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

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 Ca2+, 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.

(S)-lacosamide inhibition of CRMP2 phosphorylation reduces postoperative and neuropathic pain behaviors through distinct classes of sensory neurons identified by constellation pharmacology

“…2C). This value is in agreement with an IC 50 value of ~2.8 µM for inhibition of Ca 2+ influx for a membrane-tethered CRMP2 peptide that uncouples the CRMP2-CaV2.2 interaction that we recently reported [29]. Moreover, this value is better than TROX-1, a small-molecule, state-dependent blocker of CaV2 channels, which reduced the Ca 2+ transient with an estimated IC 50 value of 2.1 µM [1].…”

“…2B). Concurrent inhibition with 200µM ( S )-LCM and a Cdk5 inhibitor (AT7519, 100 nM) or a CRMP2/CaV2.2 blocking peptide (myr-tat-CBD3, 10 µM [28]) did not produce any additional inhibition of K + -evoked Ca 2+ responses (Supplementary Fig. 1A).…”

“…We have recently advanced an innovative strategy targeting protein interactions modulating CaV2.2 as an alternative to direct channel block [8; 24; 29; 76; 79]. Targeting channel regulation may potentially lessen many of the adverse side effects associated with direct channel block.…”

“…Targeting channel regulation may potentially lessen many of the adverse side effects associated with direct channel block. Supporting this concept, we reported that targeting CaV2.2 indirectly with a peptide derived from the collapsin response mediator protein 2 (CRMP2) peptide did not affect memory retrieval, motor coordination, depression-associated behaviors [8] and was not rewarding/addictive [29]. …”

CRMP2 and voltage-gated ion channels: potential roles in neuropathic pain

“…Several evolutionary progressions of the CBD3 peptide were synthesized, with each mutation of the original peptide sequence intended to improve its cell penetrance and structural stability [23, 90–94]. Antinociceptive effects of one such peptide were reversible in a rodent model of neuropathic pain, and nociceptive behaviors readily resurfaced once peptide administration ceased [90].…”