Mutational Insight into Allosteric Regulation of Kir Channel Activity


Potassium (K+) channels are regulated in part by allosteric communication between the helical bundle crossing, or inner gate, and the selectivity filter, or outer gate. This network is triggered by gating stimuli. In concert, there is an allosteric network which is a conjugated set of interactions which correlate long-range structural rearrangements necessary for channel function. Inward-rectifier K+ (Kir) channels favor inward K+ conductance, are ligand-gated, and help establish resting membrane potentials. KirBac1.1 is a bacterial Kir (KirBac) channel homologous to human Kir (hKir) channels. Additionally, KirBac1.1 is gated by the anionic phospholipid ligand phosphatidylglycerol (PG). In this study, we use site-directed mutagenesis to investigate residues involved in the KirBac1.1 gating mechanism and allosteric network we previously proposed using detailed solid-state NMR (SSNMR) measurements. Using fluorescence-based K+ and sodium (Na+) flux assays, we identified channel mutants with impaired function that do not alter selectivity of the channel. In tandem, we performed coarse grain molecular dynamics simulations, observing changes in PG-KirBac1.1 interactions correlated with mutant channel activity and contacts between the two transmembrane helices and pore helix tied to this behavior. Lipid affinity is closely tied to the proximity of two tryptophan residues on neighboring subunits which lure anionic lipids to a cationic pocket formed by a cluster of arginine residues. Thus, these simulations establish a structural and functional basis for the role of each mutated site in the proposed allosteric network. The experimental and simulated data provide insight into key functional residues involved in gating and lipid allostery of K+ channels. Our findings also have direct implications on the physiology of hKir channels due to conservation of many of the residues identified in this work from KirBac1.1.


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Potassium channels, allosteric regulation, helical bundle crossing, selectivity filter, inward-rectifier K+ (Kir) channels, phosphatidylglycerol (PG), solid-state NMR, molecular dynamics simulations


Maryam Yekefallah, Carver A. Rasberry, Evan J. van Aalst, Holley P. Browning, Reza Amani, Derek B. Versteeg, and Benjamin J. Wylie ACS Omega 2022 7 (48), 43621-43634 DOI: 10.1021/acsomega.2c04456