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

在哺乳动物体内，电信号控制着重要的过程，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。跨膜电压的变化发生在不同的离子种类通过专门的整合膜蛋白-离子通道从膜的一侧流到另一侧时。例如，钠离子流负责沿着神经轴突传播动作电位，钙离子流进入心脏细胞将触发心跳。钾（K+）电流是大多数细胞电事件的突破点，当与内在电压和化学传感机制结合时，为许多细胞类型提供负反馈。因此，有一个大的生物技术驱动器，以确定化学试剂，调节K+选择性通道。一种特定类别的激活剂（尼可地尔，米诺地尔）在临床上用于松弛血管平滑肌，从而降低血压，而抑制剂（格列齐特）是2型糖尿病最广泛的治疗方法之一。离子通道是拟议项目的主题，是细胞内离子调节的K+通道家族，由四个a亚基组装而成。该家族的第一个成员（Sloa1）组装形成一个众所周知的和特征性的钙激活K+通道，具有独特的药理学特征。它的兄弟姐妹Sloa2.2，a2.2和a3知之甚少，在过去的六年里，只有同样多的研究出版物描述了它们的行为。与其他多基因K+通道家族不同，单个成员在细胞内离子调节其行为、离子流过通道孔的速率、合成后到达细胞膜的能力以及改变活性的化学试剂方面具有完全不同的性质。该项目的目的是证明更多的离子通道多样性可以通过一种以上类型的Sloa亚基的共组装来实现。每个单独的亚基可以赋予蛋白质复合物的性质可以通过记录来自细胞的K+电流来确定，所述细胞已经被工程化以从外源核酸合成蛋白质。通过使用这种方法，还可以引入修饰的离子通道DNA，这将使我们能够识别蛋白质的哪些结构域负责不同的行为。由于不同Sloa亚基的分子结构相似，因此可以通过将DNA片段从一个基因移植到另一个基因来交换结构域。电生理学实验将确定功能特性是否也被转移。已知4种膜蛋白Slob1 - 4的单独家族与由4种Sloa1亚基组成的通道共组装，从而在不同程度上改变通道的功能和药理学性质。目前还不知道它们是否能够与其他Sloa亚基组装并调节，这也将得到解决。总之，目的是研究通过Sloa和B亚基的不同组合可以获得的功能和药理学K+通道特性的范围。这将使我们能够预测膜电流时，它是已知的这8个基因中的哪一个特定的细胞类型表达。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，通过了解异聚体（亚基的混合物）Slo通道的药理学，我们将能够定义更多的细胞特异性药物靶标。这是因为如果药物靶向一个单独的亚基的同源组装，则其他细胞类型具有相同亚基组合的机会将低于它们仅具有一个亚基的机会。

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.