Biogenesis of voltage-gated K+ channels

我们的研究项目旨在了解电压门控钾(Kv)通道是如何形成的。这是一个 需要获取局部二级、三级和四级结构的复杂的多步骤过程， 顺序地或作为耦合事件。这是如何发生的，在很大程度上是未知的。然而，它的影响 这些步骤意义深远，往往会产生病理性后果。我们把注意力集中在人类四聚体上 Kv1.3通道，特别是其早期折叠，以及调节这些折叠事件的决定因素 核糖体和内质网膜的出口通道。这项拨款提案有三个目标。的能力 形成螺旋的蛋白质是蛋白质折叠和在所有蛋白质组中发挥作用的基本前提。 然而，这一过程还没有在有限和不均匀的微环境中定义 首先制造蛋白质的核糖体出口通道。螺旋度必须在正确的时间和地点发生 翻译。未能满足这一要求会损害多肽靶向、伴侣结合和有效的双分子层 插入和齐聚。在目标1中，我们特别询问何时、何地、如何以及为什么这些关键 Kv1.3新生多肽中存在二级结构。我们将确定分子机制， 延迟或启动螺旋形成，并确定潜在的多肽-隧道相互作用是负责的。 为了做到这一点，我们使用了生化方法和冷冻EM单粒子重建多肽-核糖体。 复合体。在目标2中，我们探索了一个令人兴奋的新领域，对蛋白质是如何形成的具有根本重要性， 也就是说，一个肽的序列如何产生微牛顿的力，从而微调Kv肽的伸长率 和折叠效率。我们使用实验方法和分子动力学模拟来识别 产生力的多肽-隧道相互作用的类型、力的性质及其作为 多肽是拉长的。鉴于人类Kv1.3在神经细胞和免疫细胞中表达，并受到损害 表达会导致慢性炎症性疾病和自身免疫性疾病，人们不得不问 编码Kv1.3的KCNA3基因与人类疾病相关的变体引入了 折叠/组装/运输缺陷。在目标3中，我们使用最近开发的“基因组-- 确定特定KCNA3罕见变异体和生物物理学的临床后果的第一种方法 Kv1.3折叠的测定和鉴定分子缺陷的功能。我们对KV折叠的整体愿景 包括肽内片段、核糖体出口通道和内质网之间的复杂耦合事件 薄膜。我们现在通过引入两个新概念来扩展这一观点，以供进一步研究：1) 抑制子/激活子活性作为一个分子开关控制Kv螺旋的时间和隧道位置 2)共转换力的产生调节翻译速率和折叠。这两个概念 代表范式转变，揭示了对Kv通道折叠的额外监管水平，并可能概括 与其他蛋白质的生物发生有关。

Our research program aims to understand how a voltage-gated potassium (Kv) channel is made. This is a complicated multi-step process that requires acquisition of local secondary, tertiary, and quaternary structures, either sequentially or as coupled events. How this happens is, for the most part, unknown. Yet the impact of these steps is profound, often with pathological consequences. We focus our attention on the human tetrameric Kv1.3 channel, particularly its early-stage folding, and the determinants that regulate these folding events in the exit tunnel of the ribosome and the ER membrane. Three Aims comprise this grant proposal. The ability of a protein to form helices is a fundamental prerequisite for protein folding and function in all proteomes. However, this process has not been defined in the confined and heterogeneous microenvironment of the ribosome exit tunnel where a protein is first made. Helicity must occur at the right time and place during translation. Failure to meet this requirement impairs peptide targeting, chaperone association, efficient bilayer insertion, and oligomerization. In Aim 1, we specifically ask when, where, how, and why these critical secondary structures arise in the Kv1.3 nascent peptide. We will determine the molecular mechanisms that delay or initiate helix formation and identify the underlying peptide-tunnel interactions that are responsible. To do this, we use biochemical approaches and cryo-EM single particle reconstruction of peptide-ribosome complexes. In Aim 2, we explore an exciting new field of fundamental importance to how proteins are made, namely, how a peptide's sequence generates piconewtons of force that fine tune Kv peptide's rate of elongation and folding efficiency. We use experimental approaches and molecular dynamics simulations to identify the type of peptide-tunnel interactions giving rise to force, the nature of the force, and its consequences as the peptide is elongated. Given that human Kv1.3 is expressed in neuronal and immune cells, and impaired expression produces chronic inflammatory disease and autoimmune disorders, it is compelling to ask whether human disease-linked variants of the KCNA3, the gene that encodes Kv1.3, introduce folding/assembly/trafficking defects. In Aim 3, we address this question using the recently developed “genome- first” approach to determine the clinical consequences of specific KCNA3 rare variants and biophysical determinations of Kv1.3 folding and function to identify the molecular defects. Our overall vision of Kv folding includes complex coupled events between intrapeptide segments, the ribosome exit tunnel, and the ER membrane. We now expand this view by introducing two new concepts for further investigation: 1) repressor/activator activity acts as a molecular switch to govern the time and tunnel location of Kv helix initiation, and 2) cotranslational force generation modulates translation rates and folding. Both concepts represent paradigm shifts that reveal additional levels of regulation for Kv channel folding and may generalize to the biogenesis of other proteins.