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

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K+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）的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。虽然Kv通道的打开和关闭（门控）是由膜电压控制的，但越来越多的证据表明，它们也受到施加在细胞膜上的机械力的调节。然而，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组分。 假设：Kv1.5的细胞外定位的S1-S2接头代表了Kv1.5调节的新型机械传感器。由机械力诱导的S1-S2接头的变形可以诱导通道的胞内定位的N-末端的构象变化，导致改变的N-末端-Src酪氨酸激酶相互作用，并因此改变通道活性。 方法：将野生型和突变型Kv1.5 cDNA分别克隆到pcDNA 3载体上。我们将用cDNA构建体转染HEK细胞。我们将使用生物化学技术测定蛋白质表达，并使用膜片钳方法记录Kv1.5通道电流。我们将通过将细胞培养基的渗透压从315 mOsm/L降低至211 mOsm/L（低渗透压，LO）来增加细胞体积，从而增加膜张力，并检查LO对野生型和突变型Kv1.5通道的影响。目的1.确定Kv1.5的S1-S2接头是机械传感器。与其他K+通道相比，Kv1.5的S1-S2接头异常长（54个氨基酸残基对5-27个氨基酸残基），具有许多（12个）额外的脯氨酸残基。我们将确定LO是否选择性地增加Kv1.5电流（与Kv1.1和Kv4.3等其他Kv通道相比）。 目标2.确定LO导致的S1-S2接头变形可影响Kv1.5通道N-末端的构象。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功能。Src酪氨酸激酶通过靶向基序65-RPLPPLPDPGVRPLPPLP-82抑制Kv1.5电流。我们建议Kv1.5是由内源性Src酪氨酸激酶组成性抑制。我们将通过使用Src抑制剂或通过突变Src结合基序来破坏Src-Kv 1.5相互作用，并检查LO对Kv 1. 5电流的影响。重要性：这项研究是第一个确定Kv通道Kv1.5内的机械传感器，这是理解通道功能和发现通道调节新方法的基础。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。

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.