Syntaxin 1A co-associates with native rat brain and cloned large conductance, calcium-activated potassium channels in situ
Syntaxin 1A co-associates with native rat brain and cloned large conductance, calcium-activated potassium channels in situ
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DOI：
10.1113/jphysiol.2003.051631
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发表时间：
2003-11-15
影响因子：
5.5
通讯作者：
Braun, AP
中科院分区：
文献类型：
--
作者：
Ling, SZ;Sheng, JZ;Braun, AP
大电导、钙活化钾通道（BKCa通道）受几种不同的机制调节，包括磷酸化/去磷酸化事件和蛋白质-蛋白质相互作用。在这项研究中，我们研究了BKCa通道和syntaxin 1A之间的相互作用，syntaxin 1A是一种可溶性的n -乙基马来酰亚胺敏感因子附着蛋白受体（SNARE），据报道可以调节不同类型离子通道的活性和/或定位。使用互反共免疫沉淀策略，我们观察到大鼠海马中的天然BKCa通道与syntaxin 1A共同关联，而不是与syntaxin 3密切相关的同系物。这种BKCa通道-syntaxin 1A的相互作用可以在非神经元细胞系（人胚胎肾（HEK） 293细胞）中进一步证明，在从小鼠脑或牛主动脉克隆的大鼠syntaxin 1A和BKCa通道中共同表达。然而，这些通道与syntaxin 3的共表达并没有导致可检测到的蛋白质-蛋白质相互作用。表达BKCa通道和syntaxin 1A的HEK 293细胞的免疫荧光共染色显示这两种蛋白在原位重叠分布。功能上，根据切除膜斑块的宏观电流记录，观察到BKCa通道与syntaxin 1A而不是syntaxin 3的共表达在低浓度（1-4 μ m）的游离胞质钙下增强通道门控和动力学，但在高浓度（小于或等于10 μ m）下则没有。因此，BKCa通道与邻近膜蛋白之间的相互作用可能在调节这些通道在特定细胞区室中的活性和/或分布方面发挥重要作用。
Large conductance, calcium-activated potassium channels (BKCa channels) are regulated by several distinct mechanisms, including phosphorylation/dephosphorylation events and protein-protein interactions. In this study, we have examined the interaction between BKCa channels and syntaxin 1A, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is reported to modulate the activity and/or localization of different classes of ion channels. Using a reciprocal co-immunoprecipitation strategy, we observed that native BKCa channels in rat hippocampus co-associate with syntaxin 1A, but not the closely related homologue syntaxin 3. This BKCa channel-syntaxin 1A interaction could be further demonstrated in a non-neuronat cell line (human embryonic kidney (HEK) 293 cells) following co-expression of rat syntaxin 1A and BKCa channels cloned from either mouse brain or bovine aorta. However, co-expression of these same channels with syntaxin 3 did not lead to a detectable protein-protein interaction. Immunofluorescent co-staining of HEK 293 cells expressing BKCa channels and syntaxin 1A demonstrated overlapping distribution of these two proteins in situ. Functionally, co-expression of BKCa channels with syntaxin 1A, but not syntaxin 3, was observed to enhance channel gating and kinetics at low concentrations (1-4 mum) of free cytosolic calcium, but not at higher concentrations (less than or equal to 10 mum), as judged by macroscopic current recordings in excised membrane patches. Interactions between BKCa channels and neighbouring membrane proteins may thus play important roles in regulating the activity and/or distribution of these channels within specialized cellular compartments.