Precrystallization Screening of FMRF-amide Gated Sodium Channel in Helix Aspersa

The Phe-Met-Arg-Phe-amide (FMRF-amide) gated sodium channel in Helix aspersa (garden snail) is the first peptide gated ion channel to be cloned. The FMRF-amide gated sodium channel is a homotrimer and belongs to the ENaC/degenerin family of ion channels which are involved in maintenance of sodium-water balance, taste sensation and pain. Although FMRF-amide gated sodium channel is found in snails, the protein can serve as a model for other channels of this family. The 3-dimensional structure of FMRF-amide gated sodium channel may provide an additional channel structure for the ENaC/degenerin family. Our main goal is to determine the 3-dimensional structure of FMRF-amide channel. We have designed and generated a C-terminally polyhistidine tagged FMRF-amide protein. Small scale expression experiments have been performed and produced intact trimeric FMRF-amide gated channel protein. Furthermore, detergent screens and a time course study have been performed to find suitable conditions for stabilizing homotrimer. We are analyzing the stability of protein during metal affinity chromatography and size exclusion chromatography (SEC) and have begun with the crystallization trials. From the results, we can conclude that the current FMRF-amide gated channel construct is suitable for protein production. Our western blot analysis and SEC results show that we have been getting sufficient amount of protein. We anticipate that purified protein, in milligram quantities, will be isolated and subjected to crystallization trials. Future experiments will focus on generating protein on a larger scale to determine the optimal conditions required to crystallize the protein and to determine the FMRF-amide channel’s 3-dimensional crystal structure.