Investigating the role of the inner helix bundle crossing during KIR channel gating

© 2018 Dr Katrina Anne BlackControl over potassium (K+) levels is essential to normal tissue homeostasis and a number of physiological processes such as electrolyte balance, cell signalling and cellular electrical activity. Potassium channels mediate the flux of potassium across the cell membrane in a highly selective manner. It is essential that channels be able to regulate the diffusion of ions, and so must be able to switch between a conductive and non-conductive state, a process known as gating. During gating, the channel’s pore width is thought to change, with an ‘open’ channel having a wide pore aperture, and a ‘closed’ channel a narrow one. A wide body of accessibility, mutagenesis, spectroscopic, structural and simulation-based studies have led to the conventional acceptance of this model, although there are inconsistencies between published data and the gating model (Chapter Two). This study investigates whether a conformational change in the pore aperture region is essential for normal conduction and gating in the prokaryotic KIR channel, KIRBac3.1. KIRBac3.1 consistently adopts a narrow pore aperture in crystal structures, which has been interpreted as a ‘closed’ conformation. The narrowness allows adjacent subunits to be covalently crosslinked together, thereby constraining the movement of the inner helices and preventing the aperture from widening. In Chapter Four, the steps required to design, generate and assess the formation of crosslinks at the bundle crossing of a K+ channel are presented. SDS-PAGE, X-ray crystallography, and native mass spectrometry were used to verify that crosslinks had formed correctly and efficiently. While SDS-PAGE provided preliminary assessment of crosslinking efficiency, crystallography enabled visualisation of crosslinks and any effect of mutagenesis on the global and local protein conformation. The sensitivity of native mass-spectrometry verified that the correct crosslinked species had formed. In Chapter Five, the function of wildtype, non-crosslinked mutant and crosslinked mutant channels is discussed. A novel fluorescent liposome flux assay was developed to assess the activity of crosslinked and non-crosslinked KIRBac3.1. The results indicate that a constricted pore aperture did not prevent crosslinked channels functioning as effectively as wildtype channels. Chapter Five also describes steps that were taken to verify that functional results derived from crosslinked KIRBac3.1 channels. The quantity of incompletely crosslinked material present in proteoliposomes, as well as the relationship between function and g of channels incorporated into liposomes, were analysed. Chapter Six provides discussion of the results, including the strengths and caveats associated with the methods chosen. Chapter Six also attempts to rationalise the findings of the study, as well as place the findings within a literary context. In summary, in this study, two sets of KIRBac3.1 mutants were efficiently crosslinked at the bundle crossing under three reactive conditions. All six forms of crosslinked KIRBac3.1 were as functional as wildtype or non-crosslinked channels in a liposomal flux assay. The quantity of incompletely crosslinked material was insufficient to explain the measured functional output of the assay and several controls ruled out the presence of a contaminating transporter or channel mediating function. Together, the data indicate that in KIRBac3.1 channels, constraining the pore to a narrow conformer does not preclude gating or conduction. This finding can be rationalised by suggesting that K+ ions may translocate through the narrow inner pore mouth in a partially dehydrated state