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

背景：电压门控K+(Kv)通道是一种含孔膜蛋白。它们可以根据膜电压的不同而打开或关闭。当它们打开时，它们选择性地允许钾离子流过细胞膜，产生对所有可兴奋组织的功能至关重要的电流，如大脑、心脏、肌肉和内分泌腺。KV通道由4个通常相同的亚基组成。每个亚基包含6个跨膜片段，S1到S6，以及细胞内的N-末端和C-末端。我们最近发现，一种被称为蛋白酶K(PK)的酶可以将S1-S2连接子上的Kv1.5 K+通道切割成两段：N端+S1，S2到S6+C端。耐人寻味的是，PK裂解的Kv1.5通道是有功能的。本研究探讨了Kv1.5通道的组装、表达和功能中S1片段和N-末端的作用。*假设：S1片段是Kv1.5组装和运输所必需的，而N-末端是调节通道功能的靶点。*方法：我们将在pCDNA3载体中构建野生型和突变型Kv1.5基因。我们将构建的Kv1.5基因导入HEK细胞。我们将使用生化和细胞成像技术来确定蛋白质的表达，并使用膜片钳方法记录反映通道功能的Kv1.5通道电流。*目的1.确定S1和N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片由具有孔区(S2-S6和C末端)的通道的主要部分组成。N片段由S1和N末端组成。我们的初步数据显示，N片和C片都不产生电流。然而，N和C片段的共同表达会产生稳健的电流。我们将去掉N-片段中的N-末端，以确定S1和N-末端在Kv1.5表达中的作用。*目的2.通过分析C-片段+N-片段(具有不同长度的N-末端)产生的电流的生物物理性质，以及它们是如何被N-末端靶向分子(如Src激酶)调节的，来研究N-末端和S1在Kv1.5功能和调控中的作用。我们将阐明N-末端在Kv1.5通道的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。具体地说，阐明N-末端在K+通道功能中的作用将影响对通过N-末端调节K+通道的药物/分子(例如，Src激酶)的设计/理解。此外，对Kv1.5的PK裂解的分析将揭示蛋白水解酶作为K+通道的调节机制。通过实施这一假设驱动的提议，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 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.******