Kv4.3 Gating Current: Mechanisms Underlying Closed State Inactivation

描述（由申请人提供）：电压敏感钾（Kv）通道是存在于所有可电兴奋细胞类型膜中的蛋白质。Kv1.4 （Shaker的哺乳动物同源物）和所有Kv4（沙尔型）亚基产生钾选择性电流表型，在心肌细胞中称为“Ito”，在神经元中称为“IA”。由于它们的快速激活和随后的失活动力学，这两种通道类型都可以显著调节动作电位复极化和频率相关的电信号。因此，IA/Ito表型被假设在心血管和神经系统中发挥重要的功能作用。特别是，Kv4通道最近在记忆和疼痛感知的神经机制中有重要的影响。虽然对Shaker/Kv1.4中激活、失活和恢复的分子机制有很多了解，但Kv4中相应的机制目前尚不确定。尽管如此，普遍的共识是，“传统的”震动器N型和C型失活机制不涉及。Kv4通道还显示出从预激活的关闭状态（关闭状态失活或CSI）中突出的失活，这一过程在Shaker中并不重要。因此，作为传统的Shaker/Kv1.4门控模型的替代方案，我假设Kv4电压感应域（VSD）中的转变，特别是由S4带正电的精氨酸(R)残基介导的转变，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，要么通过与振动器明显不同的机制耦合到激活。因此，Kv4恢复（从打开不激活状态和关闭不激活状态）将耦合到去激活状态。以非洲爪蟾卵母细胞为表达系统，结合诱变和功能动力学分析（两个微电极电压钳，切开卵母细胞电压钳），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亚基门控电流“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，并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、癫痫和疼痛感知。该研究将为Kv4通道的基本调控机制提供新的分子和生物物理见解，并可能因此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.