Multi-protein assembly of intracellular ion-sensitive potassium channel complexes

细胞内离子敏感钾通道复合物的多蛋白组装

• 批准号: BB/D000939/1
• 负责人: Jonathan Lippiat
• 金额: $ 29.68万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD000939%2F1
• 关键词: Multi; protein; assembly; intracellular; ion

In the mammalian organism, electrical signals control important processes such as nerve impulses, muscle contraction, and hormone secretion. The advantage of this method of communication is that the temporal resolution is higher than that of chemical signalling. Changes in transmembrane voltage occur when different ionic species flow from one side of the membrane to the other through specialist integral membrane proteins - ion channels. For example, sodium flux is responsible for the propagated action potential along a nerve axon, and the flow of calcium ions into heart cells will trigger a heartbeat. Potassium (K+) currents are a foot on the break for most cellular electrical events, and when coupled with intrinsic voltage and chemical sensing mechanisms provide negative feedback for many cell types. Thus there is a large biotechnological drive for identifying chemical agents that modulate K+-selective channels. Activators (nicorandil, minoxidil) of one particular class are used clinically to relax vascular smooth muscle and thus lower blood pressure, whilst an inhibitor (gliclazide) is one of the most wide-spread treatments for type-2 diabetes. The ion channels that are the topic of the proposed project are a family of intracellular ion-regulated K+ channels, which are formed by the assembly of four a subunits. The first member of the family (Sloa1) assembles to form a well known and characterized calcium-activated K+ channel with a unique pharmacological profile. Its siblings Sloa2.2, a2.2, and a3 are poorly understood, and over the last six years there have been only as many research publications describing their behaviour. Unlike other multi-gene families of K+ channels the individual members have quite different properties with respect to which intracellular ions modulate their behaviour, the rate of ion flow through the channel pore, the ability to reach the cell membrane following synthesis, and the chemical agents that alter activity. The aim of the project is to demonstrate that even more ion channel diversity can be achieved by the co-assembly of more than one type of Sloa subunit. The properties that each individual subunit can confer to the protein complex with be determined by recording K+ currents from cells that have been engineered to synthesise the proteins from foreign nucleic acids. By using this approach modified ion channel DNA can also be introduced, which will allow us to identify which domains of the proteins are responsible for the different behaviour. Because the molecular structures of the different Sloa subunits are similar, domains can be swapped by transplanting segments of DNA from one gene to the other. Electrophysiological experiments will determine whether the functional properties have also been transferred. A separate family of 4 membrane proteins Slob1-4 are known to co-assemble with channels comprised of 4 Sloa1 subunits, altering the functional and pharmacological properties of the channel to different extents. It is not know if they are able to assemble with and modulate the other Sloa subunits, and this will also be addressed. In summary, the aim is to investigate the range of functional and pharmacological K+ channel properties that can be obtained by different combinations of Sloa and b subunits. This will allow us to make predictions of the membrane currents when it is known which of these 8 genes a particular cell-type expresses. On the other hand, we will be able to predict which molecular subunits are likely to be present by studying the properties of the K+ current from a native mammalian cell. Furthermore, by understanding the pharmacology of heteromeric (mixture of subunits) Slo channels we will be able to define more cell-specific drug targets. This is because the chance of other cell types having the same combination of subunits will be lower than the chance of them having just the one subunit, if a drug targets a homomeric assembly of one subunit alone.

在哺乳动物生物体中，电信号控制着重要过程，例如神经冲动，肌肉收缩和激素分泌。这种通信方法的优点是时间分辨率高于化学信号传导的分辨率。当不同的离子物种通过专业的积分膜蛋白 - 离子通道从膜的一侧流到另一侧，跨膜电压的变化发生。例如，钠通量负责沿神经轴突传播的动作电位，钙离子流入心脏细胞会引发心跳。对于大多数细胞电气事件而言，钾（K+）电流是断裂的脚，当与内在电压和化学感应机制相结合时，为许多细胞类型提供了负反馈。因此，有一个较大的生物技术驱动器，用于鉴定调节K+选择通道的化学试剂。一种特定类别的活化剂（尼古兰，米诺地尔）在临床上用于放松血管平滑肌，从而降低血压，而抑制剂（胶质氮）是2型糖尿病的最广泛的治疗方法之一。拟议项目主题的离子通道是细胞内离子调节的K+通道的家族，由四个A亚基组装而成。该家族的第一个成员（SlOA1）组装出具有独特的药理学特征的钙激活的K+通道。它的兄弟姐妹Sloa2.2，A2.2和A3的理解很少，在过去的六年中，只有许多研究出版物描述了其行为。与其他K+通道的其他多基因家族不同，各个成员具有相当不同的特性，而细胞内离子调节其行为，离子流经通道孔的速率，合成后到达细胞膜的能力以及改变活性的化学剂。该项目的目的是证明，多种类型的Sloa亚基的共同组装可以实现更多的离子渠道多样性。每个单个亚基可以通过记录已经设计的细胞中的K+电流来确定的蛋白质复合物的特性，这些细胞已设计为合成外来核酸的蛋白质。通过使用这种方法修改的离子通道DNA也可以引入，这将使我们能够确定蛋白质的哪些域负责不同的行为。由于不同Sloa亚基的分子结构相似，因此可以通过将DNA的移植段从一个基因移动到另一个基因来交换结构域。电生理实验将确定功能特性是否也已转移。已知一个单独的4个膜蛋白SLOB1-4的家族与由4个SlOA1亚基组成的通道共组装，将通道的功能和药理特性更改为不同的范围。不知道他们是否能够组装并调节其他Sloa亚基，这也将得到解决。总而言之，目的是研究可以通过SLOA和B亚基的不同组合获得的功能和药理学K+通道特性的范围。这将使我们能够对膜电流进行预测，而当它已知特定细胞类型的这8个基因中的哪一个中的哪个。另一方面，我们将能够通过研究天然哺乳动物细胞的K+电流的性质来预测哪些分子亚基可能存在。此外，通过了解杂体（亚基的混合物）SLO通道的药理学，我们将能够定义更多细胞特异性的药物靶标。这是因为如果某种药物仅针对一个亚基的同源组件，那么其他具有相同亚基组合的细胞类型的机会将低于其仅具有一个亚基的机会。