A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors

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Abstract

Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2-CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3-the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.

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Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.

Cagla Eroglu, Nicola J Allen, Michael W Susman … (2009)

Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

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A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization.

Qi Li, Anthony Lau, Terence Morris … (2004)

Presynaptic Ca(V)2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive Galpha(O) G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of Ca(V)2.2 with syntaxin 1 and Galpha(O). Ca(V)2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with Ca(V)2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the Ca(V)2.2 puncta, whereas the bulk colocalized with MUNC18-1. Galpha(O,) whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca(V)2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with Ca(V)2.2 and Galpha(O), as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.

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Patterns of abuse among unintentional pharmaceutical overdose fatalities.

Aron Hall (2008)

Use and abuse of prescription narcotic analgesics have increased dramatically in the United States since 1990. The effect of this pharmacoepidemic has been most pronounced in rural states, including West Virginia, which experienced the nation's largest increase in drug overdose mortality rates during 1999-2004. To evaluate the risk characteristics of persons dying of unintentional pharmaceutical overdose in West Virginia, the types of drugs involved, and the role of drug abuse in the deaths. Population-based, observational study using data from medical examiner, prescription drug monitoring program, and opiate treatment program records. The study population was all state residents who died of unintentional pharmaceutical overdoses in West Virginia in 2006. Rates and rate ratios for selected demographic variables. Prevalence of specific drugs among decedents and proportion that had been prescribed to decedents. Associations between demographics and substance abuse indicators and evidence of pharmaceutical diversion, defined as a death involving a prescription drug without a documented prescription and having received prescriptions for controlled substances from 5 or more clinicians during the year prior to death (ie, doctor shopping). Of 295 decedents, 198 (67.1%) were men and 271 (91.9%) were aged 18 through 54 years. Pharmaceutical diversion was associated with 186 (63.1%) deaths, while 63 (21.4%) were accompanied by evidence of doctor shopping. Prevalence of diversion was greatest among decedents aged 18 through 24 years and decreased across each successive age group. Having prescriptions for a controlled substance from 5 or more clinicians in the year prior to death was more common among women (30 [30.9%]) and decedents aged 35 through 44 years (23 [30.7%]) compared with men (33 [16.7%]) and other age groups (40 [18.2%]). Substance abuse indicators were identified in 279 decedents (94.6%), with nonmedical routes of exposure and illicit contributory drugs particularly prevalent among drug diverters. Multiple contributory substances were implicated in 234 deaths (79.3%). Opioid analgesics were taken by 275 decedents (93.2%), of whom only 122 (44.4%) had ever been prescribed these drugs. The majority of overdose deaths in West Virginia in 2006 were associated with nonmedical use and diversion of pharmaceuticals, primarily opioid analgesics.