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，产生电流，这对所有可兴奋组织（如大脑、心脏、肌肉和内分泌腺）的功能至关重要。千伏通道由4个通常相同的亚基组成。每个亚基包含6个跨膜片段，S1至S6，以及细胞内N和c端。我们最近发现一种被称为蛋白酶K （PK）的酶可以将S1-S2连接体上的Kv1.5 K+通道切割成两段：n端加S1，以及S2到S6加c端。有趣的是，pk切割的Kv1.5通道是功能性的。本研究探讨了S1片段和n端如何参与Kv1.5通道的组装、表达和功能。****假设：S1片段对Kv1.5的组装和运输至关重要，n端构成通道功能调控的靶位点。****方法：在pCDNA3载体上构建野生型和突变型Kv1.5 cDNA。我们将用Kv1.5 cDNA构建体转染HEK细胞。我们将使用生化和细胞成像技术测定蛋白表达，并使用膜片钳法记录反映通道功能的Kv1.5通道电流。* * * *的目标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片由通道主体部分与孔区（S2-S6和C端）组成。n片由S1和n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片的共表达产生稳健电流。我们将移除n片段内的n端，以确定S1和n端在Kv1.5表达中的作用。* * * *的目标2。通过分析C-piece + N-piece（不同长度的n -末端）产生的电流的生物物理特性，以及它们如何受到n -末端靶向分子（如Src激酶）的调节，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。具体来说，阐明n端在K+通道功能中的作用将影响通过n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裂解分析将揭示蛋白质水解作为K+通道调节的机制。通过实施这一假设驱动的建议，HQP将在尖端技术和批判性思维技能方面得到培训。******

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.******