Biogenesis of voltage-gated K+ channels

我们的研究计划旨在了解电压门控钾（Kv）通道是如何形成的。这是一 复杂的多步骤过程，需要获得局部二级、三级和四级结构， 顺序地或作为耦合事件。这是如何发生的，在大多数情况下，是未知的。然而， 这些步骤是深刻的，往往具有病理后果。我们把注意力集中在人类的四聚体 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。三个目标包括这个赠款建议。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功能的基本先决条件。 然而，这个过程还没有被定义在有限的和异构的微环境中， 核糖体出口通道是蛋白质最初形成的地方。螺旋性必须在正确的时间和地点发生， 翻译.不满足这一要求会损害肽靶向、分子伴侣缔合、有效的双分子层结合、分子间的相互作用和分子间的相互作用。 插入和寡聚化。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 二级结构出现在Kv1.3新生肽中。我们将确定 延迟或启动螺旋形成，并确定潜在的肽-隧道相互作用。 为此，我们使用生物化学方法和肽-核糖体的冷冻-EM单颗粒重建 配合物在目标2中，我们探索了一个令人兴奋的新领域，这一领域对蛋白质的合成具有根本的重要性， 也就是说，肽序列如何产生微微牛顿的力来微调Kv肽的延伸速率 和折叠效率。我们使用实验方法和分子动力学模拟来确定 肽-隧道相互作用的类型，产生力，力的性质，及其后果， 肽被延长。考虑到人Kv1.3在神经元和免疫细胞中表达，并且受损， 表达产生慢性炎症性疾病和自身免疫性疾病，这是令人信服的问， 人类疾病相关的KCNA 3变体，编码Kv1.3的基因，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KCNA 3罕见变异和生物物理学的临床结果的“第一”方法 Kv1.3折叠和功能的测定以鉴定分子缺陷。我们对Kv折叠的总体愿景 包括肽内片段、核糖体出口通道和ER之间的复杂偶联事件 膜的我们现在通过引入两个新的概念来扩展这个观点，以供进一步研究：1） 阻遏物/激活物活性作为一个分子开关来控制Kv螺旋的时间和隧道位置 起始，和2）共翻译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KV通道折叠的额外调节水平，并可能概括 与其他蛋白质的生物合成有关。

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.