Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes.

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

CB2 agonist mitigates cocaine-induced reinstatement of place preference and modulates the inflammatory response in mice.

Chronic exposure to cocaine is known to have profound effects on the brain, leading to the dysregulation of inflammatory signalling pathways, the activation of microglia, and the manifestation of cognitive and motivational behavioural impairments. The endocannabinoid system has emerged as a potential mediator of cocaine's deleterious effects. In this study, we sought to investigate the therapeutic potential of the cannabinoid CB2 receptor agonist, JWH-133, in mitigating cocaine-induced inflammation and associated motivational behavioural alterations in an in vivo model. Our research uncovered compelling evidence that JWH-133, a selective CB2 receptor agonist, exerts a significant dampening effect on the reinstatement of cocaine-induced conditioned place preference. This effect was accompanied by notable changes in the neurobiological landscape. Specifically, JWH-133 administration was found to upregulate Δ-FOSB expression in the nucleus accumbens (Nac), elevate CX3CL1 levels in both the ventral tegmental area and prefrontal cortex (PFC), and concurrently reduce IL-1ß expression in the PFC and NAc among cocaine-treated animals. These findings highlight the modulatory role of CB2 cannabinoid receptor activation in altering the reward-seeking behaviour induced by cocaine. Moreover, they shed light on the intricate interplay between the endocannabinoid system and cocaine-induced neurobiological changes, paving the way for potential therapeutic interventions targeting CB2 receptors in the context of cocaine addiction and associated behavioural deficits.

Morphine-induced mechanical hypersensitivity in mice requires δ receptors, ß-arrestin2, and c-Src activity.

Morphine diminishes pain, but its long-term use is compromised by tolerance and hyperalgesia. Studies implicate δ receptors, ß-arrestin2 and Src kinase in tolerance. We examined whether these proteins are also involved in morphine-induced hypersensitivity (MIH). A common pathway for tolerance and hypersensitivity may provide a single target to guide improved analgesic approaches. We examined mechanical sensitivity using automated von Frey in wild-type (WT) and transgenic male and female C57Bl/6 mice before and after hind paw inflammation by complete Freund's adjuvant (CFA). CFA-evoked hypersensitivity ceased on day 7 in WT but persisted for the 15-day testing period in µ-/- . Recovery was delayed until day 13 in δ-/- . We explored the expression of opioid genes in the spinal cord using quantitative RT-PCR. Restoration to basal sensitivity in WT occurred with increased δ expression. By contrast, κ expression was reduced, while µ remained unchanged. Daily morphine reduced hypersensitivity in WT on day 3 compared to controls; however, hypersensitivity recurred on day 9 and beyond. By contrast, WT had no recurrence of hypersensitivity in the absence of daily morphine. We used ß-arrestin2-/- , δ-/- and Src inhibition by dasatinib in WT to establish whether these approaches, which diminish tolerance, also attenuate MIH. While none of these approaches affected CFA-evoked inflammation or acute hypersensitivity, all caused sustained morphine anti-hypersensitivity, abolishing MIH. Like morphine tolerance, MIH in this model requires δ receptors, ß-arrestin2 and Src activity. Our findings suggest that MIH is caused by a tolerance-induced reduction in endogenous opioid signalling. KEY POINTS: Morphine is effective for treating severe acute pain, but tolerance and hypersensitivity often develop during its use in treating chronic pain. It is unclear whether these detrimental effects share similar mechanisms; if so, it might be possible to develop a single approach to minimise both phenomena. Mice deficient in µ receptors, δ receptors or ß-arrestin2 and wild type mice treated with the Src inhibitor dasatinib exhibit negligible morphine tolerance. We show that these same approaches also prevent the development of morphine-induced hypersensitivity during persistent inflammation. This knowledge identifies strategies, such as the use of Src inhibitors, which may mitigate tolerance and morphine induced hyperalgesia.

A genome-wide association study finds genetic variants associated with neck or shoulder pain in UK Biobank.

BACKGROUND: Common types of musculoskeletal conditions include pain in the neck and shoulder areas. This study seeks to identify the genetic variants associated with neck or shoulder pain based on a genome-wide association approach using 203 309 subjects from the UK Biobank cohort and look for replication evidence from the Generation Scotland: Scottish Family Health Study (GS:SFHS) and TwinsUK. METHODS: A genome-wide association study was performed adjusting for age, sex, BMI and nine population principal components. Significant and independent genetic variants were then sent to GS:SFHS and TwinsUK for replication. RESULTS: We identified three genetic loci that were associated with neck or shoulder pain in the UK Biobank samples. The most significant locus was in an intergenic region in chromosome 17, rs12453010, having P = 1.66 × 10-11. The second most significant locus was located in the FOXP2 gene in chromosome 7 with P = 2.38 × 10-10 for rs34291892. The third locus was located in the LINC01572 gene in chromosome 16 with P = 4.50 × 10-8 for rs62053992. In the replication stage, among four significant and independent genetic variants, rs2049604 in the FOXP2 gene and rs62053992 in the LINC01572 gene were weakly replicated in GS:SFHS (P = 0.0240 and P = 0.0202, respectively). CONCLUSIONS: We have identified three loci associated with neck or shoulder pain in the UK Biobank cohort, two of which were weakly supported in a replication cohort. Further evidence is needed to confirm their roles in neck or shoulder pain.

Amino acid substitutions in the human homomeric ß3 GABAA receptor that enable activation by GABA.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α1ß2γ2 receptor is the major subtype in the brain; GABA binds at the ß2(+)α1(-) interface. The structure of the homomeric ß3 GABAAR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ß3(+)α1(-) heteromeric interface in the homomeric human ß3 GABAAR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ß3 GABAAR variants containing Y87F, Q89R, and G152T. Reversion of Phe87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABAARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ß3 GABAAR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr87 prevents inhibitory effects of GABA.

Perioperative opioid analgesia-when is enough too much? A review of opioid-induced tolerance and hyperalgesia.

Opioids are a mainstay of acute pain management but can have many adverse effects, contributing to problematic long-term use. Opioid tolerance (increased dose needed for analgesia) and opioid-induced hyperalgesia (paradoxical increase in pain with opioid administration) can contribute to both poorly controlled pain and dose escalation. Hyperalgesia is particularly problematic as further opioid prescribing is largely futile. The mechanisms of opioid tolerance and hyperalgesia are complex, involving µ opioid receptor signalling pathways that offer opportunities for novel analgesic alternatives. The intracellular scaffold protein ß-arrestin-2 is implicated in tolerance, hyperalgesia, and other opioid side-effects. Development of agonists biased against recruitment of ß-arrestin-2 could provide analgesic efficacy with fewer side-effects. Alternative approaches include inhibition of peripheral µ opioid receptors and blockade of downstream signalling mechanisms, such as the non-receptor tyrosine kinase Src or N-methyl-D-aspartate receptors. Furthermore, it is prudent to use multimodal analgesic regimens to reduce reliance on opioids during the perioperative period. In the third paper in this Series we focus on clinical and mechanism-based understanding of tolerance and opioid-induced hyperalgesia, and discuss current and future strategies for pain management.

A Structural Rationale for N-Methylbicuculline Acting as a Promiscuous Competitive Antagonist of Inhibitory Pentameric Ligand-Gated Ion Channels.

Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABAA receptors and affects other pentameric ligand-gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence-quenching assay and isothermal titration calorimetry to record low-micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine-binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Šresolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABAA receptor-bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures, but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand-gated ion channels in the CNS.

Potent Inactivation-Dependent Inhibition of Adult and Neonatal NaV1.5 Channels by Lidocaine and Levobupivacaine.

BACKGROUND: Cardiotoxic effects of local anesthetics (LAs) involve inhibition of NaV1.5 voltage-gated Na channels. Metastatic breast and colon cancer cells also express NaV1.5, predominantly the neonatal splice variant (nNaV1.5) and their inhibition by LAs reduces invasion and migration. It may be advantageous to target cancer cells while sparing cardiac function through selective blockade of nNaV1.5 and/or by preferentially affecting inactivated NaV1.5, which predominate in cancer cells. We tested the hypotheses that lidocaine and levobupivacaine differentially affect (1) adult (aNaV1.5) and nNaV1.5 and (2) the resting and inactivated states of NaV1.5. METHODS: The whole-cell voltage-clamp technique was used to evaluate the actions of lidocaine and levobupivacaine on recombinant NaV1.5 channels expressed in HEK-293 cells. Cells were transiently transfected with cDNAs encoding either aNaV1.5 or nNaV1.5. Voltage protocols were applied to determine depolarizing potentials that either activated or inactivated 50% of maximum conductance (V½ activation and V½ inactivation, respectively). RESULTS: Lidocaine and levobupivacaine potently inhibited aNaV1.5 (IC50 mean [SD]: 20 [22] and 1 [0.6] µM, respectively) and nNaV1.5 (IC50 mean [SD]: 17 [10] and 3 [1.6] µM, respectively) at a holding potential of -80 mV. IC50s differed significantly between lidocaine and levobupivacaine with no influence of splice variant. Levobupivacaine induced a statistically significant depolarizing shift in the V½ activation for aNaV1.5 (mean [SD] from -32 [4.6] mV to -26 [8.1] mV) but had no effect on the voltage dependence of activation of nNaV1.5. Lidocaine had no effect on V½ activation of either variant but caused a significantly greater depression of maximum current mediated by nNaV1.5 compared to aNaV1.5. Similar statistically significant shifts in the V½ inactivation (approximately -10 mV) occurred for both LAs and NaV1.5 variants. Levobupivacaine (1 µM) caused a significantly greater slowing of recovery from inactivation of both variants than did lidocaine (10 µM). Both LAs caused approximately 50% tonic inhibition of aNaV1.5 or nNaV1.5 when holding at -80 mV. Neither LA caused tonic block at a holding potential of either -90 or -120 mV, voltages at which there was little steady-state inactivation. Higher concentrations of either lidocaine (300 µM) or levobupivacaine (100 µM) caused significantly more tonic block at -120 mV. CONCLUSIONS: These data demonstrate that low concentrations of the LAs exhibit inactivation-dependent block of NaV1.5, which may provide a rationale for their use to safely inhibit migration and invasion by metastatic cancer cells without cardiotoxicity.

Loop G in the GABAA receptor α1 subunit influences gating efficacy.

KEY POINTS: The functional importance of residues in loop G of the GABAA receptor has not been investigated. D43 and T47 in the α1 subunit are of particular significance as their structural modification inhibits activation by GABA. While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol potentiated maximal GABA-evoked currents mediated by α1(D43C)ß2γ2 and α1(T47R)ß2γ2 receptors. Non-stationary variance analysis revealed a reduction in maximal GABA-evoked Popen , suggesting impaired agonist efficacy. Further analysis of α1(T47R)ß2γ2 receptors revealed that the efficacy of the partial agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol) relative to GABA was impaired. GABA-, THIP- and propofol-evoked currents mediated by α1(T47R)ß2γ2 receptors deactivated faster than those mediated by α1ß2γ2 receptors, indicating that the mutation impairs agonist-evoked gating. Spontaneous gating caused by the ß2(L285R) mutation was also reduced in α1(T47R)ß2(L285R)γ2 compared to α1ß2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of agonist activation. ABSTRACT: The modification of cysteine residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G is known to impair activation of α1ß2γ2 receptors by GABA and propofol. While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol (1 µm), which potentiates sub-maximal but not maximal GABA-evoked currents mediated by α1ß2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)ß2γ2 and α1(T47R)ß2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)ß2γ2 and α1(T47R)ß2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)ß2γ2 and α1(T47R)ß2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol) was used to activate WT or α1(T47R)ß2γ2 receptors. Propofol-evoked currents mediated by α1(T47R)ß2γ2 and α1(F64C)ß2γ2 receptors also exhibited faster deactivation than their WT counterparts, revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the ß2(L285R) mutation was also reduced in α1(T47R)ß2(L285R)γ2 compared to α1ß2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's ß1 strand during agonist-dependent and spontaneous gating. Immobilisation of the ß1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline.

Src Kinase Inhibition Attenuates Morphine Tolerance without Affecting Reinforcement or Psychomotor Stimulation.

BACKGROUND: Prolonged opioid administration leads to tolerance characterized by reduced analgesic potency. Pain management is additionally compromised by the hedonic effects of opioids, the cause of their misuse. The multifunctional protein ß-arrestin2 regulates the hedonic effects of morphine and participates in tolerance. These actions might reflect µ opioid receptor up-regulation through reduced endocytosis. ß-Arrestin2 also recruits kinases to µ receptors. We explored the role of Src kinase in morphine analgesic tolerance, locomotor stimulation, and reinforcement in C57BL/6 mice. METHODS: Analgesic (tail withdrawal latency; percentage of maximum possible effect, n = 8 to 16), locomotor (distance traveled, n = 7 to 8), and reinforcing (conditioned place preference, n = 7 to 8) effects of morphine were compared in wild-type, µ, µ, and ß-arrestin2 mice. The influence of c-Src inhibitors dasatinib (n = 8) and PP2 (n = 12) was examined. RESULTS: Analgesia in morphine-treated wild-type mice exhibited tolerance, declining by day 10 to a median of 62% maximum possible effect (interquartile range, 29 to 92%). Tolerance was absent from mice receiving dasatinib. Tolerance was enhanced in µ mice (34% maximum possible effect; interquartile range, 5 to 52% on day 5); dasatinib attenuated tolerance (100% maximum possible effect; interquartile range, 68 to 100%), as did PP2 (91% maximum possible effect; interquartile range, 78 to 100%). By contrast, c-Src inhibition affected neither morphine-evoked locomotor stimulation nor reinforcement. Remarkably, dasatinib not only attenuated tolerance but also reversed established tolerance in µ mice. CONCLUSIONS: The ability of c-Src inhibitors to inhibit tolerance, thereby restoring analgesia, without altering the hedonic effect of morphine, makes c-Src inhibitors promising candidates as adjuncts to opioid analgesics.

A role for loop G in the ß1 strand in GABAA receptor activation.

KEY POINTS: The role of the ß1 strand in GABAA receptor function is unclear. It lies anti-parallel to the ß2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the ß1 strand of the GABAA ß3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47 in the GABAA α1 subunit showed that D43C and T47C reduced the apparent potency of GABA. F45C caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Cys43 and Cys47 were accessible to 2-aminoethyl methanethiosulphonate (MTSEA) modification, whereas Cys45 was not. Both GABA and the allosteric agonist propofol reduced MTSEA modification of Cys43 and Cys47. By contrast, modification of Cys64 in the ß2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of ß1 strand loop G residues during agonist activation of the GABAA receptor. ABSTRACT: The GABAA receptor α subunit ß1 strand runs anti-parallel to the ß2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the ß1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of ß1 strand residues in the GABAA ß3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the α1 subunit have an alternating pattern of high and low SASA consistent with a ß strand structure. We investigated the functional role of these ß1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1ß2γ2 receptors by 50-fold and eight-fold, respectively, whereas the F45C substitution caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Receptors with D43C or T47C substitutions were sensitive to 2-aminoethyl methanethiosulphonate (MTSEA) modification. However, GABA-evoked currents mediated by α1(F45C)ß2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)ß2γ2 and α1(T47C)ß2γ2 receptors, indicating movement of the ß1 strand even during allosteric activation. This is in contrast to α1(F64C)ß2γ2 receptors, where only GABA, but not propofol, reduced MTSEA modification. These findings provide the first functional evidence for movement of the ß1 strand during gating of the receptor and identify residues that are critical for maintaining GABAA receptor function.

The minimum M3-M4 loop length of neurotransmitter-activated pentameric receptors is critical for the structural integrity of cytoplasmic portals.

The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Šgap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.

Mutagenic analysis of the intracellular portals of the human 5-HT3A receptor.

Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg(436) and Arg(440)) within the MA helix of 5-hydroxytryptamine type 3A (5-HT3A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT3A receptor model. We probed the majority of residues within the MA helix of the human 5-HT3A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.

Early-life adversity increases morphine tolerance and persistent inflammatory hypersensitivity through upregulation of δ opioid receptors in mice.

ABSTRACT: Exposure to severely stressful events during childhood is associated with poor health outcomes in later life, including chronic pain and substance use disorder. However, the mediators and mechanisms are unclear. We investigated the impact of a well-characterized mouse model of early-life adversity, fragmented maternal care (FC) between postnatal day 2 and 9, on nociception, inflammatory hypersensitivity, and responses to morphine. Male and female mice exposed to FC exhibited prolonged basal thermal withdrawal latencies and decreased mechanical sensitivity. In addition, morphine had reduced potency in mice exposed to FC and their development of tolerance to morphine was accelerated. Quantitative PCR analysis in several brain regions and the spinal cords of juvenile and adult mice revealed an impact of FC on the expression of genes encoding opioid peptide precursors and their receptors. These changes included enhanced abundance of δ opioid receptor transcript in the spinal cord. Acute inflammatory hypersensitivity (induced by hind paw administration of complete Freund's adjuvant) was unaffected by exposure to FC. However, after an initial recovery of mechanical hypersensitivity, there was a reappearance in mice exposed to FC by day 15, which was not seen in control mice. Changes in nociception, morphine responses, and hypersensitivity associated with FC were apparent in males and females but were absent from mice lacking δ receptors or ß-arrestin2. These findings suggest that exposure to early-life adversity in mice enhances δ receptor expression leading to decreased basal sensitivity to noxious stimuli coupled with accelerated morphine tolerance and enhanced vulnerability to persistent inflammatory hypersensitivity.

Cross-sectional study examining the epidemiology of chronic pain in Nepal.

Introduction: The World Health Organization recognizes chronic pain as a global public health concern; however, there is a bias towards research conducted in relatively affluent nations. There is a dearth of large-scale epidemiological studies in Nepal using rigorously validated, cross-culturally adapted instruments. Objectives: The aim of this study was to examine the prevalence of both chronic pain and chronic pain of predominantly neuropathic origin and their associations with a range of sociodemographic and psychosocial characteristics. Methods: We conducted a cross-sectional study of adults (≥18 years) in all households in Ranipani, Baluwa Village Development Committee, Nepal. All adults (n = 887) were approached, and those consenting, who met the inclusion criteria (n = 520, 58.6%), participated. Questionnaires validated in Nepali were used to examine several constructs: demographics; chronic pain; neuropathic pain; pain catastrophizing; resilience, pain intensity; pain interference; sleep disturbance; and depression. Results: The point prevalence of chronic pain was 53.3% (n = 277). The point prevalence of chronic pain of predominantly neuropathic origin was 12.7% (n = 66). Chronic pain was associated with female gender, older age, and manual labour occupations. Using standardized scoring techniques, compared with available population estimates from other countries, those with chronic pain were associated with lower pain intensity and resilience scores and higher pain catastrophizing, pain interference, and depression scores. Conclusion: These findings are broadly comparable to epidemiological studies from other countries, and these indicate areas for targeting interventions (eg, occupational and mental health). For comparison, more data are needed, from larger population samples in this region.

Influences on blockade by t-butylbicyclo-phosphoro-thionate of GABA(A) receptor spontaneous gating, agonist activation and desensitization.

Picrotoxin and t-butylbicyclophosphorothionate (TBPS) are GABA(A) receptor (GABA(A)R) open channel blockers. However, picrotoxin displaceable [(35)S]TBPS binding to α1ß2γ2 GABA(A)Rs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 ± 3% and 85 ± 5%, respectively) GABA-evoked currents mediated by α1ß2γ2 receptors. We used two approaches to inhibit spontaneous GABA(A)R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the α1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that α1(K278M)ß2γ2 receptors have negligible spontaneous gating. Application of bicuculline to α1ß2γ2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [(35)S]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, α1(K278M)ß2γ2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced α1ß2γ2 receptor blockade (to 98% in both cases). The GABA-dependent component of TBPS blockade accounts for the stimulation of [(35)S]TBPS binding to α1ß2γ2 receptors seen with GABA (1 µm) application. Moreover, application of GABA at concentrations that cause significant steady-state desensitization reduced [(35)S]TBPS binding. The α1(K278M) subunit slowed desensitization kinetics and increased the rate of deactivation of GABA-evoked currents. Furthermore, there was a marked increase in the GABA EC(50) for desensitization of α1(K278M)ß2γ2 receptors associated with a large increase in the GABA-dependent stimulation of [(35)S]TBPS binding. These data establish a relationship between GABA(A)R function and the three phases of [(35)S]TBPS binding seen in the absence and the presence of GABA.

Fluorophore assisted light inactivation (FALI) of recombinant 5-HT3A receptor constitutive internalization and function.

Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin3 A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT3A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT3A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking.

Traumatic needle damage to nerves during regional anesthesia: presentation of a novel mechanotransduction hypothesis.

Despite advances in needle positioning techniques, nerve damage still occurs after regional anesthesia. Recognized causes include local anesthetic toxicity, subperineural injection, high subepineural fluid injection pressures and subepineural hematoma after forceful needle--nerve contact.We hypothesize that subperineural injection is still possible, but less likely to be the cause of nerve damage because needle penetration of fascicles and mechanical damage is difficult to achieve. High-resolution (75 µm) 40 MHz micro-ultrasound images of pig axillae show short-bevelled 22 g, 0.7 mm wide block needles that are three times larger than the average fascicle. Fascicular bundles are extremely difficult to puncture because they spin away on needle contact. Histology from fresh cadavers after supposed intrafascicular injection shows fluid spread within perineurium and intrafascicular perineural septae, but no breach of endoneurium or axons.We propose that mechanotransduction, the cellular changes that occur in response to force, contributes to nerve damage. Piezo ion channel proteins transduce force into electrical activity by rapid entry of cations into cells. Excessive Ca2+ influx into cells has the potential to inhibit nerve regeneration. Cellular changes include regulation of gene expression. The forces associated with purposeful needle insertion are generally unknown. Our experiments in the soft embalmed Thiel cadaver showed a lognormal range of forces between 0.6 N and 16.8 N on epineural penetration.We hypothesize that forceful needle injury may cause nerve damage by activation of Piezo receptors and release of intracellular Ca2.

Validation of the Nepali Version of the Self-reported Leeds Assessment of Neuropathic Symptoms and Signs (S-LANSS) in Adults With Chronic Pain and Predominantly Low-literacy Levels.

Neuropathic pain research and clinical care is limited in low- and middle-income countries with high prevalence of chronic pain such as Nepal. We translated and cross-culturally adapted the Self-report version of the Leeds Assessment of Neuropathic Symptoms and Signs (S-LANSS)-a commonly used, reliable and valid instrument to screen for pain of predominantly neuropathic origin (POPNO)-into Nepali (S-LANSS-NP) and validated it using recommended guidelines. We recruited 30 patients with chronic pain in an outpatient setting for cognitive debriefing and recruited 287 individuals with chronic pain via door-to-door interviews for validation. For known-group validity, we hypothesized that the POPNO group would report significantly more pain intensity and pain interference than the chronic pain group without POPNO using a cut-off score of ≥10/24. The S-LANSS-NP was comprehensible based on the ease of understanding the questionnaire and lack of missing responses. The validation sample consisted of predominantly low-levels of literacy (81% had 5 years or less education); 23% were classified as having POPNO. Internal consistency was good (alpha = .80). Known-group validity was supported (chronic pain with POPNO reported significantly greater pain intensity than those without). The S-LANSS-NP is a comprehensible, unidimensional, internally consistent, and valid instrument to screen POPNO in individuals with chronic pain with predominantly low-levels of literacy for clinical and research use. PERSPECTIVE: This paper shows that the Nepali version of the S-LANSS is comprehensible, reliable and valid in adults with chronic pain and predominantly low-levels of literacy in rural Nepal. The study could potentially develop research and clinical care of neuropathic pain in this resource-limited setting where chronic pain is a significant problem.

Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking ß-arrestin 2.

Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of µ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs) over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including µ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of ß-arrestin 2 (ß-arr2) augments the constitutive coupling of µ receptors to voltage-activated Ca²+ channels in primary afferent dorsal root ganglion neurons from ß-arr2⁻/⁻ mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type µ receptors in neurons. We administered these agents to ß-arr2⁻/⁻ mice to explore the role of constitutive µ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive µ receptor activity in vivo in ß-arr2⁻/⁻ mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in ß-arr2⁻/⁻ and ß-arr2+/+ mice, suggesting that hedonic tone was unaffected.