Regulation of N-type voltage-gated calcium channels (Cav2.2) and transmitter release by collapsin response mediator protein-2 (CRMP-2) in sensory neurons.
Collapsin response mediator proteins (CRMPs) mediate signal transduction of neurite outgrowth and axonal guidance during neuronal development. Voltage-gated Ca(2+) channels and interacting proteins are essential in neuronal signaling and synaptic transmission during this period. We recently identified the presynaptic N-type voltage-gated Ca(2+) 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 Ca(2+) 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 approximately 70%, whereas siRNA targeting CRMP-2 significantly reduced release of iCGRP by approximately 54% compared with control cultures. These findings support a novel role for CRMP-2 in the regulation of N-type Ca(2+) channels and in transmitter release.
Chi
Xian Xuan
XX
Pharmacology and Toxicology Department, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Schmutzler
Brian S
BS
Brittain
Joel M
JM
Wang
Yuying
Y
Hingtgen
Cynthia M
CM
Nicol
Grant D
GD
Khanna
Rajesh
R
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D002478:Cells, Cultured; D004594:Electrophysiology; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011984:Sensory Receptor Cells / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology
D020864:Calcium Channels, N-Type; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; C095114:collapsin response mediator protein-2; D002118:Calcium