Elucidating the role of S1 and N-terminus in the trafficking and regulation of Kv1.5 potassium channels

阐明 S1 和 N 末端在 Kv1.5 钾通道运输和调节中的作用

• 批准号: RGPIN-2018-05037
• 负责人: Zhang, Shetuan
• 金额: $ 2.62万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=669709
• 关键词: Elucidating; role; S1; terminus; trafficking

BACKGROUND: Voltage-gated K+ (Kv) channels are pore-containing membrane proteins. They can be open or closed depending on membrane voltage. When open, they selectively allow potassium ions to flow across the membrane, generating electrical currents that are critical for the function of all excitable tissues such as the brain, the heart, muscles, and endocrine glands. Kv channels are formed by 4, often identical, subunits. Each subunit contains 6 transmembrane segments, S1 to S6, and intracellular N- and C-termini. We recently discovered that an enzyme known as proteinase K (PK) can cleave Kv1.5 K+ channels at the S1-S2 linker into two pieces: N-terminus plus S1, and S2 to S6 plus C-terminus. Intriguingly, the PK-cleaved Kv1.5 channels are functional. This proposal investigates how the S1 segment and the N-terminus contribute to the assembly, expression, and function of Kv1.5 channels.****HYPOTHESIS: The S1 segment is essential for Kv1.5 assembly and trafficking, and the N-terminus constitutes a target site for the regulation of channel function.****METHODS: We will construct wild type and mutant Kv1.5 cDNA in a pCDNA3 vector. We will transfect HEK cells with Kv1.5 cDNA constructs. We will use biochemical and cell imaging techniques to determine protein expression, and use the patch clamp method to record Kv1.5 channel current, which reflects channel function.****OBJECTIVE 1. Identify the role of S1 and the N-terminus in Kv1.5 expression. We will create two channel pieces to mimic the PK-cleaved products of Kv1.5 channels. The C-piece consists of the main part of the channel with the pore region (S2-S6 and the C terminus). The N-piece consists of S1 and the N-terminus. Our preliminary data show that neither the N-piece nor the C-piece generates current. However, co-expression of both N and C pieces generates robust current. We will remove the N-terminus within the N-piece to identify the role of S1 and the N-terminus in Kv1.5 expression.****OBJECTIVE 2. Investigate the role of the N-terminus and S1 in Kv1.5 function and regulation By analyzing the biophysical properties of the currents produced by C-piece + N-piece (with different lengths of N-termini), and how they are regulated by N-terminus-targeting molecules (e.g. Src kinase), we will elucidate the role of the N-terminus in the function and regulation of Kv1.5 channels.****SIGNIFICANCE: This study will obtain new knowledge that may influence the field. Specifically, elucidating the role of the N-terminus in K+ channel function would impact design/understanding of agents/molecules (e.g. Src kinase) that regulate K+ channels through the N-terminus. In addition, analysis of PK cleavage of Kv1.5 would bring to light proteolysis as a mechanism for K+ channel regulation. By implementing this hypothesis-driven proposal, HQP will be trained in cutting-edge technologies and critical-thinking skills.******

背景：电压门控性钾离子通道是一类含孔膜蛋白。它们可以根据膜电压打开或关闭。当打开时，它们选择性地允许钾离子流过膜，产生对所有可兴奋组织（如大脑，心脏，肌肉和内分泌腺）功能至关重要的电流。KV通道由4个通常相同的亚基形成。每个亚基含有6个跨膜片段，S1至S6，以及细胞内N-和C-末端。我们最近发现，一种称为蛋白酶K（PK）的酶可以将Kv1.5 K+通道在S1-S2接头处切割成两段：N-末端加S1，和S2至S6加C-末端。有趣的是，PK切割的Kv1.5通道是功能性的。本提案研究了S1片段和N末端如何有助于Kv1.5通道的组装、表达和功能。*假设：S1片段对于Kv1.5的组装和运输是必需的，并且N-末端构成调节通道功能的靶位点。方法：将野生型和突变型Kv1.5cDNA分别构建于pCDNA 3载体中。我们将用Kv1.5 cDNA构建体转染HEK细胞。我们将使用生物化学和细胞成像技术来确定蛋白质表达，并使用膜片钳方法来记录反映通道功能的Kv1.5通道电流。目的1.确定S1和N-末端在Kv1.5表达中的作用。我们将创建两个通道片段来模拟Kv1.5通道的PK切割产物。C段由通道的主要部分和孔区（S2-S6和C末端）组成。N-片段由S1和N-末端组成。我们的初步数据表明，N型和C型都不产生电流。然而，N片段和C片段两者的共表达产生稳健的电流。我们将去除N-片段内的N-末端以鉴定S1和N-末端在Kv1.5表达中的作用。****目标2.研究N端和S1在Kv1.5通道功能和调节中的作用通过分析C端和N端（不同长度的N端）产生的电流的生物物理特性，以及它们如何被N端靶向分子（如Src激酶）调节，我们将阐明N端在Kv1.5通道功能和调节中的作用。*意义：本研究将获得可能影响该领域的新知识。具体而言，阐明N-末端在K+通道功能中的作用将影响通过N-末端调节K+通道的试剂/分子（例如Src激酶）的设计/理解。此外，对Kv1.5的PK切割的分析将揭示蛋白水解作为K+通道调节的机制。通过实施这一假设驱动的提案，HQP将接受尖端技术和批判性思维技能的培训。**