Pharmacology and Toxicology Department, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
J Cell Sci. 2009 Dec 1;122(Pt 23):4351-62. doi: 10.1242/jcs.053280. Epub 2009 Nov 10.
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.
collapsin 反应介质蛋白 (CRMPs) 在神经元发育过程中介导神经突生长和轴突导向的信号转导。电压门控 Ca(2+) 通道和相互作用蛋白在神经元信号转导和突触传递过程中至关重要。我们最近确定了突触前 N 型电压门控 Ca(2+) 通道 (Cav2.2) 是 CRMP-2 的相互作用伙伴。在这里，我们研究了 CRMP-2 与感觉神经元中的 Cav2.2 功能关联的影响。Cav2.2 与未成熟突触和生长锥、成熟突触和背根神经节 (DRG) 神经元的胞体中的 CRMP-2 共定位。共免疫沉淀实验表明，CRMP-2 从 DRG 裂解物中与 Cav2.2 结合。通过核转染在 DRG 神经元中过表达与增强型绿色荧光蛋白 (EGFP) 融合的 CRMP-2 会导致 Cav2.2 电流密度显著增加与表达 EGFP 的细胞相比。CRMP-2 的操作改变了 Cav2.2 的表面水平。由于 CRMP-2 定位于致密 DRG 培养物中突触小泡蛋白阳性的斑点中，我们测试了这种 CRMP-2 介导的 Ca(2+) 电流变化是否导致突触传递的变化。短暂的高 K(+) 诱导刺激后，这些斑点中充满了 FM4-64 染料。在 EGFP 和表达 CRMP-2-EGFP 的神经元中，观察到类似密度的 FM 加载斑点。最后，DRG 中 CRMP-2 的过表达使免疫反应性神经递质降钙素基因相关肽 (iCGRP) 的释放增加了约 70%，而针对 CRMP-2 的 siRNA 使 iCGRP 的释放减少了约 54%与对照培养物相比。这些发现支持 CRMP-2 在调节 N 型 Ca(2+) 通道和递质释放中的新作用。
