Expression of ryanodine receptors in human embryonic kidney (HEK293) cells.
Expression of ryanodine receptors in human embryonic kidney (HEK293) cells.
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兰尼碱受体在人胚肾 (HEK293) 细胞中的表达。
DOI：
10.1042/bj3340079
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Geiger,JD
中科院分区：
文献类型：
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作者：
Querfurth,HW;Haughey,NJ;Greenway,SC;Yacono,PW;Golan,DE;Geiger,JD
先前已经表明，咖啡因敏感性细胞内钙（Ca 2 + 1）储存的动员增加了从转染的人胚胎肾细胞（HEK 293）释放淀粉样蛋白β-肽（Aβ）[Querfurth，Jiang，盖革和Selkoe（1997）神经化学杂志（J.Neurochem.69），1580-1591]。本研究采用[3 H]兰尼碱受体结合、钙离子荧光显像、免疫细胞荧光、免疫沉淀和PCR技术，验证咖啡因/Aβ反应是由于与特定亚型的兰尼碱受体（RyR）相互作用的假说。[3 H]Ryanodine与一类高亲和力咖啡因敏感位点结合（Kd= 9.9±1.6 nM，Bmax= 25±4 fmol/mg蛋白质）。RyR以点状网状线性模式进行免疫修饰。SDS/PAGE和逆转录酶-PCR的结果表明内源性表达的1型（骨骼）和2型（心脏）RyR。HEK 293细胞RyR具有功能活性，因为（i）在应用5-15 mM咖啡因后，[Ca 2 +] i比基线增加了2.8倍，（ii）观察到[Ca 2 +] i的重复加标增加，以及（iii）获得了使用依赖性阻滞的证据。这些发现中的一些被扩展到包括HeLa和人成纤维细胞系，这表明对上皮样谱系细胞的更广泛的适用性。β-淀粉样前体蛋白在阿尔茨海默病和钙通道研究使用转染HEK 293细胞的加工的影响进行了讨论。
It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid β-peptide (Aβ) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem.69, 1580–1591]. The present study was to test the hypothesis that the caffeine/Aβ responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd= 9.9±1.6 nM,Bmax= 25±4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]iincreased 2.8-fold over baseline following applications of 5–15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]iwere observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the β-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.