Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABA B receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABA B receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapyinduced neuropathic pain.pain | chemotherapy | alpha9 | nicotinic

Angiotensin signaling promotes interactions between AKAP150, PKC, and TRPV4 channels to form signaling domains that control Ca2+ influx into arterial myocytes.

TRPV2 is a member of the transient receptor potential family of ion channels involved in chemical and thermal pain transduction. Unlike the related TRPV1 channel, TRPV2 does not appear to bind either calmodulin or ATP in its N-terminal ankyrin repeat domain. In addition, it does not contain a calmodulin-binding site in the distal C-terminal region, as has been proposed for TRPV1. We have found that TRPV2 channels transiently expressed in F-11 cells undergo Ca 2ϩ -dependent desensitization, similar to the other TRPVs, suggesting that the mechanism of desensitization may be conserved in the subfamily of TRPV channels. TRPV2 desensitization was not altered in whole-cell recordings in the presence of calmodulin inhibitors or on coexpression of mutant calmodulin but was sensitive to changes in

For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABA A receptor ␣ 1 (K219, R220, K221) and ␤ 2 (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type ␤ 2 or ␣ 1 in Xenopus oocytes. Cysteine substitution of ␤ 2 R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3 H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto ␣ 1 K219C, ␤ 2 K213C, and ␤ 2 K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of ␣ 1 K219C, ␤ 2 K213C, and ␤ 2 K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified ␤ 2 K213C and ␤ 2 K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of ␤ 2 K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3'-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.

Plasmid vectors used for mammalian expression or for in vitro cRNA translation can differ substantially and are rarely cross-compatible. To make comparisons between mammalian and Xenopus oocyte expression systems, it would be advantageous to use a single vector without the need for shuttle vectors or subcloning. We have designed such a vector, designated pUNIV for universal, with elements that will allow for in vitro or ex vivo expression in multiple cell types. We tested the expression of pUNIV-based cDNA cassettes using enhanced green fluorescent protein and two forms of the type A γ-aminobutyric acid receptor (GABA A R) and compared pUNIV to vectors optimized for expression in either Xenopus oocytes or mammalian cells. In HEK293 cells, radioligand binding was robust, and patch clamp experiments showed that subtle macroscopic GABA A R kinetics were indistinguishable from our previous results. In Xenopus oocytes, agonist median effective concentration measurements matched previous work using a vector optimized for oocyte expression. Furthermore, we found that expression using pUNIV was significantly enhanced in oocytes and was remarkably long-lasting in both systems.

␥-Aminobutyric acid (GABA) binding to GABA A receptors (GABA A Rs) triggers conformational movements in the ␣ 1 and ␤ 2 pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABA A R channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABA A R pre-M1 regions. ␣ 1 ␤ 2 GABA A Rs containing cysteine substitutions in the pre-M1 ␣ 1 (K219C, K221C) and ␤ 2 (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC 50 values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto ␣ 1 K219C and ␣ 1 K221C affected GABA-and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For ␣ 1 K221C␤ 2 receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For ␣ 1 ␤ 2 K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA-and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA-and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.Ligand-gated ion channels (LGICs) 2 are integral membrane proteins that mediate fast synaptic transmission between cells in the brain and at the neuromuscular junction. The type A ␥-aminobutyric acid receptor (GABA A R) is the main inhibitory LGIC in the brain and is the target for a wide range of therapeutic agents such as benzodiazepines, barbiturates, and anesthetics. Barbiturates, such as pentobarbital (PB), have three distinct effects on GABA A R activity. At low concentrations, PB modulates GABA-mediated Cl Ϫ current (I GABA ). At higher concentrations, PB directly activates the GABA A R in the absence of GABA, and at still higher concentrations, PB blocks channel activity (1). Little is known, however, about the structural rearrangements underlying these functional effects.Single channel studies from mouse spinal neurons (2-4) and from rat hippocampal neurons (5) have shown that currents evoked by PB are similar in conductance as those evoked by GABA, suggesting that the open state structures stabilized by PB binding are similar to those stabilized by GABA. However, GABA and PB bind to distinct sites on the GABA A...

Voltage-gated, dihydropyridine-sensitive L-type Ca2+ channels are multmeric proteins composed of a pore-forming transmembrane α1 subunit (CaV1.2) and accessory β, α2δ and γ subunits. Ca2+ entry via CaV1.2 channels shapes the action potential (AP) of cardiac myocytes and is required for excitation-contraction coupling. Two de novo point mutations of CaV1.2 glycine residues, G406R and G402S, cause a rare multi-system disorder called Timothy syndrome (TS). Here, we discuss recent work on the mechanisms by which CaV1.2 channels bearing TS mutations display slowed inactivation that leads to increased Ca2+ influx, prolonging the cardiac AP and promoting lethal arrhythmias. Based on these studies, we propose a model in which the scaffolding protein AKAP79/150 stabilizes the open conformation of CaV1.2-TS channels and facilitates physical interactions among adjacent channels via their C-tails, increasing the activity of adjoining channels and amplifying Ca2+ influx.