Ion channel crystallisation for structural studies

Ion channels control the movement of ions allowing specific ions to pass across biological membranes. This apparently simple process is used to control many essential physiological processes including the generation and propagation of neuronal excitation, regulation of insulin secretion, heartbeat and cell volume. Ion channels are able to do this by having multiple means of controlling the opening and closing of the ion conduction pathway. Several recent ion channel structures have shed light on the important process of ion selectivity as well as providing clues on channel gating. However, it is not yet completely clear how ion channels control the passage of ions across the membrane. My work is centred on understanding the gating process through structural analysis mostly using X-ray crystallography. In order to get to this point, crystals of the target ion channels need to be grown. This thesis presents four projects that were aimed at developing the techniques for over-expression, purification and crystallisation of ion channels or transporters making them amenable for structural studies. One of the difficult steps in the structure determination pathway is obtaining well-ordered crystals of the target membrane protein. Crystals of the EcCLC channel (now found to be a transporter) from E. coli were initially unacceptable for structure determination. It was shown that the process of dehydration improved the quality and diffraction limit of the EcCLC crystals. This simple method provides a possible procedure for improving the resolution limit of soluble and membrane protein crystals. Inward rectifier potassium channels (Kir channels) are a distinct subfamily that preferentially allows K+ ions to move into the cell. Two bacterial homologues of Kir channels, KirBac1.1 and KirBac3.1, were crystallised in 3-D and 2-D forms, respectively. As the crystal structure revealed the full-length model provided an excellent starting point for understanding the gating process in these channels. The 3-D structure of KirBac1.1 was captured in the closed state. This K+ channel prevents ion movement by using hydrophobic residues to occlude the ion conduction pathway, altering the conformation of selectivity filter, misaligning the pore helices and decreasing the volume of the cavity. The 2-D projection maps of KirBac3.1 were obtained in both closed and a potentially open conformations. These two maps provided evidence as to how the structural elements of the inner, outer, slide helices and the C-terminal domains change relative to one another as the channel moves between these two gating states. These motions were modelled as rigid body movements. They included bending of the inner helices, a rotation of the outer helices as they straighten in the membrane, translocation of the slide helices and C-terminal domain rotations. It is hoped that the information gained from these projects, the plasmids construction, the expression systems and the protocols that were established for crystallisation of the ion channels and transporter will provide guidelines for future structural studies of membrane proteins.