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

点击翻译按钮获取中文摘要

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.