Different KChIPs Compete for Heteromultimeric Assembly with Pore-Forming Kv4 Subunits
Different KChIPs Compete for Heteromultimeric Assembly with Pore-Forming Kv4 Subunits
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不同的 KChIP 与成孔 Kv4 亚基竞争异源多聚体组装
DOI：
10.1016/j.bpj.2015.04.024
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发表时间：
2015
影响因子：
3.4
通讯作者：
Wang KeWei
中科院分区：
文献类型：
--
作者：
Zhou Jingheng;Tang Yiquan;Zheng Qin;Li Meng;Yuan Tianyi;Chen Liangyi;Huang Zhuo;Wang KeWei
辅助 Kv 通道相互作用蛋白 1-4 (KChIPs1-4) 与成孔 Kv4α 亚基共同组装，形成体树突阈下 A 型电流下方的通道复合物，调节神经元兴奋性。据推测，不同的 KChIP 可以竞争性地与 Kv4α 亚基结合，形成可变通道复合物，该复合物可以表现出不同的生物物理特性来调节神经功能。在本研究中，我们利用全内反射荧光显微镜进行单分子亚基计数，并结合电生理学和生物化学来研究辅助 KChIP、KChIP4a 和 KChIP4bl 的不同亚型是否可以竞争结合 Kv4.3，以共组装异源多聚体通道复合物来调节通道功能。为了仅从细胞膜计算光漂白步骤的数量，我们利用膜束缚的 k-ras-CAAX 肽，将胞质 KChIP4 蛋白锚定到表面以减少背景噪音。单分子亚基计数表明，Kv4.3-KChIP4 复合物中 KChIP4 亚型的数量可能会根据 KChIP4 表达水平而变化。增加 KChIP4bl 的量逐渐减少 KChIP4a 亚型蛋白的漂白步骤，反之亦然。对不同 Kv4-KChIP4 亚基组成的通道门控动力学的进一步分析证实，KChIP4a 和 KChIP4bl 都可以在共组装时调节通道复合体功能。综上所述，我们的研究结果表明，辅助 KChIP 可以以竞争性方式与 Kv4 异质组装，形成异多聚体 Kv4-KChIP4 通道复合物，该复合物在生物物理上不同，并在生理或病理条件下受到调节。
Auxiliary Kv channel-interacting proteins 1–4 (KChIPs1–4) coassemble with pore-forming Kv4α-subunits to form channel complexes underlying somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4α-subunit to form variable channel complexes that can exhibit distinct biophysical properties for modulation of neural function. In this study, we use single-molecule subunit counting by total internal reflection fluorescence microscopy in combinations with electrophysiology and biochemistry to investigate whether different isoforms of auxiliary KChIPs, KChIP4a, and KChIP4bl, can compete for binding of Kv4.3 to coassemble heteromultimeric channel complexes for modulation of channel function. To count the number of photobleaching steps solely from cell membrane, we take advantage of a membrane tetheredk-ras-CAAX peptide that anchors cytosolic KChIP4 proteins to the surface for reduction of background noise. Single-molecule subunit counting reveals that the number of KChIP4 isoforms in Kv4.3-KChIP4 complexes can vary depending on the KChIP4 expression level. Increasing the amount of KChIP4bl gradually reduces bleaching steps of KChIP4a isoform proteins, and vice versa. Further analysis of channel gating kinetics from different Kv4-KChIP4 subunit compositions confirms that both KChIP4a and KChIP4bl can modulate the channel complex function upon coassembly. Taken together, our findings show that auxiliary KChIPs can heteroassemble with Kv4 in a competitive manner to form heteromultimeric Kv4-KChIP4 channel complexes that are biophysically distinct and regulated under physiological or pathological conditions.