Journal: Nature medicine
Article Title: Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca 2+ channel complex
Figure Lengend Snippet: A CRMP-2 peptide suppresses the CaV2.2-CRMP-2 interaction in vitro. (a) Cartoon illustrating the main hypothesis. (b) Summary of normalized binding of CaV2.2 to 15 amino acid peptides (overlapping by 12 amino acids) encompassing full-length CRMP-2 overlaid with spinal cord lysates. Sequence of peptide #96, designated CBD3, is shown. (c) Immunoprecipitation (IP) with CRMP-2 antibody from spinal cord lysates in the presence of scramble or CBD3 peptides failed to pull-down CaV2.2 (top) and CRMP-2 (middle) but not β-tubulin (bottom). (d) Sensorgram of CBD3 (1/3/5 μM; solid traces) or scramble peptide (1/3/5 μM; dotted traces) binding to immobilized cytosolic loop 1 (L1) and distal C-terminus (Ct-dis) of CaV2.2. Dissociation was monitored for 4 min. RU, resonance units. (e) In vitro binding of L1-GST and Ct-dis-GST fusion proteins to CRMP-2 in the presence of scramble or CBD3 peptides (10 μM). CRMP-2 bound to L1 and Ct-dis was probed with a CRMP-2 antibody. CaV2.2 is detected on the surface of CAD cells (f) but not when CBD3 fused to GFP is over-expressed (g). Below, normalized surface intensity (SI) between arrows demarcating the surface of cells shown in f and g. (h) Summary of the percent of cells exhibiting surface CaV2.2 expression (n>100). (i) Immunoblots of biotinylated (surface) fractions of CAD cells expressing vector (scramble), an N-terminal region of CRMP-2 (CBD1), or CBD3 probed with a CaV2.2 antibody (n=3). (j) Top, voltage protocol. Bottom, exemplar traces from hippocampal neurons overexpressing vector (EGFP), CRMP-2 or CRMP-2 + CBD3. (k) Peak current density (pA/pF), at +10 mV, for CRMP-2- and CRMP-2 + CBD3-transfected neurons. *, p <0.05 versus CRMP-2, Student's t-test.
Article Snippet: However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects that result from inhibited physiological functions of these channels.
Techniques: In Vitro, Binding Assay, Sequencing, Immunoprecipitation, Expressing, Western Blot, Plasmid Preparation, Transfection