Proteolytic activation of calcium channels: potential target for channel - directed therapeutics in arterial smooth muscle cells

钙离子进入可兴奋细胞是人体生理过程中最普遍的电化学触发。钙进入是脑细胞（神经元）、心脏细胞（心肌细胞）收缩和动脉血压平滑肌细胞（SMCs）之间信号传播的常见上游信息。在所有这些类型的细胞中，激发引起的钙进入是由一类特殊的蛋白质复合物控制的，称为电压门控钙通道（VGCCs）。vgc在不可渗透的细胞膜上形成一个特殊的孔，以选择性地调节钙在其他带电离子中进入可兴奋细胞的通量。即使是心脏和血管中正常vgc功能的微小改变也会导致严重的人类疾病。因此，vgc调控是临床上重要的小分子药物的靶点，用于降低动脉血压和治疗心功能障碍。然而，它们的广泛分布往往导致脱靶效应，迫切需要寻找新的治疗方法。为了形成一个功能齐全的复合物，VGCCs需要几个蛋白质（亚基）的共同组装，一个形成钙选择孔的中心蛋白质（称为a1亚基），以及至少两个调节VGCCs特性的辅助蛋白质（β和a2d亚基）。尽管它们具有病理相关性，但a2d亚基调控vgc通道的结构和功能机制仍然知之甚少。了解这种调控的分子基础具有基本的生物学重要性和医学重要性，因为a2d亚基被认为是开发新型vgc定向治疗方法的潜在途径。我将采用多学科方法，结合电生理、生化、成像和光学技术，研究a2d1亚基调控vgc的分子机制。我将重点关注一种新的生理途径来控制由a2d蛋白的酶切决定的vgc功能。我将研究这一机制的结构和功能方面，以确定vgc感知和响应电信号的能力是如何由a2d通过新的酶机制控制的。此外，我将评估通过抑制a2d酶切作用的新型vgc定向抑制剂的功效。我将研究这一途径对动脉SMCs中a2d1调节的影响。我将受益于与几个领先实验室的现有合作，这些实验室利用光学技术（加州大学洛杉矶分校的Riccardo Olcese教授）和结构生物学（伦敦大学学院的Mathew Gold博士）在vgc的电灵敏度研究方面具有互补的专业知识。此外，我将与主办机构的动脉SMCs研究专家（Iain Greenwood / Anthony Albert教授，sgl）分享专业知识，并与Annette Dolphin教授（UCL）分享a2d敲除小鼠的研究经验。我们的研究可以在理解a2d亚基调控VGGCs的分子基础方面开辟新天地。通过研究这一调控途径对动脉SMCs中a2d生理功能的影响，我们可以为未来针对病理性vgcc功能障碍（如高血压）的治疗方法的发展提供理论依据。除此之外，我们的实验可以提供适用于其他生物系统的概念进展，这些生物系统具有不同组成的VGCCs通道亚型，这些亚型也受与不同治疗领域（例如慢性疼痛）相关的a2d亚单位（如大脑和感觉神经元）的调节。

The entry of Calcium Ions in excitable cells is the most ubiquitous electro-chemical trigger for vital processes in human physiology. Calcium entry is the common upstream message for the propagation of signals between brain cells (neurons), the contraction of the heart cells (cardiac myocytes), and the arterial blood pressure smooth muscle cells (SMCs). The excitation - evoked Calcium entry in all these cell types is controlled by a specialized class of protein complexes, called Voltage - Gated Calcium channels (VGCCs). VGCCs form a specialised pore in the otherwise impermeable cell membrane to regulate selectively the flux of Calcium among other charged ions into the excitable cells. Even minimal alterations from the normal VGCCs functions in the heart and blood vessels can lead to severe human diseases. As a consequence, VGCCs regulation is targeted by clinically important small - molecule drugs prescribed to reduce arterial blood pressure and treat cardiac dysfunctions. However, their wide distribution often results in off-target effects and there is a pressing need to find novel therapeutic approaches. To form a fully functional complex VGCCs requires co-assembly of several proteins (subunits) a central protein forming the calcium selective pore (called a1 subunit), and at least two auxiliary proteins (beta and a2d subunits) modulating VGCCs properties. Despite their pathological relevance, the structural and functional mechanisms underlying the regulation of VGCCs channels by a2d subunits remains poorly understood. Understanding of the molecular basis this regulation is of basic biological importance, and of medical importance, as a2d subunits are recognised as a potential route for developing novel VGCCs - directed therapeutic approaches.I will pursue multidisciplinary approach by combining electrophysiological, biochemical, imaging and optical techniques to investigate the molecular mechanisms underlying the regulation of VGCCs by a2d1 subunits. I will focus on a novel physiological pathway for control VGCCs functionality determined by the enzymatic cleavage of a2d proteins. I will investigate both structural and functional aspects of this mechanism to determine how VGCCs ability to sense and respond to electrical signals is controlled by a2d by the novel enzymatic mechanism. In addition, I will assess the efficacy of novel VGCCs - directed inhibitors that act by supressing the enzymatic cleavage of a2d. I will examine the implications of targeting this pathway for the regulation of a2d1 in arterial SMCs.I will benefit from building on my existing collaborations with several leading laboratories of complementary expertise in studies of electrical sensitivity of VGCCs using optical techniques (Professor Riccardo Olcese, UCLA) and structural biology (Dr. Mathew Gold, UCL). In addition, I will share expertise with specialists in studies of arterial SMCs in the host institution (Professors Iain Greenwood / Anthony Albert, SGUL) and a2d -knock out mice with Professor Annette Dolphin (UCL).Our studies can break new ground in the understanding of the molecular basis of VGGCs regulation by a2d subunits that remains unresolved. By investigating the implications of this regulatory pathway for the physiological functions of a2d in arterial SMCs, we can provide rationale for development of future therapeutic approaches targeting pathological VGCCs dysfunctions in (such as hypertension). In addition to this, our experiments can provide conceptual advances applicable to other biological systems with different composition of VGCCs channel subtypes that are also regulated by a2d subunits (such as brain, and sensory neurons) relevant to different therapeutic areas (for example chronic pain).