Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-Dependent K+ channels in neuroendocrine islet β-cells through an interaction with the channel N terminus
Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-Dependent K+ channels in neuroendocrine islet β-cells through an interaction with the channel N terminus
复制标题
DOI：
10.1210/me.2002-0058
复制
发表时间：
2002-11-01
影响因子：
--
通讯作者：
Gaisano, HY
中科院分区：
文献类型：
--
作者：
MacDonald, PE;Wang, GT;Gaisano, HY
胰岛素分泌由涉及膜去极化和Ca 2+进入的离子事件启动，而胞吐SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）蛋白介导胞吐作用本身。在本研究中，我们表征了SNARE蛋白SNAP-25（25 kDa的突触体相关蛋白）与β细胞电压依赖性K+通道Kv2.1的相互作用。通过蛋白质印迹法在人胰岛裂解物中检测到Kv2.1、SNAP-25和突触融合蛋白1A的表达，并且免疫共沉淀研究表明异源表达的SNAP-25和突触融合蛋白1A与Kv2.1相关。SNAP-25使来自重组Kv2.1通道的电流减少约70%，而不影响通道定位。这种抑制作用可以通过共透析Kv 2. 1 N-末端肽而不是Kv 2. 1C-末端肽来部分减轻，所述肽可以在体外结合SNAP-25。类似地，SNAP-25阻断大鼠β细胞的电压依赖性外向K+电流约40%，该作用通过Kv2.1N片段的共透析完全逆转。最后，SNAP-25对β细胞中的外向K+电流没有影响，其中Kv2.1通道已经使用显性负性方法功能性敲除，表明与其他β细胞Kv通道相比，这种相互作用对Kv2.1通道是特异性的。这项研究表明，SNAP-25可以通过在通道N末端的相互作用来调节Kv2.1，并支持SNARE蛋白通过参与调节膜离子通道以及胞外膜融合来调节分泌的假设。
Insulin secretion is initiated by ionic events involving membrane depolarization and Ca2+ entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K+ channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K+ currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K+ currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.