G12/13. Most analgesics that target GPCRs activate Gi/o-coupled receptors including opioid, cannabinoid, alpha2-adrenergic, somatostatin, muscarinic acetylcholine, gamma-aminobutyric acid B (GABAB), and groups II and III metabotropic glutamate receptors . It is important to note that GPCRs can also couple to other signal transduction molecules in addition to G proteins.
Originally thought to function only as receptor monomers, it is now known that GPCRs also function as homo- and heter-odimeric complexes (between different receptor subtypes) and perhaps even as higher order oligomers . Here, the term receptor homomer or heteromer will be used to refer to dimeric or higher order complexes. Receptor heteromers frequently display unique signaling characteristics and pharmacological properties that differ from those of the individual protomers and thus can be viewed as distinct receptor entities [30,31,32,33,34,35]. It has been suggested that these unique or “signature” signaling and pharmacological characteristics can be used to determine receptor heteromer involvement in a given physiological function such as pain modulation . Further, an important feature of receptor heteromers from a pharmacological standpoint is that allosteric interactions can occur between the individual protomers. As a result, the presence of one protomer can alter the affinity and/or efficacy of ligands that bind to the other protomer. Moreover, conformational changes produced by ligand occupancy of one protomer can alter ligand affinity/efficacy at the second protomer [6,36,37,38]. Such allosteric interactions between protomers may contribute to the distinct signaling characteristics of receptor heteromers. Although there is evidence for heteromerization for all of the receptors mentioned above, given the significance of opioid receptors in pain modulation, here we will focus on heteromers that include opioid receptors.
2.1. Opioid Receptor Heteromers
All three major opioid receptors are involved in modulation of various pain states and are expressed in brain, spinal cord as well as dorsal root (DRG) and trigeminal ganglion (TG) sensory neurons [39,40]. There are several lines of evidence that support the existence of opioid receptor heromers. First, a pre-requisite for heteromer formation is cellular co-localization and all three opioid receptor subtypes have been found to co-express (to varying extents) in spinal cord and in peripheral sensory neurons [41,42,43]. Second, pharmacological characteristics of opioid ligands tested in vivo have not always matched those obtained with individual opioid receptors expressed in heterologous expression systems [44,45]. Many of these pharmacological differences can be accounted for by receptor heteromerization . Third, biochemical studies both in vitro and in vivo have shown that MOR, DOR and KOR form receptor heteromers with each other as well as with other GPCR targets for analgesic drugs, including cannabinoid and alpha adrenergic receptors.
2.2. MOR-DOR Receptor Heteromers
Many early pharmacological studies have shown that co-administration of DOR agonists as well as antagonists increases analgesic efficacy of morphine while decreasing unwanted side effects including tolerance and the dependence liability (for a review see ). Subsequently, studies done with DOR null-mutant (KO) mice and experiments using antisense oligonucleotides to reduce DOR expression have shown that the presence of DOR alters the analgesic efficacy of morphine [47,48]. Similarly, DOR-mediated antihyperalgesia appears to require the presence of MOR, because in MOR KO mice the analgesic effects of DOR agonists were lost . Such in vivo studies have provided evidence for interactions between MOR and DOR that appear to be important for the efficacy of opioid analgesics.
Heteromer receptor formation between MOR and DOR has been demonstrated directly in vitro with transfected COS-7 cells  and HEK 293 cells . Although there has been some controversy as to whether MOR and DOR receptors co-localize in DRG neurons and spinal cord , this debate has largely been put to rest by reports of Devi and coworkers  and Hokfelt and coworkers  showing that the opioid receptors do indeed co-localize to nociceptive sensory neurons in the DRG and spinal cord. Furthermore, heteromer formation between MOR and DOR has been demonstrated directly in spinal cord neurons , DRG sensory neurons  as well as rostral ventral medulla (RVM) neurons , a key relay nucleus for pain perception.
In heterologous expression systems, novel pharmacology and G protein coupling is observed for the MOR-DOR heteromer that is distinct from that of activation of either MOR or DOR alone. Agonist, but not antagonist, binding affinity to MOR is decreased in the presence of DOR co-expression [50,55] Occupancy of DOR with either an agonist (deltorphin II), antagonist (TIPPψ) or inverse agonist (ICI174864) enhanced the maximal binding of [3H]-DAMGO (selective MOR ligand) with little or no change in [3H]-DAMGO binding affinity [51,54]. In addition, the maximal binding of the selective DOR agonist, [3H]-deltorphin II, was increased in the presence of the selective MOR antagonist, CTOP  indicating that changes in ligand binding are reciprocal between the protomers of the MOR-DOR heteromer. In a recent study, Gomes et al.  demonstrated that the dissociation kinetics of MOR and DOR radioligands were altered by ligands for the opposite protomer. Changes in dissociation kinetics are a hallmark of allosterism [57,58] and strongly suggest that reciprocal allosteric interactions occur between the MOR and DOR protomers of the MOR-DOR heteromer which may contribute to the unique MOR and DOR ligand pharmacology of the receptor heteromer [57,58].
Distinct signaling characteristics of MOR-DOR heteromer receptor activation may also be evident in vivo. Thus, DOR antagonists have been shown to enhance MOR agonist-mediated GTPγs binding in spinal cord membranes. ß-arrestins were originally identified as molecules involved in desensitization of GPCRs following prolonged or repeated activation , which leads to drug tolerance. In contrast to the individual receptors, MOR-DOR heteromerization in CHO cells recruits β-arrestin-2 constitutively, leading to differences in monomer/homomer versus receptor heteromer-mediated ERK activation in vitro . Moreover, ß-arrestin recruitment to the MOR-DOR heteromer is disrupted by MOR or DOR ligands. These data suggest that MOR-DOR receptor heteromers adopt conformations favorable to arrestin recruitment. However, destabilization of this receptor conformation by ligand occupancy of either protomer in the MOR-DOR heteromer leads to a switch from arrestin-dependent to arrestin-independent signaling.
The kinetics of agonist-stimulated ERK activation also differs between the MOR-DOR heteromer and the individual receptors. In cells expressing MOR alone, the kinetics of DAMGO-mediated ERK activation is fairly rapid, with peak activity within 3–5 min of agonist administration. However, in cells expressing MOR-DOR heteromers, DAMGO-mediated ERK activation occurs in a sustained manner with peak activity 10–20 min following agonist administration. Similar differences in kinetics of ERK activation were found to occur with DOR agonists at DOR versus the MOR-DOR heteromer . Understanding the coupling of ß-arrestin to opioid receptors is important given that in β-arrestin-2 knock out (KO) mice, morphine-mediated analgesia is enhanced and development of tolerance is decreased . MOR-DOR heteromer receptors also appear to have altered G protein coupling preferences in comparison to MOR or DOR monomers/homomers. Agonist activation of MOR or DOR homomers/monomers typically leads to activation of pertussis toxin sensitive Gi/o proteins. However, in given cell types, activation of MOR-DOR heteromer receptors have been shown to preferentially activate the pertussis toxin insensitive G protein, Gz [50,62,63].
Changes in functional responses also occur upon MOR-DOR heteromerization. In an elegant study by Lakshmi Devi’s group, a potential role for MOR-DOR heteromer receptors in chronic pain has been identified . Using a subtractive immunization strategy, monoclonal antibodies were generated that selectively recognize MOR-DOR heteromers but not individual MOR or DOR. The antibody was able to block heteromer-mediated ligand binding and signaling, but not binding and signaling at MOR or DOR expressed singly. Interestingly after chronic, but not acute, morphine treatment immunocytochemical staining with the heteromer-selective antibody was increased in key areas of the CNS (RVM) as well as in the periphery (DRG), suggesting that chronic morphine increases the number of MOR-DOR heteromers in areas that are involved in pain processing. Given that MOR-DOR heteromer receptors constitutively recruit and signal via arrestins, these data suggest that increased expression of heteromers may contribute to the development of tolerance under prolonged opioid (e.g., morphine) treatment conditions. Antibodies targeting GPCR heteromers will be a very useful tool to investigate endogenous heteromer involvement in effects of opioid analgesics.
In addition to antibodies, ligands have been developed which appear to selectively target MOR-DOR heteromer receptors. Portoghese and colleagues have developed a series of bivalent ligands called MDANs ((M)-delta-opioid receptor (D)-agonist (A)-antagonists (N) which contain a MOR agonist (oxymorphone) connected, through varying spacer lengths (16–25 atoms), to a DOR antagonist (naltrindole) . These bivalent ligands have higher affinity for MOR-DOR receptor heteromers than monomer/homomers and are more potent than morphine for producing antinociception in the tail flick assay. Interestingly, neither tolerance nor dependence developed for ligands with spacers-19 and -21 in length whereas tolerance/dependence did develop in response to ligands with shorter spacer lengths . Importantly, the effects on antinociception by these bivalent ligands are consistent with findings (discussed above) that ligand occupancy of both protomers in the MOR-DOR heteromer leads to a switch in signaling mechanisms such that antinociception is enhanced, but the development of tolerance, dependence and also perhaps rewarding behavior is decreased. In summary, the distinct signaling properties of MOR-DOR heteromers suggest that these receptor heteromers may be important therapeutic targets for treatment of chronic pain with a reduction in both tolerance and dependence liabilities.
2.3. DOR-KOR Heteromers
The first direct demonstration of opioid receptor heteromer formation was between DOR and KOR in vitro . In vivo, the DOR-KOR heteromer is expressed in spinal cord [67,68] and trigeminal ganglia (TG) sensory neurons . For DOR-KOR receptor heteromers, simply the presence of one receptor can alter the affinity and/or efficacy of a ligand at the second receptor [66,67]. Further, the ligand-receptor conformation of one protomer in the DOR-KOR heteromeric pair can influence the affinity and/or efficacy of a ligand at the other protomer [68,69,70,71]. In addition, functional responses can be either enhanced or reduced depending upon the DOR-KOR ligand pairs. For example, in a recent study by Berg et al. (2011) the selective KOR antagonist, nor-BNI, differentially altered the potency and/or efficacy of DOR agonists, depending upon the specific DOR agonist used .
Several ligands have been developed which recognize DOR-KOR heteromer receptors. Although originally developed as a KOR selective ligand, 6'-guanidinonaltrindole (6'-GNTI), has been reported to preferentially activate DOR-KOR heteromers [67,72]. In rats, 6'-GNTI produces analgesia only when administered into the spinal cord and not into brain . Further, the antinociceptive potency of 6'-GNTI is greatly reduced in DOR KO mice . In addition to activity in the spinal cord, 6'-GNTI was also found to produce antinociceptive responses acting in the periphery. In the rat hindpaw model of thermal allodynia, 6'-GNTI produced a more profound reduction in PGE2-mediated-allodynia than that seen with DOR agonists  or KOR agonists . Further, the analgesic response to 6'-GNTI is blocked by both selective DOR and KOR antagonists, suggesting preferential targeting of DOR-KOR heteromer receptors in vivo . Taken together, these data are consistent with the idea that DOR-KOR heteromer receptors may be tissue-specific as well as able to be activated selectively.
A bivalent antagonist as well as a bivalent agonist have also been developed which preferentially interacts with DOR-KOR heteromer receptors [68,71]. The bivalent antagonist, KDN-21, which has antagonist properties at both DOR and KOR, displays the highest binding affinity by an order of magnitude in membranes prepared from cells co-expressing both DOR and KOR . The bivalent ligand, KDAN-18, which has KOR agonist activity but antagonist properties at DOR, also displays higher affinity, by approximately 100-fold, in membranes from cells co-expressing DOR and KOR receptors .
In addition to selective ligands, using a subtractive immunization strategy, antibodies which recognize selectively DOR-KOR heteromers have also been generated. These monoclonal antibodies recognize an epitope in cells co-expressing DOR-KOR receptors, but not other heteromer receptors (e.g., MOR-DOR), or either DOR or KOR alone . Interestingly, in vivo, this antibody, which had no effect on its own, was found to potentiate the antinociceptive effects of the DOR agonist, DPDPE, in a rat model of thermal allodynia . In the presence of this antibody, a sub-threshold (i.e., ineffective) dose of DPDPE became capable of not only inhibiting thermal allodynia, but producing close to the maximal possible antinociceptive response in this system.
2.4. MOR-KOR Heteromers
There are several reports that MOR can form heteromers with KOR, and that MOR-KOR receptor heteromers can modulate pain, particularly with drug administration into the spinal cord [75,76]. Heteromer receptor formation between MOR and KOR was demonstrated directly, in rat spinal cord membranes, using receptor subtype selective antibodies and co-immunoprecipitation techniques .
A selective ligand N-naphthoyl-à-naltrexamine (NNTA), which preferentially targets MOR-KOR receptor heteromers has been developed by Portoghese’s group . NNTA was found to increase both intracellular calcium release and GTPγS binding with 1,000-fold enhanced potency in cells expressing MOR-KOR receptor heteromers versus cells expressing KOR alone. Interestingly, NNTA was found to be an antagonist in cells expressing MOR alone. NNTA also produces potent antinociception in the mouse tail flick assay . The NNTA-mediated antinociception was more than 100-fold greater when the ligand was administered intrathecally in comparison to intracerebroventricular administration. These data suggest that a ligand such as NNTA may be particularly effective for spinal antinociception.
MOR-KOR heteromer receptors may provide an important analgesic target in women . It is known that women are more likely than men to experience more severe levels of pain, with longer duration, as well as more recurrent pain . Further, it has been suggested that females may respond better than males to analgesics targeting KOR [79,80,81]. Interestingly, spinal morphine antinociception in female, but not male, rats requires the simultaneous activation of spinal MOR and KOR receptors . Studies suggest that the mechanism for enhanced morphine responses in female rats is formation of MOR-KOR receptor heteromers . Further, MOR-KOR receptor heteromers appear to be regulated by female sex hormones since they are more prevalent during proestrous versus diestrous . In addition, estrogen may be necessary for MOR-KOR heteromer formation in spinal cord, because blockade of membrane estrogen receptors reduced the abundance of MOR-KOR receptor heteromers detected as well as abolished KOR-mediated enhancement of spinal morphine antinociception .
2.5. MOR-Alpha Adrenergic2A Receptor Heteromers
The analgesic profile of α2 adrenergic receptor agonists has been well established . There are three subtypes of α2 adrenergic receptors, -2A, -2B and -2C. Analgesic responses to α2 agonists, particularly in the spinal cord, are mediated primarily by α2A receptors [84,85]. Results of early pharmacological studies suggest that significant functional interactions occur between MOR and α2Areceptors occur in spinal cord . For example, intrathecal injection of sub-threshold doses of morphine were found to enhance the effects of clonidine and vice versa . The combination of a subcutaneous morphine injection along with intrathecal clonidine administration produced significant antinociceptive effects, whereas either drug administered alone had no effect . Further, MOR-mediated analgesia is also enhanced by the presence of α2 receptors suggesting that effective MOR-mediated spinal analgesia is dependent upon the presence of α2A receptors .
Heteromerization between MOR and α2A receptors has been demonstrated in transfected cells as well as in primary neuronal cell cultures [89,90,91]. In vitro, signaling is increased in response to morphine in cells co-expressing receptors in comparison with cells expressing MOR alone , which suggests that heteromerization alone may lead to enhanced antinociception in response to morphine. Interestingly, differences in MOR-α2A receptor heteromer signaling profiles are observed in response to single drug administration versus co-administration of morphine and clonidine . In membranes obtained from primary cultures of rat spinal cord neurons (which co-express both receptors), GTPγS binding was reduced in the presence of both morphine and clonidine relative to morphine alone . Overall, these data are consistent with the idea that agonist occupancy of both protomers in the MOR-Aalpha2A heteromer leads to a decrease in signal transduction whereas activation of one protomer in the complex leads to enhanced responsiveness.
2.6. DOR-Alpha Adrenergic2A Receptor Heteromers
Although no direct demonstration of heteromerization between DOR and α2A receptors has been reported, it is important to note that functional interactions between DOR and α2A receptors have been shown. For example, co-incubation with the DOR agonist, deltorphin II along with clonidine produced a greater than additive reduction in K+-stimulated neuropeptide release from synaptosomes prepared from rat spinal cord . Further, DOR and α2A receptors co-localize in rat dorsal horn . Therefore, along with MOR-α2A receptor heteromers, its quite possible that DOR-α2A receptor heteromers in spinal cord may play a significant role in pain modulation.
2.7. Cannabinoid-Opioid Receptor Heteromers
Activation of either subtype of cannabinoid receptor, CB1 or CB2, which are found in areas of the brain and in the periphery involved in pain processing, can have profound antinociceptive effects (for review see [93,94]). CB2 receptors have been shown to homodimerize . However, there are no reports of receptor heteromers with CB2 receptors and other GPCRs known to be involved in pain modulation. In contrast, biochemical and pharmacological data have shown that CB1 receptors can form receptor heteromers with each of the three opioid receptors in vitro . Although direct evidence for heteromers between CB1 and opioid receptors has yet to be determined in vivo, CB1 receptors do co-localize with MOR in the dorsal horn of the spinal cord [97,98] as well as in brain regions which play an important role in antinociception (e.g., the periaqueductal gray) [99,100].
Results from a number of pharmacological studies in vivo suggest that CB1 receptors may interact with MOR in a synergistic fashion to enhance antinociceptive responses. Thus, co-administration of the CB1 receptor agonist, delta-9-tetrahydrocannabinol (THC) synergized with morphine to enhance the antinociceptive response to a thermal stimulus in the tail flick assay in mice . Further, co-administration of low doses of both CB1 and MOR agonists produced a synergistic enhancement of antinociception allowing for agonist dosing to be kept well below those which produce analgesic tolerance .
2.8. MOR-GRPR (Gastrin-Releasing Peptide) Receptor Heteromers
Scratching behavior in mice induced by diverse chemical and pharmacological agents has been shown to be mediated by activation of gastrin-releasing peptide receptor (GRPR) in the spinal cord [103,104]. The most common side effect of opioids, such as morphine, is the sensation of itch which is particularly prominent after spinal administration. Until recently, it was thought that the onset of itch was linked to the same mechanisms as the reduction in pain by morphine. However, a recent report showed that not only are the analgesic effects of morphine separable from the sensation of itch, but that morphine-induced itch is due to activation of heteromers between GRPR and MOR-1D, a splice variant of MOR, localized in spinal cord . Direct protein-protein interactions were demonstrated to occur between MOR-1D and GRPR in HEK cells as well as in native spinal cord membranes . Selective reduction of MOR-1D receptor expression by siRNA knockdown reduced morphine-induced scratching in mice whereas morphine-induced analgesia remained unaffected . Further, blocking formation of MOR1D-GRPR receptor heteromers by injection of a TAT fusion peptide (TAT-MOR1Dct) into spinal cord led to reduction of morphine-induced itch but had no effect on morphine-induced analgesia. In addition, morphine-induced scratching, but not antinociception, was blocked in the presence of a GRP receptor antagonist. Taken together, these data strongly suggest that activation of MOR1D-GRPR receptor heteromers by morphine in mice mediates itch whereas activation of MOR mediates antinociception.
3. Ligand-Gated Channel Heteromers as Therapeutic Targets in Pain Pathway
Except for certain pore-forming peptides, all ion channels function as multimers, composed of varying numbers of subunits. Thus, ligand-gated channels can function as homomeric or heteromeric. These multimers form either tetrameric or pentameric structures. Throughout the last decade, the functions of these heteromeric channels in pain pathways were extensively studied. In this section, we will provide an overview of the functional and regulatory consequences of heteromerization for ligand-gated channels that have been implicated in the pain pathway. It should be noted here that a majority of voltage-gated channels also function as homo- or heteromers. However, their roles in the pain pathway have not been assessed in detail as of yet.
3.1. GABAA Channels
Classical “gate control theory” of pain proposed by Melzack and Wall postulated that inhibitory dorsal horn neurons would control nociceptive signals coming from the periphery . Time has passed and the nature of this inhibitory control has been attributed to γ-aminobutyric acid (GABA) and glycine channels. Subsequently, it was demonstrated that pharmacological blockade of GABA or glycine channels in the dorsal horn provokes pain in both animals and humans . It has also been shown that a reduction in GABAergic and glycinergic neurotransmission occurs during inflammatory and neuropathic pain . Accordingly, facilitation of inhibitory neurotransmission in the dorsal horn could form the basis for the treatment of chronic pain (i.e., antihyperalgesia and allodynia). As such, GABAA receptors are among the most successfully exploited drug targets. Benzodiazepine-sensitive GABAA receptors are expressed in pivotal structures of the pain pathway, including sensory neurons as well as the superficial layers of the dorsal horn . Local administration of diazepam or midazolam is antinociceptive for animals  and is successfully utilized in human patients . However, this approach is not without its drawbacks, as there are severe side effects associated with GABAA agonists . What are possible strategies in overcoming these difficulties? One feasible approach could be based on the hypothesis that subtypes of GABAA receptors involved in anti-hyperalgesia differ from those GABAA receptors involved in other CNS functions. Indeed, GABAA is a classic example of a channel-heteromer, which cannot function as a monomer or homomer. GABAA channels are heteropentameric chloride channels assembled from a repertoire of α, β, γ, δ, ε, θ, π, and ρ subunits. Benzodiazepine-sensitive GABAA channels are assembled from two α (α1, α2, α3 or α5) subunits, two β subunits, and one γ2 subunit. All benzodiazepine-sensitive GABAA channels contain a conserved histidine residue in the N-terminal domain, while the benzodiazepine-insensitive subunits (α4 and α6) carry an arginine at the same site . Rudolph and Mohler used an elegant approach to attribute the different in vivo actions of benzodiazepines to molecularly defined GABAA receptor subtypes . They generated knock-in point mutations for all four benzodiazepine-sensitive GABAA receptor α subunits (α1(H101R), α2(H101R), α3(H126R), and α5(H105R)). Using these mice, it was demonstrated that the spinal antihyperalgesic effects of diazepam were mediated mainly through GABAA channels containing α2, but not α1, α3 and α5 subunits [115,116]. Furthermore, analysis of these mice and mice carrying mutations in β1 and β3 subunits also indicated that the GABAA subtypes responsible for antihyperalgesia were different from those that mediate sedation . This is an important observation, as these subtypes could provide the foundation for benzodiazepine-site ligands with a better side-effect profile. At present, the two relevant questions are: (1) Can other unwanted effects besides sedation also be avoided with subtype-selective agonists? (2) Does the concept of a benzodiazepine-mediated antihyperalgesia also work in humans? Both questions are at present difficult to answer. The second question has to await the availability of subtype-selective agonists suitable for clinical trials in humans. A subtype-specific GABAA agonist L-838,417 possesses undesirable pharmacokinetics in man, while TPA023 and SL651498 appear more favorable . It is, however, not yet clear whether these compounds would be suitable for human pain studies. TPA023 has low intrinsic activity , while SL651498 activates α1 subunits and could cause significant sedation when given at antihyperalgesic doses . The question of whether other relevant side effects apart from sedation such as tolerance development, addiction, and memory impairment can be avoided with subtype-selective agonists is also not yet resolved. Nevertheless, some potentially important information was obtained from studies on the function of differing GABAA heteromers. Analysis of α1 (H101R) point-mutated mice demonstrated that facilitation of GABAAα1 subunits underlies the amnestic effects of benzodiazepines . However, hippocampus-dependent learning also involves α5 GABAA receptors . Physical dependence does not occur with several α1-sparing agents . L-838,417 tolerance against antihyperalgesia was completely absent during a 9-day treatment period—in striking contrast to morphine, which had lost its analgesic activity after 6 days . In summary, recent evidence obtained either with genetically modified mice or with subtype-selective GABAA ligands indicates that the antihyperalgesic activity of GABAA channels could be potentially separated from other unwanted side effects of the receptor. Novel subtype-selective GABAA agonists are in development which not only lack undesired sedation but also avoid the other GABAA agonist side effects such as memory impairment, physical dependence, and addiction.
3.2. Nicotinic Channels
It has recently been recognized that antagonists against nicotinic acetylcholine receptors (nAChRs) are not only able to generate acute analgesia, but are also capable of producing long lasting anti-hyperalgesia . nAChRs are classic heteromeric-pentameric channels. However, unlike GABAA channels, they can function as homomers. nAChRs are assembled from one or more α subunits (α1–α10) either alone or together with one or more non-α subunits (β-subunit, (β1–β4), γ, δ or ε) . nAChR subunits may be divided into two broad classes. Alpha subunits have a defining “cysteine loop”, whereas non-alpha subunits lack this cysteine loop. Alpha subunits may be further divided into two groups: one group consists of α7, α9 and α10 subunits which are all potently blocked by α-bungarotoxin . Both the α7 and the α9 subunit can assemble into a functional homopentamer, while the α10 subunit has only been functionally co-expressed with an α9 subunit [127,128]. Individual subtypes of nAChRs each have unique expression patterns as well as pharmacological and biophysical properties. This characteristic allows for the possibility that a variety of nAchR heteromers and homomers could each have distinctive physiological functions [128,129]. Therefore, subtype selective compounds may have distinct therapeutic applications with a restricted set of side effects. Originally, the generation of mice lacking one or more subunits of nAChRs has been utilized to examine the function of different nAChR subtypes. However, this approach is less effective, as one particular subtype of nicotinic channel can be involved in many types of nAChR. For example, there are α4β2, α4α5β2, α4β4 and α6α4β2β3 nAChR’s, deletion of the α4 nAChR subunit would eliminate all of these nAChR subtypes. This illustrates why compounds that can differentiate between these individual receptor subtypes are urgently needed. Compounds can be either chemically synthesized or isolated as toxins from different animals. In the case of nAChRs, nature has presented a unique opportunity. A bio-diverse lineage of the cone snail (500–700 different species) produces an exogenous family of toxins targeting subtypes of the nAChR family. These toxins comprise the α-conotoxin family, which contain >1,500 different α-conotoxins. Careful combination of molecular genetics, peptide chemistry and molecular phylogeny allowed exploration of the subtype selectivity of this large inventory of α-conotoxin sequences. During the last decade, a significant number of subtype specific ligands for individual nAChR isoforms were discovered. There are already detailed and comprehensive reviews on this subject . In this review, we will focus on subtype-specific α-conotoxins and other AChRs antagonists which are involved in the pain pathway. Chronic injury leads to significant over-expression of key subunits including the α4, 5 and 7 subunits. This has supported the therapeutic potential of AChRs in pain control . Thus, nicotinic channel agonists which are selective for the α4, 5 and 7 subunits (epibatidine, ABT-594), particularly compared to α4β2, have demonstrated antinociception in several models of pain including neuropathic pain. Several sites of action have been proposed including supraspinal sites via descending pathways and actions on sensory neurons. Earlier clinical evaluation of ABT-594 revealed inadequate tolerability, but more recent compounds such as TC6499 (GSK/Targacept) and ABT-894 (Abbott) are progressing forward for their use in diabetic neuropathy pain, and are currently in clinical phases 1 and 2, respectively . α-Conotoxin RgIA is the most selective α9α10 antagonist yet reported . One additional peptide that blocks α9α10 nAChRs is α-conotoxin Vc1.1 (or ACV1) . The subcutaneous or intramuscular administration of α-Conotoxin RgIA and Vc1.1 acutely alleviates pain resulting from traumatic, inflammatory, or metabolic neuronal injury . Thus subcutaneous administration of Vc1.1 in rats dose-dependently reverses chronic constriction injury-induced mechanical allodynia by approximately 54–80%, with higher concentrations having an extended effect lasting up to 24 hours post-administration . α9α10 nAChR antagonists also show anti-hyperalgesia in the partial sciatic nerve ligation model and diabetic neuropathy . Vc1.1 is an effective analgesic against pain resulting from a purely inflammatory insult generated by Complete Freund’s Adjuvant (CFA). Recent reports suggest that the anti-hyperalgesic effects of α-conotoxins Vc1.1 and RgIA could be mediated via activation of GABAB receptors in DRG [136,137]. Yet other reports have shown that these toxins are not able to directly activate GABAB receptors, as they have no homology to nAChRs . But what are the possible off-target effects for these toxins? α-conotoxins, as charged peptides, are unlikely to cross the blood-brain-barrier in significant quantities and intrathecal administration of α-conotoxin RgIA is not analgesic in spinal nerve ligated (SNL) rats . Both α9 and α10 nAChR subunits are expressed in a wide range of peripheral tissues such as sensory ganglia, heart, skin, nasal epithelium, immune cells and so on . Precise off target actions for α9α10 nAChR-specific drugs are unknown. However, a role for α9α10 nAChR in anti-hyperalgesia was suggested . In summary, α-conotoxins Vc1.1 and RgIA as α9α10 nAChR-specific antagonists have unique pharmacological actions and analgesic effects that may serve as models for the development of additional nicotinic channel subtype-specific drugs for pain research and management.
3.3. Glutamate-Gated Channels
The excitatory transmitter glutamate plays an important role in the initiation and maintenance of chronic pain conditions. Glutamate acts through ionotropic (iGluRs) as well as metabotropic receptors. Glutamate-gated channels can be classified into three groups: N-methyl-D-aspartate (NMDA) channels, alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) channels and kainate channels [139,140]. Injections of NMDA, AMPA, and kainate produce a pro-nociceptive response, whereas the administration of iGluR antagonists attenuates pain [141,142]. Molecular cloning has identified that GluR1-4 (or GluRA-D) make up the AMPA receptors, GluR5-7 and KA1-2 comprise the kainate receptors, and NR1 together with NR2A-D form the NMDA receptors . Expression studies in various heterologous systems demonstrated that iGluR operate as tetramer homomeric or heteromeric complexes , although pentamer complexes were also suggested . The AMPA and kainate receptors can form homomeric receptors, while the NMDA receptors are obligate heteromeric receptors composed of homologous NR1, NR2, and/or NR3 subunits. A large fraction of the neuronal NMDA channels are composed of glycine binding NR1 and glutamate binding NR2 subunits . It was suggested that NMDA channels have a dimer arrangement, with each dimer having one glycine binding subunit and one glutamate binding subunit . The NMDA channel heteromers have distinctive biophysical, pharmacological and physiological properties . Thus, replacement or supplementation of NR2B by NR2A during development has been implicated in the acceleration of NMDA-EPSC decay, a phenomenon often linked with the ability of neuronal circuits to exhibit experience-dependent synaptic plasticity . This type of change was described in many areas of the brain, including visual cortical cells, cerebellar granule cells, hippocampal CA1 pyramidal cells and the anterior neostriatum. All of these cells display a change in the NMDA-EPSC kinetics consistent with a decreasing contribution of NR2B subunits and an increasing synaptic involvement of NR2A, which is responsible for faster NMDA-EPSC. These changes also lead to the significant alteration of Ca2+ signaling that could affect the synaptic conductance. How could these NMDA heteromer specific drugs be employed in pain management? NMDA antagonists show robust attenuation of pain but come with a number of side-effects (sedation, confusion, and decrease motor coordination) and thus appear to have insufficient therapeutic margin. In an attempt to avoid these side-effects, specific blockers of NMDA receptor subtypes (NR1 and NR2) are being developed. Consequently, antagonists against glycine sites could modulate the NMDA channel during sustained receptor stimulation found to occur during chronic pain. Accordingly, selective NR1-glycine site antagonists reduce pain with reduced side-effects . Unfortunately, clinical experience with this compound, GV196771 did not show efficacy against clinical pain . The NR2B receptor has a specific distribution in sensory pathways and as mentioned slows NMDA current kinetics . Blockade of this receptor has also been reported to produce antinociception (ifenprodil, traxoprodil, CP-101606) with reduced side-effects . To date, traxoprodil has advanced into phase 1 safety and efficacy studies for acute ischemic stroke and there is as of yet little information about possible developments in its use in treating pain. However, other NR2B projects including RGH 896 and EV101 have reached phase 2 and were evaluated for cognition and neuropathic pain . The roles of AMPA and kainate channel heteromers in the pain pathway have yet to be studied. However several antagonists against AMPA and kainate channels were implicated in control of nociceptive processing during chronic pain conditions [153,154].
3.4. P2X Channels
P2X channels contain seven members and belong to the non-selective Ca2+-permeable cation channel family . The P2X channel typically functions as a homomer. The number of subunits of a P2X channel has been proposed to be three  or four . However, P2X can form at least four heteromeric complexes [158,159]. Homomeric and heteromeric P2X channels display different electrophysiological and pharmacological properties . P2X4 and P2X7 were implicated in pain signaling in the dorsal horn [161,162], where activated microglia after nerve injury increased the expression of P2X4 and P2X7. The P2X4 and P2X7 receptors are activated by ATP which is presumably released from primary sensory neurons. P2X receptor activation causes a rise in the intracellular calcium, along with the release of diffusible factors, such as BDNF and other pro-inflammatory cytokines and chemokines. This affects excitatory and inhibitory neurons in the spinal cord, increasing the net hyper excitability of the dorsal horn in the pain network and may be responsible for neuropathic pain. So far, P2X4 or P2X7 heteromerization between other P2X channels and their respective role in the activation of microglia has yet to be reported. Furthermore, roles in pain and other physiological process of reported P2X heteromers are not yet clear.
3.5. TRP Channels
TRP channels are comprised of multiple subfamilies, including TRPC, TRPM, TRPV, TRPP and TRPN . TRP channels have unique characteristics in terms of cell-type expression patterns , modes of activation , pharmacological profile and biophysical properties including both voltage-dependency and ion permeability . It is now accepted that TRP channels function as homo-tetramers [7,24,166,167]. Accumulated evidence also demonstrates that TRP channels of the same subfamily are able to form heterotetrameric channel complexes . The formation of various TRP channel complexes of TRPC [168,169,170], TRPV [23,171,172,173], TRPM , or TRPP  channels has been demonstrated. Some of these heteromers could be formed in neurons involved in nociceptive signal transmission [7,10,172].
In humans and non-human primates, TRPV1 and TRPV3 are co-expressed in sensory neurons of DRG [172,175]. Like many known heteromers, the TRPV1/TRPV3 heteromer exhibits unique biophysical and functional properties [24,173,176]. Homomeric TRPV1-V4 channels assemble into a tetramer by interacting via their C-terminal domains . However, it appears that this region does not play a critical role in heteromerization between TRPV1 and TRPV3 . TRPV1/TRPV3 heteromeric channels are different from TRPV1 and TRPV3 homomeric channels in regards to sensitivities and dynamic range to thermal and chemical stimuli, but are similar in regards to changes in transmembrane voltage . These observations indicate that they serve as distinct cellular sensors to thermal and chemical stimuli, and possibly mediate different sensory responses to a variety of stimulants. Like other heteromeric channels, temperature-sensitive TRPV channels are known to be allosteric proteins . Cooperative gating among subunits shifts the sensitivity and dynamic range exhibited by subunits in homomeric channels. These properties must be carefully considered when examining the response of native neurons to heat and agonists.
Recently, formation of a heteromeric complex in sensory neurons between TRP channels belonging to different subfamilies (TRPA1 and TRPV1) has been revealed [10,22]. The heteromerization and subunit composition can influence the biophysical and regulatory properties of the resulting channel complex , revealing unique pharmacological properties [178,179]. This includes altering the stability of receptors on the plasma membrane by governing the trafficking properties of the receptors [180,181,182]. Desensitization of receptors can also be influenced by complex formation [25,183,184,185]. The sensitization modes of receptors by different cell stimulants (such as inflammatory mediators) could be regulated in a unique way within these heteromeric complexes. Based upon these findings, it is possible that a functional interaction between TRPA1 and TRPV1 channels may occur via a variety of pathways within a heteromeric complex [185,186,187,188,189].
Regulation of TRP channels within homomers by a variety of extracellular and intracellular soluble factors has been investigated in detail. The physiological roles of a majority of TRP channels are well described and assessed. However, the regulation and especially the physiological functions of heteromeric TRP complexes are less understood compared to other ligand gated channels discussed in this review. Most notably the TRPA1/TRPV1 heteromeric complex formed in the plasma membranes of sensory neurons and found in pain pathways . Here we will focus on the regulation and possible functions of this heteromeric complex. A stoichiometry of the TRPA1/TRPV1 complex is unknown, and the percentage of sensory neurons in trigeminal (TG) and dorsal root ganglia (DRG) expressing functional TRPA1/TRPV1 heteromers is also unclear. However, it is presumed that all TRPA1 and TRPV1 co-expressing sensory neurons contain functional TRPA1/TRPV1 heteromers. It has been shown that the percentage of TRPA1 and TRPV1 co-expressing neurons is up-regulated by inflammation and NGF . At physiological and un-inflamed conditions (Vh = −60 mV; 2 mM extracellular Ca2+) the biophysical properties of TRPA1 is regulated by the presence of TRPV1 channels [10,22,27]. TRPV1 also controls the density of functional TRPA1 channels on the plasma membrane . It is still not clear whether TRPA1 density is controlled by the TRPV1 channel on a transcriptional, translational and/or post-translational level. TRPV1 can also modulate the pharmacological properties of the TRPA1 channel. Thus, the TRPA1 agonists AM1241 (synthetic cannabinoid activating CB2 receptor) and AM630 (synthetic antagonist for CB2) are more potent in the presence of TRPV1 [178,179].
There are several possible mechanisms underlying the functional interaction between TRPA1 and TRPV1 channels. First, capsaicin and mustard oil pretreatments result in pharmacological [191,192] and functional [193,194] cross-desensitization between TRPA1 and TRPV1. Mustard oil desensitizes TRPV1 via a Ca2+-dependent mechanism involving the Ca2+-dependent phosphatase, calcineurin [25,192]. Mustard oil triggers Ca2+-influx into sensory neurons and activates calcineurin, which then dephosphorylates and desensitizes the TRPV1 channel. Desensitization of TRPA1 by capsaicin is also Ca2+-dependent , but this process employs Ca2+-evoked depletion of PIP2 . Additionally, in TRPA1-TRPV1 containing sensory neurons, it appears that TRPV1 also governs desensitization of TRPA1 by inhibiting the internalization process for TRPA1 . However, it is still not clear whether heteromerization between TRPA1 and TRPV1 can directly contribute to desensitization between these channels. Nevertheless, such cross-desensitization between TRPA1 and TRPV1, which plays a critical role in inflammatory hyperalgesia, could have therapeutic implications. Thus, inhibition of sensory neurons via certain cannabinoids (including WIN55, 212-2, AM1241) activating TRPA1 can lead to functional desensitization of the pain processes in sensory neurons mediated by TRPV1 [178,185].
Functional interactions between TRPA1 and TRPV1 channels may take place within a heteromeric complex. There are three possible pathways that may underlie this interaction. (1) Bradykinin (BK) can indirectly activate TRPA1, possibly via generation of diacylglycerol (DAG) accumulation . TRPV1, as a component of the complex, may act as a modulator that is responsible for sensitization of TRPA1-mediated BK responses; (2) The pharmacological desensitization of TRPA1-mediated responses in sensory neurons lacking TRPV1 is more pronounced . Therefore, it could be suggested that the absence of TRPV1 in sensory neurons may lead to a faster desensitization of BK responses in sensory neurons, and this, in turn, may suppress the development of inflammatory hyperalgesia. Indeed it was reported that an increased rate of functional desensitization of BK-induced action potentials was found in TRPV1 KO mice peripheral sensory fibers ; (3) Finally, TRPA1-mediated BK responses in sensory neurons could be modulated by a combination of TRPV1 and [Ca2+]i. Thus, [Ca2+]i may influence ligand affinity and/or signaling or other properties of a TRPA1-TRPV1 complex. In summary, to understand the mechanisms underlying functional interaction between the TRPA1 and TRPV1 channels, TRPA1 and TRPV1 in vitro and in vivo characteristics need to be studied in the presence and absence of TRPV1 and TRPA1 channels, respectively.
4. Approaches To Design Inhibitors for Heteromer Receptors and Channels
Currently, there are multiple options for the screening of drugs which are dependent on the nature of the therapeutic target and on the strategy of the laboratory or company involved in drug discovery. The existence of receptor dimers, especially of receptor and channel heteromers, gives rise to concerns about the use of monomer-based strategies. At the same time, the existence of receptor heteromers opens exciting new possibilities for drug development. Traditionally, pharmaceutical companies have utilized single receptor screening methods to identify candidate drugs. Although this approach has been successful, many of the clinical effects imparted by the drugs discovered utilizing this method have not always been predicted by the results from screening against a single receptor subtype. Further, recent analysis of the mechanism of action of some clinically used drugs has demonstrated that these drugs may be more heteromer specific and thus offer unique advantages. The atypical antipsychotic, clozapine, is an example for how targeting a GPCR heteromer may produce therapeutically effective results. Originally identified as a mixed dopamine and serotonin receptor antagonist, recently clozapine has been shown to cause dissociation of dopamine D1-D2 receptor heteromers . This unique mechanism of action of clozapine offers several advantages over a typical antipsychotic such as haloperidol and is now the preferred therapy in treatment resistant patients . Similar success has been seen with the smoking cessation drug, varenicline, which targets the α4β2 multimer of nicotinic acetylcholine receptors . Since certain pathological states may lead to increased heteromerization, a strategy for identifying molecules that specifically target heteromers may unveil drugs that result in lesser side effects. Indeed, several new companies including Cara Therapeutics and Dimerex hope to benefit from such an approach.
Early investigations in drug discovery should first identify if a drug target of interest consists of a receptor/channel monomer, homodimer or heterodimer. If receptor monomers or homodimers are the assumed target, and screening is based on second-messenger readouts, then no modifications in the current approach are required. Yet, if radioligand binding is used in the screening of drugs targeting receptor homodimers, then dimer-based binding data will differ from monomer-based models. Finally, drug discovery must consider that drug targets may in fact be receptor heteromers, with unique properties that differ from their constitutive homomers.
As mentioned above, receptor heteromers often display unique pharmacological properties in comparison with their individual receptors [5,6,35,199]. Drugs that are selective for receptor heteromers typically have higher affinity. For example, the affinity of caffeine for the A2A receptor is one order of magnitude lower for the receptor monomer/homomer than for the A2A-A1 receptor heteromer . Compound screening should be performed in parallel with cells expressing both monomers/homomers as well as heteromers. It is possible that compounds discarded as having low affinity for a given receptor may actually exhibit a higher affinity for receptor heteromers, revealing a new class of receptor heteromer-selective drugs.
Receptor heterodimers may be exploited to increase therapeutic efficacy in a variety ways. As described above, small molecules that specifically bind and activate or inhibit a heteromer could be developed. Secondly, monoclonal antibodies that inhibit heteromer formation could be designed. The success of Trastuzumab and Pertuzumab which demonstrate that monoclonal antibodies can inhibit dimerization of the HER2/neu receptor in breast cancer patients suggests that such an approach is viable . Moreover, monoclonal antibodies developed using a subtractive immunization strategy have been shown to recognize selectively a given receptor heteromer and alter (either enhance or inhibit) ligand-mediated effects [53,69]. Third, gene therapy may be employed as a useful strategy. For example, novel therapeutic approaches such as siRNAs can be utilized to target heteromers where the siRNA selectively disrupts the multimerization domain of a given receptor or channel, offering a significant advantage over nonspecific silencing of individual receptors/channels.
Fourth and finally, a heteromer specific blocker (or inhibitor) may be developed using a non-conventional strategy utilizing RNA inhibitors or aptamers from an RNA library [202,203]. This approach relies on RT-PCR to amplify desired RNA molecules by exponential enrichment of their sequences over background, through multiple iteration cycles, against a specific target receptor or channel. This approach provides a great advantage because it does not require a prior knowledge of the structure of the receptor/channel target. Furthermore, aptamers by themselves do not cross the plasma membrane, and could target the extracellular domains of receptors channels , or utilizing lipid micelles, could be used for targeting the active intracellular domains of channels such as TRPV1 .
Receptors and channels can form heteromeric complexes on the plasma membrane. We are just now beginning to understand the characteristics of different receptor and channel heteromerization in cells involved in nociceptive processing. There are functional, regulatory and pharmacological outcomes of heteromerization that are beginning to be acknowledged for a set of receptors and channels engaged in pain. Thus, these heteromeric receptors and channels display unique and specific pharmacological, biophysical, regulatory and functional characteristics. Therefore, heteromer complexes may be considered to be novel and distinct entities with the potential for playing unique physiological roles during normal and pathological conditions. Heteromers provide valid and distinct targets for agonists/antagonists development. This concept is currently overlooked by pharmaceutical companies, which concentrate on a single or homomeric receptor/channel by using expression systems in which heteromers cannot occur. In summary this area of research is well worth the effort and investment, as targeting receptor/channel heteromers may provide a completely new therapeutic strategy for the treatment of different pain conditions.
We would like to thank Kenneth Hargeaves, William Clarke, Alexandr Staruschenko and Nikita Gamper for productive and fruitful collaboration; Nathaniel Jeske, Matt Rowan, Lakshmi Devi, Blaine McGuire, Mayur Patil, Nikita Ruparel, and Margaux Salas for generating important data reviewed in this article. Research was supported by NIH grants DE017696 and DE019311 (for A.N.A.), DA024865 and DA026619 (for K.A.B.).
- Casado, V.; Cortésa, A.; Mallola, J.; Pérez-Capotea, K.; Ferréb, S.; Lluisa, C.; Francoa, R.; Canela, E.I. GPCR homomers and heteromers: A better choice as targets for drug development than GPCR monomers? Pharmacol. Ther. 2009, 124, 248–257, doi:10.1016/j.pharmthera.2009.07.005.
- Mathie, A.; Clarke, C.E.; Ranatunga, K.M.; Veale, E.L. What are the roles of the many different types of potassium channel expressed in cerebellar granule cells? Cerebellum 2003, 2, 11–25, doi:10.1080/14734220310015593.
- Dunah, A.W.; Yasuda, R.P.; Luo, J.H.; Wang, Y.H.; Prybylowski, K.L.; Wolfe, B.B. Biochemical studies of the structure and function of the N-methyl-D-aspartate subtype of glutamate receptors. Mol. Neurobiol. 1999, 19, 151–179.
- Akopian, A.N. Regulation of nociceptive transmission at the periphery via TRPA1-TRPV1 interactions. Curr. Pharm. Biotechnol. 2011, 12, 89–94, doi:10.2174/138920111793937952.
- Ferre, S.; Baler, R.; Bouvier, M.; Caron, M.G.; Devi, L.A.; Durroux, T.; Fuxe, K.; George, S.R.; Javitch, J.A.; Lohse, M.J.; et al. Building a new conceptual framework for receptor heteromers. Nat. Chem. Biol. 2009, 5, 131–134, doi:10.1038/nchembio0309-131.
- Smith, N.J.; Milligan, G. Allostery at G protein-coupled receptor homo- and heteromers: Uncharted pharmacological landscapes. Pharmacol. Rev. 2010, 62, 701–725, doi:10.1124/pr.110.002667.
- Schaefer, M. Homo- and heteromeric assembly of TRP channel subunits. Pflugers Arch. 2005, 451, 35–42, doi:10.1007/s00424-005-1467-6.
- Gotti, C.; Moretti, M.; Gaimarri, A.; Zanardi, A.; Clementi, F.; Zoli, M. Heterogeneity and complexity of native brain nicotinic receptors. Biochem. Pharmacol. 2007, 74, 1102–1111, doi:10.1016/j.bcp.2007.05.023.
- Franco, R.; Lluis, C.; Canela, E.I.; Mallol, J.; Agnati, L.; Casadó, V.; Ciruela, F.; Ferré, S.; Fuxe, K. Receptor-receptor interactions involving adenosine A1 or dopamine D1 receptors and accessory proteins. J. Neural. Transm. 2007, 114, 93–104, doi:10.1007/s00702-006-0566-7.
- Salas, M.M.; Hargreaves, K.M.; Akopian, A.N. TRPA1-mediated responses in trigeminal sensory neurons: Interaction between TRPA1 and TRPV1. Eur. J. Neurosci. 2009, 29, 1568–1578, doi:10.1111/j.1460-9568.2009.06702.x.
- Enz, R.; Cutting, G.R. Molecular composition of GABAC receptors. Vision Res. 1998, 38, 1431–1441, doi:10.1016/S0042-6989(97)00277-0.
- Dingledine, R.; Borges, K.; Bowie, D.; Traynelis, S.F. The glutamate receptor ion channels. Pharmacol. Rev. 1999, 51, 7–61.
- Paoletti, P. Molecular basis of NMDA receptor functional diversity. Eur. J. Neurosci. 2011, 33, 1351–1365, doi:10.1111/j.1460-9568.2011.07628.x.
- Gavva, N.R.; Tamir, R.; Qu, Y.S.; Klionsky, L.; Zhang, T.J.; Immke, D.; Wang, J.; Zhu, D.; Vanderah, T.W.; Porreca, F.; et al. AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo [b][1,4] dioxin-6-yl)acrylamide], a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties. J. Pharmacol. Exp. Ther. 2005, 313, 474–484.
- McGaraughty, S.; Chu, K.L.; Faltynek, C.R.; Jarvis, M.F. Systemic and site-specific effects of A-425619, a selective TRPV1 receptor antagonist, on wide dynamic range neurons in CFA-treated and uninjured rats. J. Neurophysiol. 2006, 95, 18–25.
- Wang, H.L.; Katon, J.; Balan, C.; Bannon, A.W.; Bernard, C.; Doherty, E.M.; Dominguez, C.; Gavva, N.R.; Gore, V.; Ma, V.; et al. Novel vanilloid receptor-1 antagonists: 3. The identification of a second-generation clinical candidate with improved physicochemical and pharmacokinetic properties. J. Med. Chem. 2007, 50, 3528–3539.
- Gavva, N.R. Body-temperature maintenance as the predominant function of the vanilloid receptor TRPV1. Trends Pharmacol. Sci. 2008, 29, 550–557, doi:10.1016/j.tips.2008.08.003.
- Starowicz, K.; Cristino, L.; Di Marzo, V. TRPV1 receptors in the central nervous system: Potential for previously unforeseen therapeutic applications. Curr. Pharm. Des. 2008, 14, 42–54, doi:10.2174/138161208783330790.
- Palazzo, E.; Luongo, L.; de Novellis, V.; Berrino, L.; Rossi, F.; Maione, S. Moving towards supraspinal TRPV1 receptors for chronic pain relief. Mol. Pain 2011, 6, 66.
- Cui, M.; Honore, P.; Zhong, C.; Gauvin, D.; Mikusa, J.; Hernandez, G.; Chandran, P.; Gomtsyan, A.; Brown, B.; Bayburt, E.K.; et al. TRPV1 receptors in the CNS play a key role in broad-spectrum analgesia of TRPV1 antagonists. J. Neurosci. 2006, 26, 9385–9393.
- Gavva, N.R.; Bannon, A.W.; Surapaneni, S.; Hovland, D.N., Jr.; Lehto, S.G.; Gore, A.; Juan, T.; Deng, H.; Han, B.; Klionsky, L.; et al. The vanilloid receptor TRPV1 is tonically activated in vivo and involved in body temperature regulation. J. Neurosci. 2007, 27, 3366–3374.
- Staruschenko, A.; Jeske, N.A.; Akopian, A.N. Contribution of TRPV1-TRPA1 interaction to the single channel properties of the TRPA1 channel. J. Biol. Chem. 2010, 285, 15167–15177, doi:10.1074/jbc.M110.106153.
- Hellwig, N.; Albrecht, N.; Harteneck, C.; Schultz, G.; Schaefer, M. Homo- and heteromeric assembly of TRPV channel subunits. J. Cell. Sci. 2005, 118, 917–928, doi:10.1242/jcs.01675.
- Cheng, W.; Sun, C.; Zheng, J. Heteromerization of TRP channel subunits: Extending functional diversity. Protein Cell 2010, 1, 802–810, doi:10.1007/s13238-010-0108-9.
- Akopian, A.N.; Ruparel, N.B.; Jeske, N.A.; Hargreaves, K.M. Transient receptor potential TRPA1 channel desensitization in sensory neurons is agonist dependent and regulated by TRPV1-directed internalization. J. Physiol. 2007, 583, 175–193, doi:10.1113/jphysiol.2007.133231.
- Hasbi, A.; O'Dowd, B.F.; George, S.R. Dopamine D1-D2 receptor heteromer signaling pathway in the brain: Emerging physiological relevance. Mol. Brain 2010, 4, 26.
- Patil, M.J.; Jeske, N.A.; Akopian, A.N. Transient receptor potential V1 regulates activation and modulation of transient receptor potential A1 by Ca2+. Neuroscience 2010, 171, 1109–1119, doi:10.1016/j.neuroscience.2010.09.031.
- Jacoby, E.; Bouhelal, R.; Gerspacher, M.; Seuwen, K. The 7 TM G-protein-coupled receptor target family. ChemMedChem 2006, 1, 760–782, doi:10.1002/cmdc.200600134.
- Pan, H.L.; Wu, Z.-Z.; Zhou, H.-Y.; Chen, S.-R.; Zhang, H.-M.; Li, D.-P. Modulation of pain transmission by G-protein-coupled receptors. Pharmacol. Ther. 2008, 117, 141–161, doi:10.1016/j.pharmthera.2007.09.003.
- Vischer, H.F.; Watts, A.O.; Nijmeijer, S.; Leurs, R. G protein-coupled receptors: Walking hand-in-hand, talking hand-in-hand? Br. J. Pharmacol. 2011, 163, 246–260, doi:10.1111/j.1476-5381.2011.01229.x.
- Rozenfeld, R.; Devi, L.A. Receptor heteromerization and drug discovery. Trends Pharmacol. Sci. 2010, 31, 124–130, doi:10.1016/j.tips.2009.11.008.
- Milligan, G. G protein-coupled receptor hetero-dimerization: Contribution to pharmacology and function. Br. J. Pharmacol. 2009, 158, 5–14, doi:10.1111/j.1476-5381.2009.00169.x.
- Milligan, G. The role of dimerisation in the cellular trafficking of G-protein-coupled receptors. Curr. Opin. Pharmacol. 2010, 10, 23–29, doi:10.1016/j.coph.2009.09.010.
- Fuxe, K.; Marcellino, D.; Borroto-Escuela, D.O.; Frankowska, M.; Ferraro, L.; Guidolin, D.; Ciruela, F.; Agnati, L.F. The changing world of G protein-coupled receptors: From monomers to dimers and receptor mosaics with allosteric receptor-receptor interactions. J. Recept. Signal Transduct. Res. 2010, 30, 272–283, doi:10.3109/10799893.2010.506191.
- Rozenfeld, R.; Devi, L.A. Exploring a role for heteromerization in GPCR signalling specificity. Biochem. J. 2011, 433, 11–18, doi:10.1042/BJ20100458.
- Han, Y.; Moreira, I.S.; Urizar, E.; Weinstein, H.; Javitch, J.A. Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation. Nat. Chem. Biol. 2009, 5, 688–695, doi:10.1038/nchembio.199.
- Keov, P.; Sexton, P.M.; Christopoulos, A. Allosteric modulation of G protein-coupled receptors: A pharmacological perspective. Neuropharmacology 2011, 60, 24–35.
- Fuxe, K.; Marcellino, D.; Leo, G.; Agnati, L.F. Molecular integration via allosteric interactions in receptor heteromers. A working hypothesis. Curr. Opin. Pharmacol. 2010, 10, 14–22, doi:10.1016/j.coph.2009.10.010.
- Stein, C.; Clarkb, J.D.; Ohc, U.; Vaskod, M.R.; Wilcoxe, G.L.; Overlande, A.C.; Vanderahf, T.W.; Spencer, R.H. Peripheral mechanisms of pain and analgesia. Brain Res. Rev. 2009, 60, 90–113, doi:10.1016/j.brainresrev.2008.12.017.
- Stein, C.; Lang, L.J. Peripheral mechanisms of opioid analgesia. Curr. Opin. Pharmacol. 2009, 9, 3–8, doi:10.1016/j.coph.2008.12.009.
- Ji, R.R.; Zhang, Q.; Law, P.Y.; Low, H.H.; Elde, R.; Hokfelt, T. Expression of mu-, delta-, and kappa-opioid receptor-like immunoreactivities in rat dorsal root ganglia after carrageenan-induced inflammation. J. Neurosci. 1995, 15, 8156–8166.
- Rau, K.K.; Caudle, R.M.; Cooper, B.Y.; Johnson, R.D. Diverse immunocytochemical expression of opioid receptors in electrophysiologically defined cells of rat dorsal root ganglia. J. Chem. Neuroanat. 2005, 29, 255–264, doi:10.1016/j.jchemneu.2005.02.002.
- Wang, H.B.; Zhao, B.; Zhong, Y.Q.; Li, K.C.; Li, Z.Y.; Wang, Q.; Lu, Y.J.; Zhang, Z.N.; He, S.Q.; Zheng, H.C.; et al. Coexpression of delta- and mu-opioid receptors in nociceptive sensory neurons. Proc. Natl. Acad. Sci. USA 2010, 107, 13117–13122.
- Raynor, K.; Kong, H.; Chen, Y.; Yasuda, K.; Yu, L.; Bell, G.I.; Reisine, T. Pharmacological characterization of the cloned kappa-, delta-, and mu-opioid receptors. Mol. Pharmacol. 1994, 45, 330–334.
- Traynor, J.R.; Elliott, J. delta-Opioid receptor subtypes and cross-talk with mu-receptors. Trends Pharmacol. Sci. 1993, 14, 84–86, doi:10.1016/0165-6147(93)90068-U.
- Dietis, N.; Rowbotham, D.J.; Lambert, D.G. Opioid receptor subtypes: Fact or artifact? Br. J. Anaesth. 2011, 107, 8–18, doi:10.1093/bja/aer115.
- Abdelhamid, E.E.; Sultana, M.; Portoghese, P.S.; Takemori, A.E. Selective blockage of delta opioid receptors prevents the development of morphine tolerance and dependence in mice. J. Pharmacol. Exp. Ther. 1991, 258, 299–303.
- Zhu, Y.; King, M.A.; Schuller, A.G.P.; Nitsche, J.F.; Reidl, M.; Elde, R.P.; Unterwald, E.; Pasternak, G.W.; Pintar, J.E. Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice. Neuron 1999, 24, 243–252, doi:10.1016/S0896-6273(00)80836-3.
- Gendron, L.; Pintar, J.E.; Chavkin, C. Essential role of MU opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia. Neuroscience 2007, 150, 807–817, doi:10.1016/j.neuroscience.2007.09.060.
- George, S.R.; Fan, T.; Xie, Z.; Tse, R.; Tam, V.; Varghese, G.; O'Dowd, B.F. Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties. J. Biol. Chem. 2000, 275, 26128–26135.
- Gomes, I.; Jordan, B.A.; Gupta, A.; Trapaidze, N.; Nagy, V.; Devi, L.A. Heterodimerization of mu and delta opioid receptors: A role in opiate synergy. J. Neurosci. 2000, 20, RC110.
- Scherrer, G.; Imamachi, N.; Cao, Y.-Q.; Contet, C.; Mennicken, F.; O'Donnell, D.; Kieffer, B.L.; Basbaum, A.I. Dissociation of the opioid receptor mechanisms that control mechanical and heat pain. Cell 2009, 137, 1148–1159, doi:10.1016/j.cell.2009.04.019.
- Gupta, A.; Mulder, J.; Gomes, I.; Rozenfeld, R.; Bushlin, I.; Ong, E.; Lim, M.; Maillet, E.; Junek, M.; Cahill, C.M.; et al. Increased abundance of opioid receptor heteromers after chronic morphine administration. Sci. Signal. 2010, 3, ra54, doi:10.1126/scisignal.2000807.
- Gomes, I.; Gupta, A.; Filipovska, J.; Szeto, H.H.; Pintar, J.E.; Devi, L.A. A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. Proc. Natl. Acad. Sci. USA 2004, 101, 5135–5139.
- Law, P.Y.; Erickson-Herbrandson, L.J.; Zha, Q.Q.; Solberg, J.; Chu, J.; Sarre, A.; Loh, H.H. Heterodimerization of mu- and delta-opioid receptors occurs at the cell surface only and requires receptor-G protein interactions. J. Biol. Chem. 2005, 280, 11152–11164.
- Gomes, I.; IJzerman, A.P.; Ye, K.; Maillet, E.L.; Devi, L.A. G protein-coupled receptor heteromerization: A role in allosteric modulation of ligand binding. Mol. Pharmacol. 2011, 79, 1044–1052, doi:10.1124/mol.110.070847.
- Christopoulos, A.; Kenakin, T. G protein-coupled receptor allosterism and complexing. Pharmacol. Rev. 2002, 54, 323–374, doi:10.1124/pr.54.2.323.
- Gregory, K.J.; Sexton, P.M.; Christopoulos, A. Overview of receptor allosterism. Curr. Protoc. Pharmacol. 2010, 51, 1.21.1–1.21.34.
- Pierce, K.L.; Premont, R.T.; Lefkowitz, R.J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 2002, 3, 639–650, doi:10.1038/nrm908.
- Rozenfeld, R.; Devi, L.A. Receptor heterodimerization leads to a switch in signaling: beta-Arrestin2-mediated ERK activation by mu-delta opioid receptor heterodimers. FASEB J. 2007, 21, 2455–2465, doi:10.1096/fj.06-7793com.
- Bohn, L.M.; Lefkowitz, R.J.; Caron, M.G. Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice. J. Neurosci. 2002, 22, 10494–10500.
- Fan, T.; Varghese, G.; Nguyen, T.; Tse, R.; O'Dowd, B.F.; George, S.R. A role for the distal carboxyl tails in generating the novel pharmacology and G protein activation profile of mu and delta opioid receptor hetero-oligomers. J. Biol. Chem. 2005, 280, 38478–38488.
- Hasbi, A.; Nguyen, T.; Fan, T.; Cheng, R.; Rashid, A.; Alijaniaram, M.; Rasenick, M.M.; O'Dowd, B.F.; George, S.R. Trafficking of preassembled opioid mu-delta heterooligomer-Gz signaling complexes to the plasma membrane: Coregulation by agonists. Biochemistry 2007, 46, 12997–13009.
- Daniels, D.J.; Lenard, N.R.; Etienne, C.L.; Law, P.-Y.; Roerig, S.C.; Philip, S. Portoghese Opioid-induced tolerance and dependence in mice is modulated by the distance between pharmacophores in a bivalent ligand series. Proc. Natl. Acad. Sci. USA 2005, 102, 19208–19213.
- Lenard, N.R.; Daniels, D.J.; Portoghese, P.S.; Roerig, S.C. Absence of conditioned place preference or reinstatement with bivalent ligands containing mu-opioid receptor agonist and delta-opiold receptor antagonist pharmacophores. Eur. J. Pharmacol. 2007, 566, 75–82, doi:10.1016/j.ejphar.2007.02.040.
- Jordan, B.A.; Devi, L.A. G-protein-coupled receptor heterodimerization modulates receptor function. Nature 1999, 399, 697–700, doi:10.1038/21441.
- Waldhoer, M.; Fong, J.; Jones, R.M.; Lunzer, M.M.; Sharma, S.K.; Kostenis, E.; Portoghese, P.S.; Whistler, J.L. A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers. Proc. Natl. Acad. Sci. USA 2005, 102, 9050–9055.
- Ansonoff, M.A.; Portoghese, P.S.; Pintar, J.E. Consequences of opioid receptor mutation on actions of univalent and bivalent kappa and delta ligands. Psychopharmacology (Berl.) 2010, 210, 161–168, doi:10.1007/s00213-010-1826-7.
- Berg, K.A.; Rowan, M.P.; Gupta, A.; Sanchez, T.A.; Silva, M.; Gomes, I.; McGuire, B.A.; Portoghese, P.S.; Hargreaves, K.M.; Devi, L.A.; et al. Allosteric Interactions between delta and kappa opioid receptors in peripheral sensory neurons. Mol. Pharmacol. 2011, doi:10.1124/mol.111.072702.
- Ansonoff, M.A.; Wen, T.; Pintar, J.E. Kappa2 opioid receptor subtype binding requires the presence of the DOR-1 gene. Front. Biosci. (Schol. Ed.) 2010, 2, 772–780.
- Xie, Z.; Bhushan, R.G.; Daniels, D.J.; Portoghese, P.S. Interaction of bivalent ligand KDN21 with heterodimeric delta-kappa opioid receptors in human embryonic kidney 293 cells. Mol. Pharmacol. 2005, 68, 1079–1086, doi:10.1124/mol.105.012070.
- Sharma, S.K.; Jones, R.M.; Metzger, T.G.; Ferguson, D.M.; Portoghese, P.S. Transformation of a kappa-opioid receptor antagonist to a kappa-agonist by transfer of a guanidinium group from the 5'- to 6'-position of naltrindole. J. Med. Chem. 2001, 44, 2073–2079.
- Rowan, M.P.; Ruparel, N.B.; Patwardhan, A.M.; Berg, K.A.; Clarke, W.P.; Hargreaves, K.M. Peripheral delta opioid receptors require priming for functional competence in vivo. Eur. J. Pharmacol. 2009, 602, 283–287, doi:10.1016/j.ejphar.2008.11.028.
- Berg, K.A.; Rowan, M.P.; Sanchez, T.A.; Silva, M.; Patwardhan, A.M.; Milam, S.B.; Hargreaves, K.M.; Clarke, W.P. Regulation of kappa-opioid receptor signaling in peripheral sensory neurons in vitro and in vivo. J. Pharmacol. Exp. Ther. 2011, 338, 92–99, doi:10.1124/jpet.110.177493.
- Chakrabarti, S.; Liu, N.J.; Gintzler, A.R. Formation of mu-/kappa-opioid receptor heterodimer is sex-dependent and mediates female-specific opioid analgesia. Proc. Natl. Acad. Sci. USA 2010, 107, 20115–20119.
- Yekkirala, A.S.; Kalyuzhny, A.E.; Portoghese, P.S. Standard opioid agonists activate heteromeric opioid receptors: Evidence for morphine and D-Ala(2)-MePhe(4)-Glyol(5) enkephalin as selective mu-delta agonists. ACS Chem. Neurosci. 2010, 1, 146–154, doi:10.1021/cn9000236.
- Liu, N.J.; Chakrabarti, S.; Schnell, S.; Wessendorf, M.; Gintzler, A.R. Spinal synthesis of estrogen and concomitant signaling by membrane estrogen receptors regulate spinal kappa- and mu-opioid receptor heterodimerization and female-specific spinal morphine antinociception. J. Neurosci. 2011, 31, 11836–11845, doi:10.1523/JNEUROSCI.1901-11.2011.
- Fillingim, R.B.; Gear, R.W. Sex differences in opioid analgesia: Clinical and experimental findings. Eur. J. Pain 2004, 8, 413–425, doi:10.1016/j.ejpain.2004.01.007.
- Gear, R.W.; Gordon, N.C.; Heller, P.H.; Paul, S.; Miaskowski, C.; Levine, J.D. Gender difference in analgesic response to the kappa-opioid pentazocine. Neurosci. Lett. 1996, 205, 207–209, doi:10.1016/0304-3940(96)12402-2.
- Gear, R.W.; Miaskowski, C.; Gordon, N.C.; Paul, S.M.; Heller, P.H.; Levine, J.D. Kappa-opioids produce significantly greater analgesia in women than in men. Nat. Med. 1996, 2, 1248–1250.
- Gear, R.W.; Miaskowski, C.; Gordon, N.C.; Paul, S.M.; Heller, P.H.; Levine, J.D. The kappa opioid nalbuphine produces gender- and dose-dependent analgesia and antianalgesia in patients with postoperative pain. Pain 1999, 83, 339–345, doi:10.1016/S0304-3959(99)00119-0.
- Liu, N.J.; von Gizycki, H.; Gintzler, A.R. Sexually dimorphic recruitment of spinal opioid analgesic pathways by the spinal application of morphine. J. Pharmacol. Exp. Ther. 2007, 322, 654–660, doi:10.1124/jpet.107.123620.
- Fairbanks, C.A.; Stone, L.S.; Wilcox, G.L. Pharmacological profiles of alpha 2 adrenergic receptor agonists identified using genetically altered mice and isobolographic analysis. Pharmacol. Ther. 2009, 123, 224–238, doi:10.1016/j.pharmthera.2009.04.001.
- Stone, L.S.; MacMillan, L.B.; Kitto, K.F.; Limbird, L.E.; Wilcox, G.L. The alpha2a adrenergic receptor subtype mediates spinal analgesia evoked by alpha2 agonists and is necessary for spinal adrenergic-opioid synergy. J. Neurosci. 1997, 17, 7157–7165.
- Mansikka, H.; Lähdesmäki, J.; Scheinin, M.; Pertovaara, A. Alpha(2A) adrenoceptors contribute to feedback inhibition of capsaicin-induced hyperalgesia. Anesthesiology 2004, 101, 185–190, doi:10.1097/00000542-200407000-00029.
- Spaulding, T.C.; Fielding, S.; Venafro, J.J.; Lal, H. Antinociceptive activity of clonidine and its potentiation of morphine analgesia. Eur. J. Pharmacol. 1979, 58, 19–25, doi:10.1016/0014-2999(79)90335-2.
- Wilcox, G.L.; Carlsson, K.-H.; Jochim, A.; Jurna, I. Mutual potentiation of antinociceptive effects of morphine and clonidine on motor and sensory responses in rat spinal cord. Brain Res. 1987, 405, 84–93, doi:10.1016/0006-8993(87)90992-9.
- Drasner, K.; Fields, H.L. Synergy between the antinociceptive effects of intrathecal clonidine and systemic morphine in the rat. Pain 1988, 32, 309–312, doi:10.1016/0304-3959(88)90042-5.
- Jordan, B.A.; Gomes, I.; Rios, C.; Filipovska, J.; Devi, L.A. Functional interactions between mu opioid and alpha 2A-adrenergic receptors. Mol. Pharmacol. 2003, 64, 1317–1324, doi:10.1124/mol.64.6.1317.
- Zhang, Y.Q.; Limbird, L.E. Hetero-oligomers of alpha2A-adrenergic and mu-opioid receptors do not lead to transactivation of G-proteins or altered endocytosis profiles. Biochem. Soc. Trans. 2004, 32, 856–860, doi:10.1042/BST0320856.
- Vilardaga, J.P.; Nikolaev, V.O.; Lorenz, K.; Ferrandon, S.; Zhuang, Z.; Lohse, M.J. Conformational cross-talk between alpha2A-adrenergic and mu-opioid receptors controls cell signaling. Nat. Chem. Biol. 2008, 4, 126–131, doi:10.1038/nchembio.64.
- Riedl, M.S.; Schnell, S.A.; Overland, A.C.; Chabot-Doré, A.J.; Taylor, A.M.; Ribeiro-da-Silva, A.; Elde, R.P.; Wilcox, G.L.; Stone, L.S. Coexpression of alpha 2A-adrenergic and delta-opioid receptors in substance P-containing terminals in rat dorsal horn. J. Comp. Neurol. 2009, 513, 385–398, doi:10.1002/cne.21982.
- Clapper, J.R.; Moreno-Sanz, G.; Russo, R.; Guijarro, A.; Vacondio, F.; Duranti, A.; Tontini, A.; Sanchini, S.; Sciolino, N.R.; Spradley, J.M.; et al. Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism. Nat. Neurosci. 2010, 13, 1265–1270.
- Vigano, D.; Rubino, T.; Parolaro, D. Molecular and cellular basis of cannabinoid and opioid interactions. Pharmacol. Biochem. Behav. 2005, 81, 360–368, doi:10.1016/j.pbb.2005.01.021.
- Filppula, S.; Yaddanapudi, S.; Mercier, R.; Xu, W.; Pavlopoulos, S.; Makriyannis, A. Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system. J. Pept. Res. 2004, 64, 225–236, doi:10.1111/j.1399-3011.2004.00188.x.
- Rios, C.; Gomes, I.; Devi, L.A. mu opioid and CB1 cannabinoid receptor interactions: Reciprocal inhibition of receptor signaling and neuritogenesis. Br. J. Pharmacol. 2006, 148, 387–395.
- Salio, C.; Fischer, J.; Franzoni, M.F.; Mackie, K.; Kaneko, T.; Conrath, M. CB1-cannabinoid and mu-opioid receptor co-localization on postsynaptic target in the rat dorsal horn. Neuroreport 2001, 12, 3689–3692, doi:10.1097/00001756-200112040-00017.
- Hohmann, A.G.; Briley, E.M.; Herkenham, M. Pre- and postsynaptic distribution of cannabinoid and mu opioid receptors in rat spinal cord. Brain Res. 1999, 822, 17–25, doi:10.1016/S0006-8993(98)01321-3.
- Rodriguez, J.J.; Mackie, K.; Pickel, V.M. Ultrastructural localization of the CB1 cannabinoid receptor in mu-opioid receptor patches of the rat Caudate putamen nucleus. J. Neurosci. 2001, 21, 823–833.
- Pickel, V.M.; Chan, J.; Kash, T.L.; Rodríguez, J.J.; MacKie, K. Compartment-specific localization of cannabinoid 1 (CB1) and mu-opioid receptors in rat nucleus accumbens. Neuroscience 2004, 127, 101–112, doi:10.1016/j.neuroscience.2004.05.015.
- Cichewicz, D.L.; McCarthy, E.A. Antinociceptive synergy between delta(9)-tetrahydrocannabinol and opioids after oral administration. J. Pharmacol. Exp. Ther. 2003, 304, 1010–1015, doi:10.1124/jpet.102.045575.
- Cichewicz, D.L. Synergistic interactions between cannabinoid and opioid analgesics. Life Sci. 2004, 74, 1317–1324, doi:10.1016/j.lfs.2003.09.038.
- Sun, Y.G.; Chen, Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature 2007, 448, 700–703.
- Sun, Y.G.; Zhao, Z.-Q.; Meng, X.-L.; Yin, J.; Liu, X.-Y.; Chen, Z.-F. Cellular basis of itch sensation. Science 2009, 325, 1531–1534, doi:10.1126/science.1174868.
- Liu, X.Y.; Liu, Z.-C.; Sun, Y.-G.; Ross, M.; Kim, S.; Tsai, F.-F.; Li, Q.-F.; Jeffry, J.; Kim, J.-Y.; Loh, H.H.; et al. Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids. Cell 2011, 147, 447–458.
- Melzack, R.; Wall, P.D. Pain mechanisms: A new theory. Science 1965, 150, 971–979.
- Yaksh, T.L. Behavioral and autonomic correlates of the tactile evoked allodynia produced by spinal glycine inhibition: Effects of modulatory receptor systems and excitatory amino acid antagonists. Pain 1989, 37, 111–123, doi:10.1016/0304-3959(89)90160-7.
- Zeilhofer, H.U. Loss of glycinergic and GABAergic inhibition in chronic pain--contributions of inflammation and microglia. Int. Immunopharmacol. 2008, 8, 182–187, doi:10.1016/j.intimp.2007.07.009.
- Bohlhalter, S.; Weinmann, O.; Mohler, H.; Fritschy, J.M. Laminar compartmentalization of GABAA-receptor subtypes in the spinal cord: An immunohistochemical study. J. Neurosci. 1996, 16, 283–297.
- Scholz, J.; Broom, D.C.; Youn, D.-H.; Mills, C.D.; Kohno, T.; Suter, M.R.; Moore, K.A.; Decosterd, I.; Coggeshall, R.E.; Woolf, C.J. Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury. J. Neurosci. 2005, 25, 7317–7323.
- Tucker, A.P.; Mezzatesta, J.; Nadeson, R.; Goodchild, C.S. Intrathecal midazolam II: Combination with intrathecal fentanyl for labor pain. Anesth. Analg. 2004, 98, 1521–1527.
- Jasmin, L.; Wu, M.V.; Ohara, P.T. GABA puts a stop to pain. Curr. Drug. Targets CNS Neurol. Disord. 2004, 3, 487–505, doi:10.2174/1568007043336716.
- Wieland, H.A.; Luddens, H.; Seeburg, P.H. A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. J. Biol. Chem. 1992, 267, 1426–1429.
- Rudolph, U.; Mohler, H. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 2006, 6, 18–23, doi:10.1016/j.coph.2005.10.003.
- Knabl, J.; Witschi, R.; Hösl, K.; Reinold, H.; Zeilhofer, U.B.; Ahmadi, S.; Brockhaus, J.; Sergejeva, M.; Hess, A.; Brune, K.; et al. Reversal of pathological pain through specific spinal GABAA receptor subtypes. Nature 2008, 451, 330–334.
- Witschi, R.; Punnakkal, P.; Paul, J.; Walczak, J.S.; Cervero, F.; Fritschy, J.M.; Kuner, R.; Keist, R.; Rudolph, U.; Zeilhofer, H.U. Presynaptic alpha2-GABAA receptors in primary afferent depolarization and spinal pain control. J. Neurosci. 2011, 31, 8134–8142.
- Yanovsky, Y.; Schubring, S.; Fleischer, W.; Gisselmann, G.; Zhu, X.R.; Lübbert, H.; Hatt, H.; Rudolph, U.; Haas, H.L.; Sergeeva, O.A. GABA(A) receptors involved in sleep and anaesthesia: β1- versus β3-Containing assemblies. Pflugers Arch. 2011, 463, 187–199.
- Rudolph, U.; Knoflach, F. Beyond classical benzodiazepines: Novel therapeutic potential of GABAA receptor subtypes. Nat. Rev. Drug Discov. 2011, 10, 685–697, doi:10.1038/nrd3502.
- Atack, J.R.; Wafford, K.A.; Tye, S.J.; Cook, S.M.; Sohal, B.; Pike, A.; Sur, C.; Melillo, D.; Bristow, L.; Bromidge, F.; et al. TPA023 [7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluor ophenyl)-1,2,4-triazolo[4,3-b]pyridazine], an agonist selective for alpha2- and alpha3-containing GABAA receptors, is a nonsedating anxiolytic in rodents and primates. J. Pharmacol. Exp. Ther. 2006, 316, 410–422.
- Griebel, G.; Perrault, G.; Simiand, J.; Cohen, C.; Granger, P.; Depoortere, H.; Françon, D.; Avenet, P.; Schoemaker, H.; Evanno, Y.; et al. SL651498, a GABAA receptor agonist with subtype-selective efficacy, as a potential treatment for generalized anxiety disorder and muscle spasms. CNS Drug Rev. 2003, 9, 3–20.
- Sonner, J.M.; Cascio, M.; Xing, Y.; Fanselow, M.S.; Kralic, J.E.; Morrow, A.L.; Korpi, E.R.; Hardy, S.; Sloat, B.; Eger, E.I., II; et al. Alpha 1 subunit-containing GABA type A receptors in forebrain contribute to the effect of inhaled anesthetics on conditioned fear. Mol. Pharmacol. 2005, 68, 61–68.
- Caraiscos, V.B.; Elliott, E.M.; You-Ten, K.E.; Cheng, V.Y.; Belelli, D.; Newell, J.G.; Jackson, M.F.; Lambert, J.J.; Rosahl, T.W.; Wafford, K.A.; et al. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha5 subunit-containing gamma-aminobutyric acid type A receptors. Proc. Natl. Acad. Sci. USA 2004, 101, 3662–3667.
- Mirza, N.R.; Nielsen, E.O. Do subtype-selective gamma-aminobutyric acid A receptor modulators have a reduced propensity to induce physical dependence in mice? J. Pharmacol. Exp. Ther. 2006, 316, 1378–1385.
- Finnerup, N.B.; Otto, M.; McQuay, H.J.; Jensen, T.S.; Sindrup, S.H. Algorithm for neuropathic pain treatment: An evidence based proposal. Pain 2005, 118, 289–305, doi:10.1016/j.pain.2005.08.013.
- Albuquerque, E.X.; Pereira, E.F.R.; Alkondon, M.; Rogers, S.W. Mammalian nicotinic acetylcholine receptors: From structure to function. Physiol. Rev. 2009, 89, 73–120, doi:10.1152/physrev.00015.2008.
- Johnson, D.S.; Martinez, J.; Elgoyhen, A.B.; Heinemann, S.F.; McIntosh, J.M. alpha-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: Preferential inhibition of homomeric alpha 7 and alpha 9 receptors. Mol. Pharmacol. 1995, 48, 194–199.
- Elgoyhen, A.B.; Johnson, D.S.; Boulter, J.; Vetter, D.E.; Heinemann, S. alpha 9: An acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell 1994, 79, 705–715, doi:10.1016/0092-8674(94)90555-X.
- Elgoyhen, A.B.; Vetter, D.E.; Katz, E.; Rothlin, C.V.; Heinemann, S.F.; Boulter, J. alpha10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc. Natl. Acad. Sci. USA 2001, 98, 3501–3506.
- Gotti, C.; Riganti, L.; Vailati, S.; Clementi, F. Brain neuronal nicotinic receptors as new targets for drug discovery. Curr. Pharm. Des. 2006, 12, 407–428, doi:10.2174/138161206775474486.
- Olivera, B.M.; Quik, M.; Vincler, M.; McIntosh, J.M. Subtype-selective conopeptides targeted to nicotinic receptors: Concerted discovery and biomedical applications. Channels (Austin) 2008, 2, 143–152, doi:10.4161/chan.2.2.6276.
- Decker, M.W.; Meyer, M.D.; Sullivan, J.P. The therapeutic potential of nicotinic acetylcholine receptor agonists for pain control. Expert Opin. Investig. Drugs 2001, 10, 1819–1830, doi:10.1517/135437220.127.116.119.
- Dray, A. Neuropathic pain: Emerging treatments. Br. J. Anaesth. 2008, 101, 48–58, doi:10.1093/bja/aen107.
- Ellison, M.; Haberlandt, C.; Gomez-Casati, M.E.; Watkins, M.; Elgoyhen, A.B.; McIntosh, J.M.; Olivera, B.M. alpha-RgIA: A novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR. Biochemistry 2006, 45, 1511–1517.
- Sandall, D.W.; Satkunanathan, N.; Keays, D.A.; Polidano, M.A.; Liping, X.; Pham, V.; Down, J.G.; Khalil, Z.; Livett, B.G.; Gayler, K.R. A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo. Biochemistry 2003, 42, 6904–6911.
- Vincler, M.; Wittenauer, S.; Parker, R.; Ellison, M.; Olivera, B.M.; McIntosh, J.M. Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. USA 2006, 103, 17880–17884.
- Callaghan, B.; Haythornthwaite, A.; Berecki, G.; Clark, R.J.; Craik, D.J.; Adams, D.J. Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation. J. Neurosci. 2008, 28, 10943–10951, doi:10.1523/JNEUROSCI.3594-08.2008.
- Callaghan, B.; Adams, D.J. Analgesic alpha-conotoxins Vc1.1 and RgIA inhibit N-type calcium channels in sensory neurons of alpha9 nicotinic receptor knockout mice. Channels (Austin) 2011, 4, 51–54, doi:10.4161/chan.4.1.10281.
- McIntosh, J.M.; Absalom, N.; Chebib, M.; Elgoyhen, A.B.; Vincler, M. Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochem. Pharmacol. 2009, 78, 693–702, doi:10.1016/j.bcp.2009.05.020.
- Watkins, J.C.; Evans, R.H. Excitatory amino acid transmitters. Annu. Rev. Pharmacol. Toxicol. 1981, 21, 165–204, doi:10.1146/annurev.pa.21.040181.001121.
- Watkins, J.C.; Davies, J.; Evans, R.H.; Francis, A.A.; Jones, A.W. Pharmacology of receptors for excitatory amino acids. Adv. Biochem. Psychopharmacol. 1981, 27, 263–273.
- Szekely, J.I.; Torok, K.; Mate, G. The role of ionotropic glutamate receptors in nociception with special regard to the AMPA binding sites. Curr. Pharm. Des. 2002, 8, 887–912, doi:10.2174/1381612024607126.
- Neugebauer, V. Glutamate receptor ligands. Handb. Exp. Pharmacol. 2007, 177, 217–249, doi:10.1007/978-3-540-33823-9_8.
- Hollmann, M.; Heinemann, S. Cloned glutamate receptors. Annu. Rev. Neurosci. 1994, 17, 31–108, doi:10.1146/annurev.ne.17.030194.000335.
- Mano, I.; Teichberg, V.I. A tetrameric subunit stoichiometry for a glutamate receptor-channel complex. Neuroreport 1998, 9, 327–331, doi:10.1097/00001756-199801260-00027.
- Premkumar, L.S.; Auerbach, A. Stoichiometry of recombinant N-methyl-D-aspartate receptor channels inferred from single-channel current patterns. J. Gen. Physiol. 1997, 110, 485–502, doi:10.1085/jgp.110.5.485.
- Cull-Candy, S.; Brickley, S.; Farrant, M. NMDA receptor subunits: Diversity, development and disease. Curr. Opin. Neurobiol. 2001, 11, 327–335, doi:10.1016/S0959-4388(00)00215-4.
- Furukawa, H.; Singh, S.K.; Mancusso, R.; Gouaux, E. Subunit arrangement and function in NMDA receptors. Nature 2005, 438, 185–192.
- Constantine-Paton, M.; Cline, H.T. LTP and activity-dependent synaptogenesis: The more alike they are, the more different they become. Curr. Opin. Neurobiol. 1998, 8, 139–148, doi:10.1016/S0959-4388(98)80017-2.
- Quartaroli, M.; Fasdelli, N.; Bettelini, L.; Maraia, G.; Corsi, M. GV196771A, an NMDA receptor/glycine site antagonist, attenuates mechanical allodynia in neuropathic rats and reduces tolerance induced by morphine in mice. Eur. J. Pharmacol. 2001, 430, 219–227, doi:10.1016/S0014-2999(01)01278-X.
- Wallace, M.S.; Rowbotham, M.C.; Katz, N.P.; Dworkin, R.H.; Dotson, R.M.; Galer, B.S.; Rauck, R.L.; Backonja, M.M.; Quessy, S.N.; Meisner, P.D. A randomized, double-blind, placebo-controlled trial of a glycine antagonist in neuropathic pain. Neurology 2002, 59, 1694–1700.
- Gogas, K.R. Glutamate-based therapeutic approaches: NR2B receptor antagonists. Curr. Opin. Pharmacol. 2006, 6, 68–74, doi:10.1016/j.coph.2005.11.001.
- Taniguchi, K.; Shinjo, K.; Mizutani, M.; Shimada, K.; Ishikawa, T.; Menniti, F.S.; Nagahisa, A. Antinociceptive activity of CP-101,606, an NMDA receptor NR2B subunit antagonist. Br. J. Pharmacol. 1997, 122, 809–812, doi:10.1038/sj.bjp.0701445.
- Sang, C.N.; Ramadan, N.M.; Wallihan, R.G.; Chappell, A.S.; Freitag, F.G.; Smith, T.R.; Silberstein, S.D.; Johnson, K.W.; Phebus, L.A.; Bleakman, D.; et al. LY293558, a novel AMPA/GluR5 antagonist, is efficacious and well-tolerated in acute migraine. Cephalalgia 2004, 24, 596–602, doi:10.1111/j.1468-2982.2004.00723.x.
- Sang, C.N.; Hostetter, M.P.; Gracely, R.H.; Chappell, A.S.; Schoepp, D.D.; Lee, G.; Whitcup, S.; Caruso, R.; Max, M.B. AMPA/kainate antagonist LY293558 reduces capsaicin-evoked hyperalgesia but not pain in normal skin in humans. Anesthesiology 1998, 89, 1060–1067.
- North, R.A. Molecular physiology of P2X receptors. Physiol. Rev. 2002, 82, 1013–1067.
- Jiang, L.H.; Kim, M.; Spelta, V.; Bo, X.; Surprenant, A.; North, R.A. Subunit arrangement in P2X receptors. J. Neurosci. 2003, 23, 8903–8910.
- Ding, S.; Sachs, F. Inactivation of P2X2 purinoceptors by divalent cations. J. Physiol. 2000, 522, 199–214, doi:10.1111/j.1469-7793.2000.t01-1-00199.x.
- Lewis, C.; Neidhart, S.; Holy, C.; North, R.A.; Buell, G.; Surprenant, A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature 1995, 377, 432–435.
- Torres, G.E.; Haines, W.R.; Egan, T.M.; Voigt, M.M. Co-expression of P2X1 and P2X5 receptor subunits reveals a novel ATP-gated ion channel. Mol. Pharmacol. 1998, 54, 989–993.
- Jarvis, M.F.; Khakh, B.S. ATP-gated P2X cation-channels. Neuropharmacology 2009, 56, 208–215, doi:10.1016/j.neuropharm.2008.06.067.
- Chessell, I.P.; Hatcher, J.P.; Bountra, C.; Michel, A.D.; Hughes, J.P.; Green, P.; Egerton, J.; Murfin, M.; Richardson, J.; Peck, W.L.; et al. Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain. Pain 2005, 114, 386–396, doi:10.1016/j.pain.2005.01.002.
- Tsuda, M.; Shigemoto-Mogami, Y.; Koizumi, S.; Mizokoshi, A.; Kohsaka, S.; Salter, M.W.; Inoue, K. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 2003, 424, 778–783.
- Clapham, D.E. TRP channels as cellular sensors. Nature 2003, 426, 517–524, doi:10.1038/nature02196.
- Jordt, S.E.; McKemy, D.D.; Julius, D. Lessons from peppers and peppermint: The molecular logic of thermosensation. Curr. Opin. Neurobiol. 2003, 13, 487–492, doi:10.1016/S0959-4388(03)00101-6.
- Ramsey, I.S.; Delling, M.; Clapham, D.E. An introduction to trp channels. Annu. Rev. Physiol. 2006, 68, 619–647, doi:10.1146/annurev.physiol.68.040204.100431.
- Garcia-Sanz, N.; Fernández-Carvajal, A.; Morenilla-Palao, C.; Planells-Cases, R.; Fajardo-Sánchez, E.; Fernández-Ballester, G.; Ferrer-Montiel, A. Identification of a tetramerization domain in the C terminus of the vanilloid receptor. J. Neurosci. 2004, 24, 5307–5314.
- Zhang, F.; Liu, S.; Yang, F.; Zheng, J.; Wang, K.W. Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels. J. Biol. Chem. 2011, 286, 15308–15316.
- Hofmann, T.; Schaefer, M.; Schultz, G.; Gudermann, T. Subunit composition of mammalian transient receptor potential channels in living cells. Proc. Natl. Acad. Sci. USA 2002, 99, 7461–7466.
- Strubing, C.; Krapivinsky, G.; Krapivinsky, L.; Clapham, D.E. Formation of novel TRPC channels by complex subunit interactions in embryonic brain. J. Biol. Chem. 2003, 278, 39014–39019.
- Goel, M.; Sinkins, W.G.; Schilling, W.P. Selective association of TRPC channel subunits in rat brain synaptosomes. J. Biol. Chem. 2002, 277, 48303–48310.
- Rutter, A.R.; Ma, Q.P.; Leveridge, M.; Bonnert, T.P. Heteromerization and colocalization of TrpV1 and TrpV2 in mammalian cell lines and rat dorsal root ganglia. Neuroreport 2005, 16, 1735–1739, doi:10.1097/01.wnr.0000185958.03841.0f.
- Smith, G.D.; Gunthorpe, M.J.; Kelsell, R.E.; Hayes, P.D.; Reilly, P.; Facer, P.; Wright, J.E.; Jerman, J.C.; Walhin, J.P.; Ooi, L.; et al. TRPV3 is a temperature-sensitive vanilloid receptor-like protein. Nature 2002, 418, 186–190.
- Cheng, W.; Yang, F.; Liu, S.; Colton, C.K.; Wang, C.; Cui, Y.Y.; Cao, X.; Zhu, M.X.; Sun, C.S.; Wang, K.W.; et al. Heteromeric heat-sensitive TRP channels exhibit distinct temperature and chemical response. J. Biol. Chem. 2011, doi:10.1074/jbc.M111.305045.
- Chubanov, V.; Waldegger, S.; Schnitzler, M.M.; Vitzthum, H.; Sassen, M.C.; Seyberth, H.W.; Konrad, M.; Gudermann, T. Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia. Proc. Natl. Acad. Sci. USA 2004, 101, 2894–2899.
- Xu, H.; Ramsey, I.S.; Kotecha, S.A.; Moran, M.M.; Chong, J.A.; Lawson, D.; Ge, P.; Lilly, J.; Silos-Santiago, I.; Xie, Y.; et al. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 2002, 418, 181–186.
- Cheng, W.; Yang, F.; Takanishi, C.L.; Zheng, J. Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties. J. Gen. Physiol. 2007, 129, 191–207, doi:10.1085/jgp.200709731.
- Latorre, R.; Brauchi, S.; Orta, G.; Zaelzer, C.; Vargas, G. ThermoTRP channels as modular proteins with allosteric gating. Cell Calcium 2007, 42, 427–438, doi:10.1016/j.ceca.2007.04.004.
- Akopian, A.N.; Ruparel, N.B.; Patwardhan, A.; Hargreaves, K.M. Cannabinoids desensitize capsaicin and mustard oil responses in sensory neurons via TRPA1 activation. J. Neurosci. 2008, 28, 1064–1075.
- Patil, M.; Patwardhan, A.; Salas, M.M.; Hargreaves, K.M.; Akopian, A.N. Cannabinoid receptor antagonists AM251 and AM630 activate TRPA1 in sensory neurons. Neuropharmacology 2011, 61, 778–788, doi:10.1016/j.neuropharm.2011.05.024.
- Bobanovic, L.K.; Royle, S.J.; Murrell-Lagnado, R.D. P2X receptor trafficking in neurons is subunit specific. J. Neurosci. 2002, 22, 4814–4824.
- Arniges, M.; Fernández-Fernández, J.M.; Albrecht, N.; Schaefer, M.; Valverde, M.A. Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. J. Biol. Chem. 2006, 281, 1580–1586.
- Ambudkar, I.S. Trafficking of TRP channels: Determinants of channel function. Handb. Exp. Pharmacol. 2007, 179, 541–557, doi:10.1007/978-3-540-34891-7_32.
- Pfeiffer, M.; Kirscht, S.; Stumm, R.; Koch, T.; Wu, D.; Laugsch, M.; Schröder, H.; Höllt, V.; Schulz, S. Heterodimerization of substance P and mu-opioid receptors regulates receptor trafficking and resensitization. J. Biol. Chem. 2003, 278, 51630–51637.
- Haas, K.F.; Macdonald, R.L. GABAA receptor subunit gamma2 and delta subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. J. Physiol. 1999, 514, 27–45, doi:10.1111/j.1469-7793.1999.027af.x.
- Akopian, A.N.; Ruparel, N.B.; Jeske, N.A.; Patwardhan, A.; Hargreaves, K.M. Role of ionotropic cannabinoid receptors in peripheral antinociception and antihyperalgesia. Trends Pharmacol. Sci. 2009, 30, 79–84, doi:10.1016/j.tips.2008.10.008.
- Bautista, D.M.; Jordt, S.E.; Nikai, T.; Tsuruda, P.R.; Read, A.J.; Poblete, J.; Yamoah, E.N.; Basbaum, A.I.; Julius, D. TRPA1 Mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 2006, 124, 1269–1282, doi:10.1016/j.cell.2006.02.023.
- Kang, K.; Pulver, S.R.; Panzano, V.C.; Chang, E.C.; Griffith, L.C.; Theobald, D.L.; Garrity, P.A. Analysis of drosophila TRPA1 reveals an ancient origin for human chemical nociception. Nature 2010, 464, 597–600.
- Gracheva, E.O.; Ingolia, N.T.; Kelly, Y.M.; Cordero-Morales, J.F.; Hollopeter, G.; Chesler, A.T.; Sánchez, E.E.; Perez, J.C.; Weissman, J.S.; Julius, D. Molecular basis of infrared detection by snakes. Nature 2010, 464, 1006–1011.
- Patil, M.J.; Belugin, S.; Akopian, A.N. Chronic alteration in phosphatidylinositol 4,5-biphosphate levels regulates capsaicin and mustard oil responses. J. Neurosci. Res. 2011, 89, 945–954, doi:10.1002/jnr.22597.
- Diogenes, A.; Akopian, A.N.; Hargreaves, K.M. NGF up-regulates TRPA1: Implications for orofacial pain. J. Dent. Res. 2007, 86, 550–555, doi:10.1177/154405910708600612.
- Patacchini, R.; Maggi, C.A.; Meli, A. Capsaicin-like activity of some natural pungent substances on peripheral endings of visceral primary afferents. Naunyn Schmiedebergs Arch. Pharmacol. 1990, 342, 72–77.
- Ruparel, N.B.; Patwardhan, A.M.; Akopian, A.N.; Hargreaves, K.M. Homologous and heterologous desensitization of capsaicin and mustard oil responses utilize different cellular pathways in nociceptors. Pain 2008, 135, 271–279, doi:10.1016/j.pain.2007.06.005.
- Jacquot, L.; Monnin, J.; Lucarz, A.; Brand, G. Trigeminal sensitization and desensitization in the nasal cavity: A study of cross interactions. Rhinology 2005, 43, 93–98.
- Simons, C.T.; Sudo, S.; Sudo, M.; Carstens, E. Mustard oil has differential effects on the response of trigeminal caudalis neurons to heat and acidity. Pain 2004, 110, 64–71, doi:10.1016/j.pain.2004.03.009.
- Bandell, M.; Story, G.M.; Hwang, S.W.; Viswanath, V.; Eid, S.R.; Petrus, M.J.; Earley, T.J.; Patapoutian, A. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004, 41, 849–857, doi:10.1016/S0896-6273(04)00150-3.
- Kollarik, M.; Undem, B.J. Activation of bronchopulmonary vagal afferent nerves with bradykinin, acid and vanilloid receptor agonists in wild-type and TRPV1−/− mice. J. Physiol. 2004, 555, 115–123, doi:10.1113/jphysiol.2003.054890.
- Dziedzicka-Wasylewska, M.; Faron-Górecka, A.; Górecki, A.; Kuśemider, M. Mechanism of action of clozapine in the context of dopamine D1-D2 receptor hetero-dimerization—A working hypothesis. Pharmacol. Rep. 2008, 60, 581–587.
- Jorenby, D.E.; Hays, J.T.; Rigotti, N.A.; Azoulay, S.; Watsky, E.J.; Williams, K.E.; Billing, C.B.; Gong, J.; Reeves, K.R. Varenicline Phase 3 Study Group. Efficacy of varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, versus placebo or sustained-release bupropion for smoking cessation: A randomized controlled trial. JAMA 2006, 296, 56–63.
- Maggio, R.; Barbier, P.; Colelli, A.; Salvadori, F.; Demontis, G.; Corsini, G.U. G protein-linked receptors: Pharmacological evidence for the formation of heterodimers. J. Pharmacol. Exp. Ther. 1999, 291, 251–257.
- Ciruela, F.; Casadó, V.; Rodrigues, R.J.; Luján, R.; Burgueño, J.; Canals, M.; Borycz, J.; Rebola, N.; Goldberg, S.R.; Mallol, J.; et al. Presynaptic control of striatal glutamatergic neurotransmission by adenosine A1-A2A receptor heteromers. J. Neurosci. 2006, 26, 2080–2087.
- Gabitzsch, E.S.; Xu, Y.N.; Balcaitis, S.; Balint, J.P., Jr.; Jones, F.R. An Ad5[E1-, E2b-]-HER2/neu vector induces immune responses and inhibits HER2/neu expressing tumor progression in Ad5 immune mice. Cancer Gene Ther. 2011, 18, 326–335, doi:10.1038/cgt.2010.82.
- Ellington, A.D.; Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818–822, doi:10.1038/346818a0.
- Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249, 505–510.
- Park, J.S.; Wang, C.Z.; Han, Y.; Huang, Z.; Niu, L. Potent and selective inhibition of a single alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit by an RNA aptamer. J. Biol. Chem. 2011, 286, 15608–15617.
- Chen, C.H.; Chernis, G.A.; Hoang, V.Q.; Landgraf, R. Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3. Proc. Natl. Acad. Sci. USA 2003, 100, 9226–9231.
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