Human Μ-opioid Receptor Research Articles

Pharmacologic Profile of Naloxegol, a Peripherally Acting µ-Opioid Receptor Antagonist, for the Treatment of Opioid-Induced Constipation

Opioid-induced constipation (OIC) is a common side effect of opioid pharmacotherapy for the management of pain because opioid agonists bind to µ-opioid receptors in the enteric nervous system (ENS). Naloxegol, a polyethylene glycol derivative of naloxol, which is a derivative of naloxone and a peripherally acting µ-opioid receptor antagonist, targets the physiologic mechanisms that cause OIC. Pharmacologic measures of opioid activity and pharmacokinetic measures of central nervous system (CNS) penetration were employed to characterize the mechanism of action of naloxegol. At the human µ-opioid receptor in vitro, naloxegol was a potent inhibitor of binding (Ki = 7.42 nM) and a neutral competitive antagonist (pA2 - 7.95); agonist effects were <10% up to 30 μM and identical to those of naloxone. The oral doses achieving 50% of the maximal effect in the rat for antagonism of morphine-induced inhibition of gastrointestinal transit and morphine-induced antinociception in the hot plate assay were 23.1 and 55.4 mg/kg for naloxegol and 0.69 and 1.14 mg/kg by for naloxone, respectively. In the human colon adenocarcinoma cell transport assay, naloxegol was a substrate for the P-glycoprotein transporter, with low apparent permeability in the apical to basolateral direction, and penetrated the CNS 15-fold slower than naloxone in a rat brain perfusion model. Naloxegol-derived radioactivity was poorly distributed throughout the rat CNS and was eliminated from most tissues within 24 hours. These findings corroborate phase 3 clinical studies demonstrating that naloxegol relieves OIC-associated symptoms in patients with chronic noncancer pain by antagonizing the µ-opioid receptor in the ENS while preserving CNS-mediated analgesia.

Brain substrates of reward processing and the μ-opioid receptor: a pathway into pain?

The processing of reward and reinforcement learning seems to be important determinants of pain chronicity. However, reward processing is already altered early in life and if this is related to the development of pain symptoms later on is not known. The aim of this study was first to examine whether behavioural and brain-related indicators of reward processing at the age of 14 to 15 years are significant predictors of pain complaints 2 years later, at 16 to 17 years. Second, we investigated the contribution of genetic variations in the opioidergic system, which is linked to the processing of both, reward and pain, to this prediction. We used the monetary incentive delay task to assess reward processing, the Children's Somatization Inventory as measure of pain complaints and tested the effects of 2 single nucleotide polymorphisms (rs1799971/rs563649) of the human μ-opioid receptor gene. We found a significant prediction of pain complaints by responses in the dorsal striatum during reward feedback, independent of genetic predisposition. The relationship of pain complaints and activation in the periaqueductal gray and ventral striatum depended on the T-allele of rs563649. Carriers of this allele also showed more pain complaints than CC-allele carriers. Therefore, brain responses to reward outcomes and higher sensitivity to pain might be related already early in life and may thus set the course for pain complaints later in life, partly depending on a specific opioidergic genetic predisposition.

Lack of Association between Human µ-Opioid Receptor (&lt;em&gt;OPRM1&lt;/em&gt;) Gene Polymorphisms and Heroin Addiction in A Sample of Southeast Iranian Population

It has been proposed that genetic factors account for 30%–50% of the risk for cocaine and heroin addiction. The present study was aimed to find out the impact of µ-opioid receptor gene ( OPRM1 ) rs1799971 A > G and rs9479757 polymorphisms on heroin dependence in a sample of southeast Iranian population. This case-control study was done on 123 heroin addicts and 140 non-addicts Iranian male. Genomic DNA was extracted from peripheral blood cells using salting out method. Genotyping of OPRM1 rs1799971 and rs9479757 polymorphisms were performed using PCR-RFLP method. Overall, our results did not support an association between OPRM1 variants and risk of heroin dependence in a sample of southeast Iranian population. Further studies with larges sample sizes and different ethnicities are required to validate our findings.

Chemists probe kratom’s neurochemistry

For recreational drug users looking for an opioidlike high without the legal problems of heroin, fentanyl, and oxycodone, the Southeast Asian plant called kratom (Mitragyna speciosa) has provided an attractive alternative. But, acting on anecdotal reports of people becoming dependent on kratom, six states, including Vermont and Indiana, have banned the sale and use of the herb. A new study provides some data to support those states’ concerns (J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b00360). A team of researchers shows for the first time that kratom’s primary constituent, mitragynine, and four related alkaloids bind to and partially activate human µ-opioid receptors (MORs), the primary targets of strong opioids in the brain, spinal cord, and gastrointestinal tract. The most potent of the related alkaloids was the mitragynine oxidation product 7-hydroxymitragynine. Its MOR potency was one-tenth that of morphine, while mitragynine’s was one-hundredth. “Mitragynine is not a particularly very potent opioid,...

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human μ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines.

Human μ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.

Mouse model of OPRM1 (A118G) polymorphism has altered hippocampal function

A single nucleotide polymorphism (SNP) in the human μ-opioid receptor gene (OPRM1 A118G) has been widely studied for its association in a variety of drug addiction and pain sensitivity phenotypes; however, the extent of these adaptations and the mechanisms underlying these associations remain elusive. To clarify the functional mechanisms linking the OPRM1 A118G SNP to altered phenotypes, we used a mouse model possessing the equivalent nucleotide/amino acid substitution in the Oprm1 gene. In order to investigate the impact of this SNP on circuit function, we used voltage-sensitive dye imaging in hippocampal slices and in vivo electroencephalogram recordings of the hippocampus following MOPR activation. As the hippocampus contains excitatory pyramidal cells whose activity is highly regulated by a dense network of inhibitory neurons, it serves as an ideal structure to evaluate how putative receptor function abnormalities may influence circuit activity. We found that MOPR activation increased excitatory responses in wild-type animals, an effect that was significantly reduced in animals possessing the Oprm1 SNP. Furthermore, in order to assess the in vivo effects of this SNP during MOPR activation, EEG recordings of hippocampal activity following morphine administration corroborated a loss-of-function phenotype. In conclusion, as these mice have been shown to have similar MOPR expression in the hippocampus between genotypes, these data suggest that the MOPR A118G SNP results in a loss of receptor function.

The Impact of Genetic Variation on Sensitivity to Opioid Analgesics in Patients with Postoperative Pain: A Systematic Review and Meta-Analysis

Background: Individual response to opioid analgesics varies among patients. Objective: This study sought to clarify the impact of distinct genetic variations on pain, opioid consumption, and opioid side effects in patients with postoperative pain. Study Design: A systematic review and meta-analysis of associations between genetic singlenucleotide polymorphisms (SNPs) and opioids used for acute postoperative pain. Setting: This meta-analysis examined all studies involving an association between genetic polymorphisms and the analgesic efficacy or clinical outcome of opioid analgesics for postoperative pain. Methods: A literature search was performed up to January 31, 2014, using the PubMed, EMBase, ISI Web of Science, and Cochrane Library databases. Results: Fifty-nine studies were included in this systematic review, and 23 studies (a total of 5,902 patients) were included in the final meta-analysis. The results showed that human μ-opioid receptor gene (OPRM1) 118G allele variant carriers consumed more opioids for analgesia (SMD = -0.17, 95% CI = [-0.25, -0.10], P &lt; 0.00001), but reported higher pain scores (MD = -0.11, 95% CI = [-0.17, -0.04], P = 0.002) and less nausea and vomiting (odds ratio = 1.30, 95% CI = [1.08, 1.55], P = 0.005) than the homozygous 118AA patients during the first 24 hour but not the 48 hour postoperative period. Moreover, CYP3A4*1G carriers consumed less opioids than homozygous CYP3A4*1/*1 patients during the first 24 hours postoperative period (MD = 45.12, 95% CI = [36.17, 54.06], P &lt; 0.00001). No significant differences were found in CYP3A5*3, ABCB1 C3435T, and G2477T/A genetic polymorphisms. Limitations: Some potential non-genetic factors can modify the effects of gene SNP on pain and opioid consumption during the postoperative period, such as age, gender, mood, anxiety, and drug-drug interactions. But further analyses could not be performed in the present meta-analysis due to limited information. Conclusion: The results indicate that among the genetic SNPs we studied which include those affecting analgesic drug metabolism, transport of analgesic agents across the blood-brain barrier, and their activity at target receptors and ion channels and in the modulation of neurotransmitter pathways, the A118G allele variant of OPRM1 has the most potent influence on pain management of postoperative patients. Opioid receptor gene information may provide valuable information for clinicians to properly manage the analgesic use of opioids individually for better pain management. Key words: Postoperative pain, meta-analysis, single-nucleotide polymorphism, opioid

Mouse model of OPRM1 (A118G) polymorphism increases sociability and dominance and confers resilience to social defeat.

A single nucleotide polymorphism (SNP) in the human μ-opioid receptor gene (OPRM1 A118G) has been widely studied for its association in drug addiction, pain sensitivity, and, more recently, social behavior. The endogenous opioid system has been shown to regulate social distress and reward in a variety of animal models. However, mechanisms underlying the associations between the OPRM1 A118G SNP and these behaviors have not been clarified. We used a mouse model possessing the human equivalent nucleotide/amino acid substitution to study social affiliation and social defeat behaviors. In mice with the Oprm1 A112G SNP, we demonstrate that the G allele is associated with an increase in home-cage dominance and increased motivation for nonaggressive social interactions, similar to what is reported in human populations. When challenged by a resident aggressor, G-allele carriers expressed less submissive behavior and exhibited resilience to social defeat, demonstrated by a lack of subsequent social avoidance and reductions in anhedonia as measured by intracranial self-stimulation. Protection from social defeat in G-allele carriers was associated with a greater induction of c-fos in a resilience circuit comprising the nucleus accumbens and periaqueductal gray. These findings led us to test the role of endogenous opioids in the A112G mice. We demonstrate that the increase in social affiliation in G carriers is blocked by pretreatment with naloxone. Together, these data suggest a mechanism involving altered hedonic state and neural activation as well as altered endogenous opioid tone in the differential response to aversive and rewarding social stimuli in G-allele carriers.

Characterization of a computationally designed water-soluble human μ-opioid receptor variant using available structural information.

The recent X-ray crystal structure of the murine μ-opioid receptor (MUR) allowed the authors to reengineer a previously designed water-soluble variant of the transmembrane portion of the human MUR (wsMUR-TM). The new variant of water-soluble MUR (wsMUR-TM_v2) was engineered based on the murine MUR crystal structure. This novel variant was expressed in Escherichia coli and purified. The properties of the receptor were characterized and compared with those of wsMUR-TM. Seven residues originally included for mutation in the design of the wsMUR-TM were reverted to their native identities. wsMUR-TM_v2 contains 16% mutations of the total sequence. It was overexpressed and purified with high yield. Although dimers and higher oligomers were observed to form over time, the wsMUR-TM_v2 stayed predominantly monomeric at concentrations as high as 7.5 mg/ml in buffer within a 2-month period. Its secondary structure was predominantly helical and comparable with those of both the original wsMUR-TM variant and the native MUR. The binding affinity of wsMUR-TM_v2 for naltrexone (K(d) approximately 70 nM) was in close agreement with that for wsMUR-TM. The helical content of wsMUR-TM_v2 decreased cooperatively with increasing temperature, and the introduction of sucrose was able to stabilize the protein. A novel functional wsMUR-TM_v2 with only 16% mutations was successfully engineered, expressed in E. coli, and purified based on information from the crystal structure of murine MUR. This not only provides a novel alternative tool for MUR studies in solution conditions but also offers valuable information for protein engineering and structure-function relations.

Overview of genetic analysis of human opioid receptors.

The human μ-opioid receptor gene (OPRM1), due to its genetic and structural variation, has been a target of interest in several pharmacogenetic studies. The μ-opioid receptor (MOR), encoded by OPRM1, contributes to regulate the analgesic response to pain and also controls the rewarding effects of many drugs of abuse, including opioids, nicotine, and alcohol. Genetic polymorphisms of opioid receptors are candidates for the variability of clinical opioid effects. The non-synonymous polymorphism A118G of the OPRM1 has been repeatedly associated with the efficacy of opioid treatments for pain and various types of dependence. Genetic analysis of human opioid receptors has evidenced the presence of numerous polymorphisms either in exonic or in intronic sequences as well as the presence of synonymous coding variants that may have important effects on transcription, mRNA stability, and splicing, thus affecting gene function despite not directly disrupting any specific residue. Genotyping of opioid receptors is still in its infancy and a relevant progress in this field can be achieved by using advanced gene sequencing techniques described in this review that allow the researchers to obtain vast quantities of data on human genomes and transcriptomes in a brief period of time and with affordable costs.

Characteristics of TRK-130 (Naltalimide), a novel opioid ligand, as a new therapeutic agent for overactive bladder.

We characterized TRK-130 (N-[(5R,6R,14S)-17-(cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6-yl]phthalimide; naltalimide), an opioid ligand, to clarify the therapeutic potential for overactive bladder (OAB). In radioligand-binding assays with cells expressing human µ-opioid receptors (MORs), δ-opioid receptors (DORs), or κ-opioid receptors (KORs), TRK-130 showed high selectivity for MORs (Ki for MORs, DORs, and KORs = 0.268, 121, and 8.97 nM, respectively). In a functional assay (cAMP accumulation) with cells expressing each human opioid receptor subtype, TRK-130 showed potent but partial agonistic activity for MORs [EC50 (Emax) for MORs, DORs, and KORs = 2.39 nM (66.1%), 26.1 nM (71.0%), and 9.51 nM (62.6%), respectively]. In isovolumetric rhythmic bladder contractions (RBCs) in anesthetized guinea pigs, TRK-130 dose-dependently prolonged the shutdown time (the duration of complete cessation of the bladder contractions) (ED30 = 0.0034 mg/kg i.v.) without affecting amplitude of RBCs. Furthermore, TRK-130 ameliorated formalin-induced frequent urination at doses of higher than 0.01 mg/kg p.o. in guinea pigs under the freely moving condition. Meanwhile, TRK-130 showed only a negligible effect on the gastrointestinal transit at doses of up to 10 mg/kg s.c. in mice. These results indicate that TRK-130 is a potent and selective human MOR partial agonist without undesirable opioid adverse effects such as constipation and enhances the storage function by suppressing the afferent limb of the micturition reflex pathway, suggesting that TRK-130 would be a new therapeutic agent for OAB.

µ-Opioid Receptor Gene Variant OPRM1 118 A&gt;G: A Summary of its Molecular and Clinical Consequences for Pain

The human µ-opioid receptor variant 118 A>G (rs1799971) has become one of the most analyzed genetic variants in the pain field. At the molecular level, the variant reduces opioid receptor signaling efficiency and expression, the latter probably via a genetic-epigenetic interaction. In experimental settings, the variant was reproducibly associated with decreased effects of exogenous opioids. However, this translates into very small clinical effects (meta-analysis of 14 studies: Cohen's d = 0.096; p = 0.008), consisting of slightly higher opioid dosing requirements in peri- and post-operative settings. An effect can neither be maintained for chronic analgesic therapy nor for opioid side effects. It seems unlikely that further studies will reveal larger effect sizes and, therefore, further analyses appear unwarranted. Thus, due to its small effect size, the SNP is without major clinical relevance as a solitary variant, but should be regarded as a part of complex genotypes underlying pain and analgesia.

The familial hemiplegic migraine type 1 mutation K1336E affects direct G protein-mediated regulation of neuronal P/Q-type Ca2+ channels

Familial hemiplegic migraine type 1 (FHM-1) is an autosomal dominant form of migraine with aura characterized by recurrent migraine, hemiparesis and ataxia. FHM-1 has been linked to missense mutations in the CACNA1A gene encoding the pore-forming subunit of the neuronal voltage-gated P/Q-type Ca(2+) channel (CaV2.1α1). Here, we explored the effects of the FHM-1 K1336E mutation on G protein-dependent modulation of the recombinant P/Q-type channel. The mutation was introduced into the human CaV2.1α1 subunit and its functional consequences investigated after heterologous expression in HEK-293 cells using patch-clamp recordings. Functional analysis of the K1336E mutation revealed a reduction of Ca(2+) current densities, a ∼10 mV left-shift in the current-voltage relationship, and the slowing of current inactivation kinetics. When co-expressed along with the human μ-opioid receptor, application of the agonist DAMGO inhibited whole-cell currents through both the wild-type and the mutant channels. Prepulse facilitation was also reduced by the K1336E mutation. Likewise, the kinetic analysis of the onset and decay of facilitation showed that the mutation affects the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex. These results suggest that the extent of G-protein-mediated inhibition is significantly reduced in the K1336E mutant CaV2.1 Ca(2+) channels. This alteration would contribute to render the neuronal network hyperexcitable, possibly as a consequence of reduced presynaptic inhibition, and may help to explain some aspects of the FHM-1 pathophysiology.

Regulation of mammalian MOR-1 gene expression after chronic treatment with morphine

Morphine is an effective analgesic that acts by binding to the μ-opioid receptor (MOR) coded in the human by the OPRM1 gene. In the present study, we investigated the regulation of μ-opioid receptor (MOR-1) mRNA levels in all-trans-retinoic acid-differentiated SH-SY5Y human neuroblastoma cells under in vitro conditions with 10 μM morphine treatment for 24 h. In addition, we measured the MOR-1 levels in recombinant Chinese hamster ovary (CHO) cells, transfected with human μ-opioid receptor gene (hMOR) with 10 μM morphine treatment for 24 h. The isolated mRNA from these cells was subjected to real-time quantitative RT-PCR analysis to determine the regulation of μ-opioid receptor gene expression. It was observed that morphine treatment did not alter MOR-1 levels in undifferentiated SH-SY5Y cells compared to undifferentiated control cells. However, the MOR-1 levels in all-trans-retinoic acid-differentiated cells were significantly higher compared to the undifferentiated cells. Morphine treatment in differentiated SH-SY5Y cells caused significant downregulation of MOR-1 expression compared to the control cells. In the morphine-treated CHO cells, the hMOR-1 mRNA levels remained the same as the untreated control. Finally, pretreatment of SH-SY5Y cells with 10 μM naloxone, the antagonist of μ-opioid receptor, for 1 h significantly blocked the downregulation of MOR-1 mRNA levels with morphine treatment. These findings suggest that regulation of MOR-1 gene expression is cell-type specific after chronic morphine treatment and provide some evidence in the understanding of morphine tolerance.

The A118G Single-Nucleotide Polymorphism of Human µ-Opioid Receptor Gene and Use of Labor Analgesia

The human µ-opioid receptor (MOR) is the major site of action of endogenous opioids and most of the clinically used opioid analgesics. The single-nucleotide polymorphism (SNP), A118G of the MOR 1 gene (OPRM1), has been associated with altered pain perception. The aim of this study was to investigate whether this polymorphism of OPRM1 is associated with a number of pain-related behaviors during labor. In this observational retrospective population-based study, pregnant women (n = 814) were recruited at gestational week 18. A plasma sample was collected from each participant and an SNP genotyping assay was performed. No differences in sociodemographic variables or labor pain-related outcomes, such as stage of cervical dilation on arrival at the delivery unit or use of any type of second-line analgesia during spontaneous labor, were found between noncarriers and G-allele carriers of OPRM1. We conclude that there is no association between the A118G polymorphism of OPRM1 regarding pain-related behavior during labor.

Brain region‐ and sex‐specific alterations in DAMGO‐stimulated [35S]GTPγS binding in mice with Oprm1A112G

The A118G single nucleotide polymorphism (SNP) of the human μ-opioid receptor (MOPR) gene (OPRM1) was associated with heightened dopamine release by alcohol intake, better treatment outcome for nicotine and alcohol addiction, and reduced analgesic responses to morphine. A mouse model that possesses the equivalent substitution (A112G) in the mouse MOPR gene (OPRM1) was generated to delineate the mechanisms of the impact of the SNP. Mice homozygous for the G112 allele (G/G) displayed lower morphine-induced antinociception than mice homozygous for the A112 allele (A/A), similar to the results in humans. In this study, we examined whether A112G SNP affected MOPR-mediated G protein activation in the mouse model. We compared A/A and G/G mice in the MOPR-selective agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO)-stimulated [(35) S]GTPγS binding in brain regions by autoradiography. When the data of males and females were combined, G/G mice exhibited lower DAMGO-stimulated [(35) S]GTPγS binding in the ventral tegmental area than A/A mice, in accord with the previously reported reduced morphine-induced hyperactivity and locomotor sensitization in G/G mice. In the nucleus accumbens (NAc) core, female G/G mice displayed lower DAMGO-stimulated [(35) S]GTPγS binding than female A/A mice, which is consistent with the previously reported deficiency in morphine-induced conditioned place preference in female G/G mice. In G/G mice, males showed higher DAMGO-stimulated [(35) S]GTPγS binding than females in the cingulate cortex, caudate putamen, NAc core, thalamus and amygdala. Thus, A112G SNP affects DAMGO-stimulated [(35) S]GTPγS binding in region- and sex-specific manners.

The human μ-opioid receptor gene polymorphism (A118G) is associated with head pain severity in a clinical cohort of female migraine with aura patients

Migraine is a painful and debilitating, neurovascular disease. Current migraine head pain treatments work with differing efficacies in migraineurs. The opioid system plays an important role in diverse biological functions including analgesia, drug response and pain reduction. The A118G single nucleotide polymorphism (SNP) in exon 1 of the μ-opioid receptor gene (OPRM1) has been associated with elevated pain responses and decreased pain threshold in a variety of populations. The aim of the current preliminary study was to test whether genotypes of the OPRM1 A118G SNP are associated with head pain severity in a clinical cohort of female migraineurs. This was a preliminary study to determine whether genotypes of the OPRM1 A118G SNP are associated with head pain severity in a clinical cohort of female migraineurs. A total of 153 chronic migraine with aura sufferers were assessed for migraine head pain using the Migraine Disability Assessment Score instrument and classified into high and low pain severity groups. DNA was extracted and genotypes obtained for the A118G SNP. Logistic regression analysis adjusting for age effects showed the A118G SNP of the OPRM1 gene to be significantly associated with migraine pain severity in the test population (P = 0.0037). In particular, G118 allele carriers were more likely to be high pain sufferers compared to homozygous carriers of the A118 allele (OR = 3.125, 95 % CI = 1.41, 6.93, P = 0.0037). These findings suggest that A118G genotypes of the OPRM1 gene may influence migraine-associated head pain in females. Further investigations are required to fully understand the effect of this gene variant on migraine head pain including studies in males and in different migraine subtypes, as well as in response to head pain medication.

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of CaV2.1 voltage-gated Ca2+ channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human CaV2.1α1 subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK‐293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant CaV2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca2+ channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.

Ets-1 Activates Overexpression of JunB and CD30 in Hodgkin's Lymphoma and Anaplastic Large-Cell Lymphoma

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.

Pharmacogenomics of the Human µ-Opioid Receptor

The µ-opioid receptor is a primary target for clinically important opioid analgesics, including morphine, fentanyl and methadone. Many genetic variations have been identified in the human µ-opioid receptor MOP gene (OPRM1), and their implications have been reported in the effects of opioid drugs and susceptibility to drug dependence. Interestingly, agonistic and antagonistic opioid effects are inversely associated with the A118G polymorphism genotype. The A118G polymorphism may also be associated with substance dependence and susceptibility to other disorders, including epilepsy and schizophrenia. The IVS1+A21573G, IVS1-T17286C, and TAA+A5359G polymorphisms in the OPRM1 gene may be associated with alcohol, opioid and tobacco dependence, respectively. However, some studies have failed to confirm the correlations between the polymorphisms and opioid effects and substance dependence. Further studies are needed to elucidate the molecular mechanisms underlying the effects of OPRM1 polymorphisms.