Differential impact of genetically modulated choline transporter expression on the release of endogenous versus newly synthesized acetylcholine.
Differential impact of genetically modulated choline transporter expression on the release of endogenous versus newly synthesized acetylcholine.
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DOI：
10.1016/j.neuint.2016.03.012
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发表时间：
2016-09
影响因子：
4.2
通讯作者：
Blakely RD
中科院分区：
文献类型：
--
作者：
Iwamoto H;Calcutt MW;Blakely RD
突触前高亲和力胆碱转运蛋白（CHT，SLC 5A 7）将胆碱有效地导入胆碱能神经末梢，决定了乙酰胆碱（ACh）的合成和释放能力。在Slc 5a 7（HET，CHT+/−）功能缺失突变杂合子小鼠中，ACh的组织水平显著降低，但在过表达的Slc 5a 7 BAC转基因小鼠（BAC）中显著升高。由于容易释放的ACh库被认为只占总ACh库的一小部分，因此这些基因型依赖性变化提出了一个问题：CHT表达或活性是否可能优先影响储备库ACh囊泡的大小。在目前的研究中，我们探讨了这个问题，通过评估CHT基因型的影响上融合的小鼠前脑片上释放乙酰胆碱。我们用升高的K+处理来自HET、BAC或野生型（WT）对照的切片，并监测新合成的和储存的ACh池的释放。新合成的乙酰胆碱产生的[3 H]胆碱摄取后，通过闪烁光谱法进行定量，而释放的内源性乙酰胆碱存储池进行定量的HPLC-MS方法，从相同的样品。而内源性乙酰胆碱释放与CHT基因剂量成比例，预载[3 H]乙酰胆碱释放显示没有显着的基因型依赖性。我们的研究结果表明，CHT蛋白水平优先影响储备池囊泡的动员所提供的乙酰胆碱释放的能力。
The efficient import of choline into cholinergic nerve terminals by the presynaptic, high-affinity choline transporter (CHT, SLC5A7) dictates the capacity for acetylcholine (ACh) synthesis and release. Tissue levels of ACh are significantly reduced in mice heterozygous for a loss of function mutation in Slc5a7 (HET, CHT+/−), but significantly elevated in overexpressing, Slc5a7 BAC-transgenic mice (BAC). Since the readily-releasable pool of ACh is thought to constitute a small fraction of the total ACh pool, these genotype-dependent changes raised the question as to whether CHT expression or activity might preferentially influence the size of reserve pool ACh vesicles. In the current study, we approached this question by evaluating CHT genotype effects on the release of ACh from suprafused mouse forebrain slices. We treated slices from HET, BAC or wildtype (WT) controls with elevated K+ and monitored release of both newly synthesized and storage pools of ACh. Newly synthesized ACh produced following uptake of [3H]choline was quantified by scintillation spectrometry whereas release of endogenous ACh storage pools was quantified by an HPLC-MS approach, from the same samples. Whereas endogenous ACh release scaled with CHT gene dosage, preloaded [3H]ACh release displayed no significant genotype dependence. Our findings suggest that CHT protein levels preferentially impact the capacity for ACh release afforded by mobilization of reserve pool vesicles.