Exploiting a novel hybrid ion channel to understand the mechanism of sodium ion selectivity

Almost every single process in the human body is controlled at some level by electrical signals, from the way our hearts beat, the way our muscles move, to the way we think. These electrical signals are generated and controlled by a family of proteins called 'ion channels' which reside in the membrane of every living cell and which act as 'electrical switches' to control the selective movement of charged ions like sodium (Na+) into the cell and and to allow potassium (K+) out of the cell. It is therefore absolutely essential that these membrane pores are able to be 'selective' for these ions i.e. to distinguish between Na+ and K+, as otherwise the movement of these ions would become mixed up and these electrical signals would not be able to happen. To understand the fundamental mechanisms which control this 'ionic selectivity' requires a detailed knowledge about the three-dimensional shape and structure of these proteins. The most common way of obtaining high-resolution structural information involves purifying the protein and then concentrating it so that it forms crystals, similar to the way a solution of table salt crystallises when it begins to dry out. These crystals are then placed in an X-ray beam and the way in which these X-ray beams are scattered by the atoms within the crystal tells us about the 3D-structure of the protein. A lot is already known about the way in which channels select for K+ ions over Na+ ions and the 2003 Nobel Prize in chemistry was awarded to Prof Rod MacKinnon (Rockefeller University) for determining this mechanism in a bacterial K+ channel. However, the mechanism by which similar channels select for Na+ over K+ remains elusive and remains one of the major challenges in ion channel biophysics. One of the problems has been that there are very few Na+ channels which produce enough protein suitable for crystallography. We have identified a possible solution to this problem as we have recently cloned a novel bacterial Na+ channel gene which is simple in structure and very similar to several other related K+ channels which have already been crystallised. We have conducted extensive preliminary investigations which demonstrate the viability of this project i.e. we show that this novel channel (KirBac9.1) expresses well and can be purified in an intact functional form. More importantly, we also demonstrate that this purified protein is capable of forming protein crystals which produce 'diffraction patterns' i.e. the data required to determine their structure. Based upon these exciting new findings we seek funding for two research assistants and associated costs to pursue this exciting new opportunity to determine the structure of this novel Na+ channel and further characterise its functional properties. This will enable us to build upon these results and move this project forwards. Achievement of these goals would have an international impact as it would not only unlock the basic molecular mechanisms of this most fundamental biological process, but would also provide a framework for understanding how this process occurs in human Na+ channels. The potential applications of this knowledge would not only benefit basic science but also have a major effect on the design of novel drugs, as Na+ channels are important therapeutic targets.

The ability of ion channels to be selectively permeable to different cations is of fundamental biological significance and in higher organisms underlies the basis of all electrical signalling processes, as well as many fluid and electrolyte transport pathways. The biophysical mechanisms which underlie ionic selectivity are therefore of fundamental biological importance. The 3D crystals structures of several different prokaryotic K+ channels have now provided an insight into how K+ selectivity is achieved. However, the mechanisms which allow similar channels to be selective for Na+ ions over K+ ions remain elusive. The principal problem is that, unlike K+ channel genes which are almost ubiquitous, prokaryotic Na+ channel genes are exceedingly rare, and those that do exist have not proven easy substrates for structural determination. We have identified a novel hybrid prokaryotic channel which has the basic structure of a simple 2 TM K+ channel, but with a selectivity filter sequence homologous to mammalian Na+ and Ca2+ channels. Our preliminary studies show that this channel functions as a Na+ channel and that specific mutations within the selectivity filter allow it to become K+ permeable. More importantly however, we have demonstrated that this channel can be expressed as a functional, membrane-bound tetramer capable of producing diffraction quality crystals. This novel hybrid channel therefore represents an exciting and realistic opportunity to obtain a 3D-structure of a Na+ channel pore. In this study we propose to a) further characterise the functional properties of this channel and the role of specific residues within the pore, b) optimise the crystallisation conditions to obtain a high-resolution structure of this channel, and c) to utilise this structural and functional information in conjunction with biomolecular modelling and simulation studies to determine the biophysical mechanism which underlie Na+ ion selectivity.