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First published online 10 November 2009
doi: 10.1242/jcs.053280

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Regulation of N-type voltage-gated calcium channels (Cav2.2) and transmitter release by collapsin response mediator protein-2 (CRMP-2) in sensory neurons

Xian Xuan Chi^1,*, Brian S. Schmutzler^1,2,*, Joel M. Brittain², Yuying Wang², Cynthia M. Hingtgen^1,2,3, Grant D. Nicol^1,2 and Rajesh Khanna^1,2,

¹ Pharmacology and Toxicology Department, Indiana University School of Medicine, Indianapolis, IN 46202, USA
² Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA
³ Neurology Department, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Author for correspondence (khanna5{at}iupui.edu)

Accepted 24 September 2009

Collapsin response mediator proteins (CRMPs) mediate signal transduction of neurite outgrowth and axonal guidance during neuronal development. Voltage-gated Ca²⁺ channels and interacting proteins are essential in neuronal signaling and synaptic transmission during this period. We recently identified the presynaptic N-type voltage-gated Ca²⁺ channel (Cav2.2) as a CRMP-2-interacting partner. Here, we investigated the effects of a functional association of CRMP-2 with Cav2.2 in sensory neurons. Cav2.2 colocalized with CRMP-2 at immature synapses and growth cones, in mature synapses and in cell bodies of dorsal root ganglion (DRG) neurons. Co-immunoprecipitation experiments showed that CRMP-2 associates with Cav2.2 from DRG lysates. Overexpression of CRMP-2 fused to enhanced green fluorescent protein (EGFP) in DRG neurons, via nucleofection, resulted in a significant increase in Cav2.2 current density compared with cells expressing EGFP. CRMP-2 manipulation changed the surface levels of Cav2.2. Because CRMP-2 is localized to synaptophysin-positive puncta in dense DRG cultures, we tested whether this CRMP-2-mediated alteration of Ca²⁺ currents culminated in changes in synaptic transmission. Following a brief high-K⁺-induced stimulation, these puncta became loaded with FM4-64 dye. In EGFP and neurons expressing CRMP-2–EGFP, similar densities of FM-loaded puncta were observed. Finally, CRMP-2 overexpression in DRG increased release of the immunoreactive neurotransmitter calcitonin gene-related peptide (iCGRP) by 70%, whereas siRNA targeting CRMP-2 significantly reduced release of iCGRP by 54% compared with control cultures. These findings support a novel role for CRMP-2 in the regulation of N-type Ca²⁺ channels and in transmitter release.

Key words: Presynaptic Ca²⁺ channels, 1B/Cav2.2, CRMP-2, DRG, CGRP, Transmitter release

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