Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Investigating the role of conformational change in gating and conduction of KIR K+ channels (#299)

Katrina Black 1 2 , David Miller 1 2 3 , Jani Bolla 4 , Paul Johnson 5 , Adam Hill 6 , Carol V Robinson 4 , Derek Laver 5 , Brian J Smith 7 , Jacqueline M Gulbisa 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria , Australia
  2. Department of Medical Biology, The University of Melbourne, Parkville, Victoria , Australia
  3. Present address: Imperial College Advanced Hackspace, Imperial College London, UK
  4. Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, UK
  5. School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, Australia
  6. Computational Cardiology Laboratory, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia
  7. Department of Biochemistry & Molecular Biology , LaTrobe University, Bundoora, VIC, Australia

Potassium (K+) channels allow rapid K+ diffusion across cell membranes. It is essential that potassium (K+) levels within cells be finely controlled for normal behaviour of cells, tissues and organisms. Potassium channels are highly selective pores that allow K+ diffusion across cell membranes and so control K+ levels in a highly regulated process. Channels rapidly switch between permissive and restrictive states in a process known as ‘gating’. Conventionally, it is thought that K+ channels gate by undergoing a conformational change, with ‘open’ channels containing a wide pore mouth, and ‘closed’ channels a constricted mouth, such that a fully hydrated K+ ion (~8 Å) cannot transit through the channel. 

We have further investigated the gating phenomenon in the KIR subfamily of potassium channels. By covalently linking adjacent subunits together, movement at the pore mouth could be constrained, limiting the channel to its narrow or ‘closed’ state. Crosslinking was inspected by a variety of biophysical techniques, including X-ray crystallography, SDS-PAGE and native mass spectrometry. The functional capacity of locked relative to non-locked channels was then assessed by means of a bulk fluorometric liposomal flux assay.

Results indicated that channels with a limited narrow pore aperture were able to function as effectively as wildtype channels, suggesting that in KIR channels, gating is inconsistent with conformational change in the pore.