Mutational Insight into Allosteric Regulation of Kir Channel Activity

Abstract

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.

Cooperative Gating of a K+ Channel by Unmodified Biological Anionic Lipids Viewed by Solid-State NMR Spectroscopy

“…CG molecular dynamics (CGMD) simulations were performed in tandem to complement the NMR data analysis. We previously observed in MD calculations that residues involved in the allosteric network could be mutated to impact PG residency time and residue–residue contacts throughout the channel . Thus, we hypothesized that we might observe interesting phenomena in CGMD PG titrations as well.…”

“…When these residues are mutated to glutamine, the channel is closed and inactive. Based on the mutational analysis in CGMD simulations, we also suggested that residues proximal to this arginine pocket may also play a role, either during the first steps of allosteric communication or as secondary residues supporting the pocket itself . However, experimentally, we did not have a complete picture of lipid–protein interactions in the open-activated state of the channel.…”

“…Based on the mutational analysis in CGMD simulations, we also suggested that residues proximal to this arginine pocket may also play a role, either during the first steps of allosteric communication or as secondary residues supporting the pocket itself. 36 However, experimentally, we did not have a complete picture of lipid− protein interactions in the open-activated state of the channel. To better identify the full set of amino acid side chain interaction with lipid headgroups, we acquired 2D z-filtered transferred echo double resonance (zf-TEDOR) 15 N− 13 C correlation spectra.…”

“…The extent of this motion was correlated to both PG residency time on gating arginines in silico and to total K + flux in vitro. 36 Here, we hypothesize that the mechanism of cooperativity, whereby lipid binding is communicated from subunit to subunit, is mediated through slide helix motion that functions to increase the accessibility of adjacent subunits' PG binding sites. It should be noted that the acyl tails for PG lipids in TEDOR samples differ from those presented in Figures 1 and 2.…”

“…Previously, we identified that gating arginine residues were directly responsible for channel activation, 16 and subsequent work showed that tryptophan side chains were also essential for anionic lipid recruitment. 36 Ultimately, however, a fundamental question was left unanswered: what is the affinity of KirBac1.1 for anionic lipids, and what role, if any, do lipid− protein cooperativity and intersubunit cooperativity play in the activation of KirBac1.1? Lipid−protein dissociation constants (K d 's) are rarely known due to complications in experimentally measuring such events in a native or native-like environment, which is unfortunate as these interactions govern and regulate a myriad of biological responses.…”