Kv4.3 Gating Current: Mechanisms Underlying Closed State Inactivation

描述(申请人提供)：电压敏感钾(Kv)通道是存在于所有可电兴奋细胞类型的膜上的蛋白质。Kv1.4(Shaker的哺乳动物同源物)和所有Kv4(Shal-type)亚基在心肌细胞中产生钾选择电流表型“Ito”，在神经元中产生“IA”。由于它们的快速激活和随后的失活动力学，这两种类型的通道都可以显著地调制动作电位复极化和依赖于频率的电信号。因此，IA/Ito表型被认为在心血管和神经系统中都扮演着重要的功能角色。特别是，Kv4通道最近被重要地涉及到记忆和痛觉的神经机制中。虽然对Shaker/Kv1.4中激活、失活和恢复的分子机制了解很多，但Kv4中的相应机制目前尚不确定。尽管如此，有一个普遍的共识，即“传统的”振动筛的N型和C型失活机制不涉及。Kv4通道也表现出从预激活的关闭状态(关闭状态失活或CSI)的显著失活，这一过程在Shaker中并不显著。因此，作为传统的Shaker/Kv1.4门控模型的替代模型，我假设Kv4电压敏感结构域(VSD)的跃迁，特别是由S4正电荷精氨酸(R)残基介导的跃迁，不仅主要负责调节激活和失活，而且还负责调节CSI和恢复。因此，CSI要么具有固有的电压依赖性，要么通过与Shaker显著不同的机制耦合到激活。因此，Kv4恢复(从打开失活和关闭失活状态)将与失活相结合。以非洲爪哇卵母细胞为表达系统，采用诱变和功能动力学分析相结合的方法(双微电极电压钳和切割开放卵母细胞电压钳)，初步了解CSI和Kv4.3亚基恢复的分子和生物物理机制。具体目标将是开发测量Kv4.3亚单位门控电流“Ig”的新方法，并确定各种S4精氨酸(R)突变如何改变它们。公共卫生相关性：Kv4通道对心脏的正常功能很重要，被认为直接参与神经系统的几种功能/功能障碍，包括学习和记忆、癫痫和痛觉。这项研究将为调节Kv4通道的基本机制提供新的分子和生物物理见解，从而可能为开发更有效的治疗方法提供基础。

DESCRIPTION (provided by applicant): Voltage-sensitive potassium (Kv) channels are proteins that exist in the membranes of all electrically excitable cell types. Kv1.4 (the mammalian homologue of Shaker) and all Kv4 (Shal-type) subunits generate potassium-selective current phenotypes designated "Ito" in cardiac myocytes and "IA" in neurons. Due to their rapid activation and subsequent inactivation kinetics, both channel types can significantly modulate action potential repolarization and frequency-dependent electrical signaling. IA/Ito phenotypes have thus been hypothesized to play important functional roles in both the cardiovascular and nervous systems. In particular, Kv4 channels have recently been importantly implicated in neural mechanisms underlying both memory and pain perception. While much is understood about molecular mechanisms underlying activation, inactivation, and recovery in Shaker/Kv1.4, the corresponding mechanisms in Kv4 are presently undetermined. Nonetheless, there is general consensus that "conventional" Shaker N- and C- type inactivation mechanisms are not involved. Kv4 channels also display prominent inactivation from pre-activated closed states (closed state inactivation or CSI), a process which is not significant in Shaker. Therefore, as an alternative to the conventional Shaker/Kv1.4 gating model, I hypothesize that transitions in the Kv4 voltage sensing domain (VSD), and in particular those mediated by S4 positively charged arginine (R) residues, are primarily responsible for regulating not only activation and deactivation, but also CSI and recovery. CSI thus either possesses inherent voltage dependence or is coupled to activation by a mechanism significantly different from that in Shaker. As a result, Kv4 recovery (from both open inactivated and closed inactivated states) will be coupled to deactivation. Using Xenopus laeavis oocytes as an expression system, a combination of mutagenic and functional kinetic analysis (two microelectrode voltage clamp, cut open oocyte voltage clamp) will be employed to gain initial insights into molecular and biophysical mechanisms underlying CSI and recovery of Kv4.3 subunits. The Specific Aim will be to develop novel methods for measurement of Kv4.3 subunit gating currents "Ig" and to determine how various S4 arginine (R) mutants alter them. PUBLIC HEALTH RELEVANCE: Kv4 channels are important for normal functioning of the heart, and are believed to be directly involved in several functions/dysfunctions of the nervous system, including learning and memory, epilepsy, and pain perception. The proposed research will provide new molecular and biophysical insights into basic mechanisms regulating Kv4 channels, and may thus provide the basis for development of more effective therapeutic treatments.