Tang Yi-Quan, Zhou Jing-Heng, Yang Fan, Zheng Jie, Wang KeWei
Department of Molecular and Cellular Pharmacology, State Key Laboratory of Natural and Biomimetic Drugs, Peking University School of Pharmaceutical Sciences, Beijing, China.
Department of Physiology and Membrane Biology, University of California School of Medicine, Davis, California.
Biophys J. 2014 Sep 2;107(5):1090-1104. doi: 10.1016/j.bpj.2014.07.038.
A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function.
A 型 Kv4 钾通道在负膜电位下会向非传导状态发生构象变化，这是一个被称为预开放关闭状态或关闭状态失活（CSI）的动态过程。CSI 导致通道活性受到抑制，而无需通道开放的前提条件，从而在静息时对神经元兴奋性、树突信号整合和突触可塑性提供动态调节。然而，Kv4 CSI 背后的结构决定因素在很大程度上仍然未知。我们最近表明，辅助性 KChIP4a 亚基包含一个 N 端 Kv4 抑制结构域（KID），它直接与 Kv4.3 通道相互作用以增强 CSI。在本研究中，我们利用 KChIP4a KID 来探究 Kv4 CSI 背后的关键结构元件。通过荧光共振能量转移双杂交图谱和基于双分子荧光互补的筛选结合电生理学方法，我们确定了细胞内四聚化（T1）结构域，其功能是抑制 CSI，并作为 KID 结合的受体。通过突变 T1 结构域 C3H1 基序内的 C110A 破坏 Kv4.3 T1-T1 相互作用界面，促进了 CSI，并消除了 KID 介导的 CSI 增强作用。此外，用 Kv1.4（没有 C3H1 基序）或 Kv2.1（有 C3H1 基序）的 T1 结构域替换 Kv4.3 T1 结构域，分别导致通道在增强或抑制的 CSI 下发挥作用。综上所述，我们的研究结果揭示了 T1 结构域在抑制 Kv4 CSI 方面的一个新（据我们所知）作用，并且 KChIP4a KID 直接与 T1 结构域相互作用以促进 Kv4.3 CSI，从而导致通道功能受到抑制。
