Calcium/calmodulin kinase II-dependent acetylcholine receptor cycling at the mammalian neuromuscular junction in vivo.
Calcium/calmodulin kinase II-dependent acetylcholine receptor cycling at the mammalian neuromuscular junction in vivo.
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DOI：
10.1523/jneurosci.3309-10.2010
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发表时间：
2010-09-15
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影响因子：
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通讯作者：
Akaaboune M
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文献类型：
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作者：
Martinez-Pena y Valenzuela I;Mouslim C;Akaaboune M
在哺乳动物骨骼神经肌肉接头处，烟碱乙酰胆碱受体 (nAChR) 的循环对于维持高突触后受体密度至关重要。然而，调节活体动物中 nAChRs 循环的机制仍然未知。利用体内延时成像、光漂白后的荧光恢复和生化下拉测定，我们证明了直接肌肉刺激和药理学诱导的细胞内钙升高都促进了内化 nAChR 循环到功能齐全和去神经突触中。大多数内化的 nAChR 直接回收到突触位点。低于静息水平的细胞内钙螯合大大降低了 nAChR 的循环。此外，我们发现钙依赖性 AChR 回收是由 Ca2+/钙调蛋白依赖性激酶 II (CaMKII) 介导的。 CaMKII 的抑制选择性地阻止再循环并导致内化 nAChR 的细胞内积累，而表面受体的内化不受影响。将 CaMKII-GFP 亚型电穿孔至胸骨乳突肌中，结果表明，肌肉特异性 CaMKIIβm 亚型在 NMJ 处高度表达，并与突触褶皱顶部的 nAChR 精确共定位，而 CaMKIIγ 和 δ 亚型在突触位点表达较差。这些结果表明 Ca2+ 和 CaMKII 活性对于受体再循环至关重要，并且可能提供体内 NMJ 处突触后 AChR 密度维持的机制。
At the mammalian skeletal neuromuscular junction, cycling of nicotinic acetylcholine receptors (nAChRs) is critical for the maintenance of a high postsynaptic receptor density. However, the mechanisms that regulate nAChRs recycling in living animals remain unknown. Using in vivo time-lapse imaging, fluorescence recovery after photobleaching, and biochemical pull down assays, we demonstrated that recycling of internalized nAChRs into fully functional and denervated synapses was promoted by both direct muscle stimulation and pharmacologically induced intracellular calcium elevations. Most of internalized nAChRs are recycled directly into synaptic sites. Chelating of intracellular calcium below resting level drastically decreased cycling of nAChRs. Furthermore we found that calcium-dependent AChR recycling is mediated by Ca2+/calmodulin-dependent kinase II (CaMKII). Inhibition of CaMKII selectively blocked recycling and caused intracellular accumulation of internalized nAChRs, whereas internalization of surface receptors remained unaffected. Electroporation of CaMKII-GFP isoforms into the sternomastoid muscle showed that muscle-specific CaMKIIβm isoform is highly expressed at NMJ and precisely co-localized with nAChRs at crests of synaptic folds while the CaMKIIγ and δ isoforms are poorly expressed in synaptic sites. These results indicate that Ca2+ along with CaMKII activity are critical for receptor recycling and may provide a mechanism by which the postsynaptic AChR density is maintained at the NMJ in vivo.