The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.

Membrane proteins and lipids coevolved to yield unique coregulatory mechanisms. Inward-rectifier K+ (Kir) channels are often activated by anionic lipids endemic to their native membranes and require accessible water along their K+ conductance pathway. To better understand Kir channel activation, we target multiple mutants of the Kir channel KirBac1.1 via solid-state nuclear magnetic resonance (SSNMR) spectroscopy, potassium efflux assays, and Förster resonance energy transfer (FRET) measurements. In the I131C stability mutant (SM), we observe an open-active channel in the presence of anionic lipids with greater activity upon addition of cardiolipin (CL). The introduction of three R to Q mutations (R49/151/153Q (triple Q mutant, TQ)) renders the protein inactive within the same activating lipid environment. Our SSNMR experiments reveal a stark reduction of lipid–protein interactions in the TQ mutant explaining the dramatic loss of channel activity. Water-edited SSNMR experiments further determined the TQ mutant possesses greater overall solvent exposure in comparison to wild-type but with reduced water accessibility along the ion conduction pathway, consistent with the closed state of the channel. These experiments also suggest water is proximal to the selectivity filter of KirBac1.1 in the open-activated state but that it may not directly enter the selectivity filter. Our findings suggest lipid binding initiates a concerted rotation of the cytoplasmic domain subunits, which is stabilized by multiple intersubunit salt bridges. This action buries ionic side chains away from the bulk water, while allowing water greater access to the K+ conduction pathway. This work highlights universal membrane protein motifs, including lipid–protein interactions, domain rearrangement, and water-mediated diffusion mechanisms.

The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K+ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K+ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional.

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.