Identification of the S1-S2 linker as a novel mechanosensor for Kv1.5 regulation

鉴定 S1-S2 连接器作为 Kv1.5 调节的新型机械传感器

• 批准号: RGPIN-2019-04878
• 负责人: Zhang, Shetuan
• 金额: $ 3.06万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=746101
• 关键词: Identification; S1; S2; linker; novel

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 K+ 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. While the opening and closing (gating) of Kv channels are controlled by membrane voltages, accumulating evidence indicates that they are also regulated by mechanical force exerted on the cell membrane. However, to date, a structural component of the channel that directly links mechanical force and Kv channel activity has not been described. Hypothesis: The extracellularly-localized S1-S2 linker of Kv1.5 represents a novel mechanosensor for Kv1.5 regulation. Deformation of the S1-S2 linker induced by mechanical force can induce a conformational change of the intracellularly-localized N-terminus of the channel, leading to an altered N-terminus-Src tyrosine kinase interaction, and consequently changed channel activity. Methods: We will construct wild type and mutant Kv1.5 cDNAs in the pcDNA3 vector. We will transfect HEK cells with the cDNA constructs. We will use biochemical techniques to determine protein expression, and use the patch clamp method to record Kv1.5 channel current. We will increase cell volume, and thus membrane tension, by lowering the osmolarity of cell culture medium from 315 to 211 mOsm/L (low osmolarity, LO) and examine the effects of LO on wild type and mutant Kv1.5 channels. Objective 1. Identify that the S1-S2 linker of Kv1.5 is a mechanosensor. Compared to other K+ channels, the S1-S2 linker of Kv1.5 is unusually long (54 versus 5-27 amino acid residues) with many (12) extra proline residues. We will determine whether LO selectively increases Kv1.5 current (compared with other Kv channels such as Kv1.1 and Kv4.3).  Objective 2. Identify that a deformation of S1-S2 linker by LO can impact the conformation of N-terminus of Kv1.5 channels. Objective 3. Identify that the S1-S2 linker regulates Kv1.5 function through altering the interaction between the N-terminus and Src kinases. Src tyrosine kinase inhibits Kv1.5 current through targeting the motif 65-RPLPPLPDPGVRPLPPLP-82. We propose Kv1.5 is constitutively inhibited by endogenous Src tyrosine kinase. We will disrupt Src-Kv1.5 interaction by using Src inhibitors or by mutating the Src binding motif, and examine the effects of LO on Kv1.5 current. Significance: This study is the first to identify a mechanosenser within a Kv channel, Kv1.5, which is fundamental to understanding channel function and discovering novel means of channel regulation. By implementing this hypothesis-driven proposal, HQP will be trained in cutting-edge technologies and critical-thinking skills.

背景：电压门控的K+（KV）通道是含孔隙的膜蛋白。它们可以根据膜电压开放或关闭。开放时，它们有选择地允许K+流过整个膜，从而产生对所有令人兴奋的时机（例如大脑，心脏，肌肉和内分泌网格）功能至关重要的电流。 KV通道由4个通常相同的亚基形成。每个亚基包含6个跨膜段，S1至S6，以及细胞内N和C-termini。尽管KV通道的开口和关闭（门控）受膜电压的控制，但积累的证据表明它们也受细胞膜上执行的机械力调节。但是，迄今为止，尚未描述直接连接机械力和KV通道活性的通道的结构成分。假设：KV1.5的细胞外S1-S2接头代表了KV1.5调节的新型机械学。机械力诱导的S1-S2接头的变形可以诱导通道的细胞内N-末端的构象变化，从而导致N-末端SRC酪氨酸激酶相互作用改变，从而改变通道活性。方法：我们将在PCDNA3载体中构建野生型和突变的KV1.5 cDNA。我们将用cDNA构建体翻译HEK细胞。我们将使用生化技术来确定蛋白质表达，并使用斑块夹方法记录KV1.5通道电流。我们将通过将细胞培养基的渗透压从315降低到211 MOSM/L（低渗透压，LO），并检查LO对LO对LO对野生型和突变体KV1.5通道的影响，从而增加细胞体积，从而增加膜张力。目标1。确定KV1.5的S1-S2链接器是一种机械学。与其他K+通道相比，KV1.5的S1-S2连接器异常长（54对5-27个氨基酸残留物），有许多（12）多个（12）额外的脯氨酸残留物。我们将确定LO是否有选择地增加KV1.5电流（与KV1.1和KV4.3等其他KV通道相比）。目标2。确定通过LO对S1-S2接头的变形会影响KV1.5通道N端的构象。目标3。确定S1-S2接头通过改变N末端和SRC激酶之间的相互作用来调节KV1.5的功能。 SRC酪氨酸激酶通过靶向基序65-RPLPPLPDPGVRPLPPLPLP-82抑制KV1.5电流。我们提出的KV1.5始终被内源性SRC酪氨酸激酶抑制。我们将通过使用SRC抑制剂或突变SRC结合基序来破坏SRC-KV1.5的相互作用，并检查LO对KV1.5电流的影响。意义：这项研究是第一个识别KV通道KV1.5中的机理启动器的研究，这对于理解通道功能并发现新颖的通道调节手段至关重要。通过实施这一假设驱动的建议，HQP将接受尖端技术和批判性思维技能的培训。