Ion channel proteins play important roles in the development and function of cells, and remarkably, they are generally neither accessible in their native membranes nor in heterologous expression systems. Thus, the characterization of ion channels is severely limited. Biochemical purification and reconstitution of ion channel proteins is tedious and time consuming, and not always successful. A reliable expression system for ion channel proteins would allow characterization of organelle ion channels, which are hardly accessible at the moment, and would tremendously promote ion channel research in general. While cell-free (in vitro) protein expression has become a routine technique for soluble proteins, cell-free expression seemed impractical for membrane proteins, which precipitate in an aqueous environment. Only very recently cell-free expression of functional membrane proteins became feasible, by adding mild detergents or liposomes. This approach opens up exciting new possibilities to develop a cell-free expression system for ion channel proteins. The objective of this project is to develop a set of protocols that will allow for the routine cell-free expression and reconstitution of functional ion channels. Plant two-pore domain potassium-channels will be used as a model in this project, because they are relatively small (100 kDa) and they have a simple architecture; homodimers form a functional channel. Cell-free expression of plant potassium-channels will be performed in the presence of added liposomes. These small proteoliposomes will be fused into giant liposomes, thus making the recombinantly expressed potassium-channels accessible to characterization by the patch-clamp technique. The most critical part of this approach, the cell-free expression, will be developed in an international collaboration with Dr. Andreas Weber (University of Düsseldorf, Germany) who is a leading expert in the functional analysis of plant membrane transporters. Once experimental protocols are established for cell-free expression and reconstitution of plant potassium-channels, these protocols can very likely be adapted to other ion channels. The project has the potential to transform the field of ion channel research. There is a certain risk that the proposed expression and reconstitution of functional plant potassium-channels might not work; this risk is acceptable, since each experimental step (cell-free expression, liposome fusion, and patch-clamp) has been shown to be viable in separate lines of experiments. This project combines these techniques and its success will set the stage for adaptation of this approach for studies of other ion channels, and could conceivably transform how ion channels are studied.
Broader Impacts
The project is designed to engage undergraduate students in the development of these techniques. In addition, this project will allow the PI to extend his ongoing outreach activities at local schools, where he routinely gives presentations covering different topics of plant science ("Meat Eating Plants", "Do Plants Move?", "Monocot/Dicot") in an age appropriate manner.
