Oral Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Ion currents through Kir potassium channels are gated by anionic lipids (#7)

Katrina A Black 1 , Ruitao Jin 2 , Sitong He 2 , Oliver B Clarke 3 , Di Wu 4 , Jani R Bolla 4 , Paul Johnson 5 , Agalya Periasamy 1 , Ahmad Wardak 1 , Peter Czabotar 1 , Peter Colman 1 , Carol Robinson 4 , Derek Laver 5 , Brian Smith 2 , Jacqueline Gulbis 1
  1. Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
  2. Latrobe Institute for Molecular Science, Latrobe University, Bundoora, Vic, Australia
  3. Biochemistry and Molecular Biophysics, Columbia University, New York, USA
  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

Potassium (K+) channels allow rapid, highly selective and finely controlled diffusion of potassium ions across cell membranes. Control over K+ flux occurs by channels rapidly switching between permissive and restrictive states in a process known as ‘gating’. Conventional thinking around gating has assumed that ion flow is regulated by conformational rearrangements that induce physical occlusion of the inner pore mouth. However, recent work1 in the Gulbis and Smith laboratories has shown that constriction of the conduction pathway is ineffective at controlling permeation.

In this work2, we present evidence for a novel, lipid-mediated model of gating in KIR channels. We utilised a broad range of methodologies in this study, including molecular dynamics simulations, crystallography, native mass spectrometry, bulk fluorometric liposomal flux assays, and single-channel electrophysiology. We show that acyl tails of phospholipids occupy structurally identified fenestrations in the wall of the channel pore. Through these fenestrations, these acyl chains interact via hydrophobic interactions with branched, aliphatic side chains to regulate the permeation pathway. We identify Leu-124 as the primary energetic barrier to permeation in the prokaryotic channel KIRBac3.1, using both experimental and computational methods. Furthermore, we show modulation of channel activity via the removal (L124M) or addition (Y132I) of aliphatic side chains at key positions along the conducting pathway. The energetic barrier to permeation at L124 is also controllable by the application of hydrocarbon chains with sufficient length to infiltrate the fenestration and interact with the side chain within the pore. Finally, we observe a tighter association between the channel and anionic phospholipids, including PE and PG, than with neutral lipids such as PC, implying that it is anionic lipids that occupy the binding site about the fenestration. In summary, we propose that binding of anionic lipids directly controls gating in KIR channels via interactions with branched hydrophobic residues in the pore.

This study offers a significant conceptual shift in our understanding of K+ channel gating. Although based on a prokaryotic channel, it is well established that many eukaryotic potassium and non-selective channels are regulated by anionic lipids. This work, therefore, provides a framework by which to interpret similar lipid-occupied fenestrations in eukaryotic channels, such as in K2P and TRP channels, or to investigate anionic lipid potentiation of KIR channels by phosphoinositide lipids.

  1. Black KA*, He S*, Jin R*, Miller DM, Bolla JR, Clarke OB, Johnson P, Windley M, Burns CJ, Hill AP, Laver D, Robinson CV, Smith BJ, Gulbis JM. A constricted opening in Kir channels does not impede potassium conduction. Nat Commun, (2020).
  2. Jin R*, He S*, Black KA*, Clarke OB, Wu D, Bolla JR, Johnson P, Periasamy A, Wardak A, Czabotar P, Colman P, Robinson CV, Laver D, Smith BJ, Gulbis JM. Ion currents through Kir potassium channels are gated by anionic lipids. Biorxiv (2021) (https://www.biorxiv.org/content/10.1101/2021.09.21.461288v1).