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Regulation of ion channel activity occurs through various mechanisms, via small molecule regulators, membrane potential, and specific interaction with lipid molecules. Inward rectifying potassium (Kir) channels conduction is controlled both by small molecules, such as polyamines blocking ion conduction, and lipid molecules that stabilize a conductive state. Advancing molecular understanding of this lipid regulation has been difficult as many structural techniques rely on detergent solubilized channels, which removes specific channel:lipid interaction. FRET, both macroscopically and in single molecules, can help surmount this issue, providing measurements of structural and dynamic changes induced by lipid regulation in a membrane environment. We have used macroscopic FRET to measure lipid specific structural changes in Kir2.1 in liposomes of defined composition. We have optimized purification and fluorophore labeling of the human Kir2.1 channel, which are reconstituted into liposomes of various lipid composition. We have measured PIP2-dependent FRET changes at numerous sites, which indicate structural movements of the intracellular domain of Kir2.1 upon PIP2 binding. This work specifically investigates how movements of the slide helix contribute to activation of the channel. Further efforts will help to define the molecular details of bulk anionic lipid and PIP2-dependent structural dynamics of Kir2.1 and help to understand more generally how lipid-protein interactions regulate membrane protein function.

Differences of ionic concentrations across lipid bilayers are some of the primary energetic driving forces for cellular electrophysiology. While macroscopic models of asymmetric ionic solutions are well-developed, their connection to ion, water, and lipid interactions at the atomic scale are much more poorly understood. In this study, we used molecular dynamics to examine a system of two chambers of equal ionic strength, but differing amounts of NaCl and KCl, separated by a lipid bilayer. Our expectation was that the net electrostatic potential difference between the two chambers should be small or zero. Contrary to our expectation, a large potential difference (−70mV) slowly evolved across the two water chambers over the course of our 172-ns simulation. This potential primarily originated from strong Na+ binding to the carbonyls of the phosphatidylcholine lipids. This ion adsorption also led to significant structural and mechanical changes in the lipid bilayer. We discuss this surprising result in the context of indirect experimental evidence for Na+ interaction with bilayers as well as potential caveats in current biomembrane simulation methodology, including force-field parameters and finite size effects.

Inwardly rectifying potassium (Kir) channels regulate cell excitability and potassium homeostasis. Our recent analyses show that Kir2.1 channels have a distinct (‘Secondary’) anionic phospholipid (PL(-)) binding site, in addition to the crystallographically determined (‘Primary’) PIP2 activating site. Docking results suggest that PL(-)s can bind to either site and therefore might compete with PIP2 at the ‘Primary’ site and inhibit. To test this prediction we performed the following assays with purified human Kir2.1 channels reconstituted in liposomes. First, Kir2.1 activity was measured with a fixed PIP2 content and with increasing content of various PL(-)s. At higher PL(-) levels, inhibition was observed that correlated well with predicted affinity at the ‘Primary’ site. The ‘Secondary’ site is generated by residues K64 and K219. K64C mutant channels are insensitive to PL(-) and only weakly PIP2-activated, but high PIP2 sensitivity is regenerated by tethering of K64C to the membrane by decyl-MTS modification. Inhibition by PL(-)s was more potent in decyl modified ‘Secondary’ site single (K64C) and double (k64C/K219A) mutant channels. It's likely that PL(-) binding at the ‘Primary’ site is augmented in these mutants as a consequence of increased effective PL(-) in the membrane as well as reduced electrostatic repulsion from the PL(-) at the ‘Secondary’ site. Finally PIP2 sensitivity was measured in the presence of increasing PL(-)s. The apparent PIP2 Kd was left-shifted at low PL(-) (as expected for the activatory effect at the ‘Secondary’ site), but shifted back to the right at higher PL(-)s, consistent with an inhibitory effect of bulk PL(-) at the ‘Primary’ site, if present at high enough levels in the membrane. Such interplay between PIP2 and other PL(-)s on Kir2.1 channel gating can be predicted by a mechanistic two-site binding model.

Inwardly rectifying potassium channels (Kir) are regulated by multiple factors, including multiple lipids. In addition to a specific requirement for phosphatidyl-4,5-bisphosphate (PI(4,5)P2) for channel activity of Kir2.1, analysis of purified proteins reconstituted into liposomes has revealed a secondary requirement for non-specific anionic lipids, which increase PI(4,5)P2 sensitivity by ∼100 fold [Cheng et al. 2011, Biophys. J. 100, 620-628]. Recent crystal structures of eukaryotic Kir channels in complex with PI(4,5)P2 reveal a common PI(4,5)P2 binding site [Hansen et al, 2011, Nature. 477, 495-498; Whorton & MacKinnon, 2011, Cell. 147, 199-208], but they have not identified the synergistic anionic lipid site.We have performed extensive docking simulations to identify potential interaction sites of different phospholipids with Kir2.1 channels. These simulations indicate two distinct binding sites; a high affinity site that corresponds to the crystallographic PI(4,5)P2 binding pocket and a lower affinity site, involving two lysine residues further towards the periphery of the cytoplasmic domain, that may correspond to the secondary anionic lipid site. When the two lysine residues are mutated to cysteine, channel activity is essentially abolished, even in the presence of PIP2. Cysteine modification of these residues by decyl-MTS, which essentially provides a ‘lipid tether' to the residue, restores channel activity in the presence of low levels of PIP2. These results point strongly to the identified site as being the site for non-specific anionic lipid interaction, and support a model in which the anionic lipid interaction (or ‘lipid tethering') pulls the Kir domain towards the membrane, facilitating PI(4,5)P2-mediated channel opening.

Ionotropic glutamate receptors are predominantly localized to neuronal synapses and are principal mediators of excitatory neurotransmission in the brain. Receptor function is regulated by multiple factors including ligands, ions, pH, posttranslational modification, subunit composition, RNA editing, and elements of the lipid environment. Analysis of channel function in native membranes has provided much quantitative information on these various regulatory features, however, investigation of lipid regulation is seriously hampered by an inability to control membrane composition in living cells. Although an inhibitory effect of free polyunsaturated fatty acids on GluK2(R) activation has been demonstrated, the specific bulk lipid requirements for normal channel operation, as well as susceptibility to fatty acid modulation, are unknown. To address this, we aim to purify rat kainate receptor 2 (GluK2), to reconstitute the purified protein, and to assess channel function under different defined lipid environments. Using Saccharomyces cerevisiae as an expression host, we have been able to express wild type GluK2 with either Gln or Arg at the editing site, and with different tags. In addition, we have expressed modified forms of GluK2 including an amino terminal domain (ATD) deletion mutant, and a double point mutation (Y590C/L572C) that allows disulfide crosslinking between ligand binding domain (LBD) dimers. We have succeeded in purifying wild type GluK2(R) through Flag affinity and subsequent size exclusion chromatography after solubilizing the protein using the detergent Foscholin-14 (F14). A major band at the expected monomer size of ∼100kDa and several bands at lower molecular weights are resolved on 1D SDS PAGE and are confirmed to be GluK2 through mass spectrometry (MS) analysis. MS analysis also reveals that a fraction of the heterologously expressed protein in yeast maintains a signal sequence at the N-terminus and is phosphorylated on a presumed extracellular residue, indicating misoriented topology.

Inwardly rectifying potassium (Kir) channels regulate cell excitability and potassium homeostasis in multiple tissues. All Kir channels absolutely require interaction of phosphatidyl-4,5-bisphosphate (PIP2) with a crystallographically identified binding site, but an additional non-specific secondary anionic phospholipid (PL(-)) is required to generate high PIP2 sensitivity of Kir2 channel gating, but the PL(-) binding site and mechanism are yet to be elucidated. We used docking simulations to identify a putative PL(-) binding site, adjacent to the PIP2 binding site, generated by two lysine residues from neighboring subunits. When either lysine is mutated to cysteine (K64C, K219C), channel activity is significantly decreased in cell membranes and in reconstituted liposomes. By directly tethering the residue to the membrane, modification of the K64C mutant with decyl MTS generates high PIP2 sensitivity in liposomes, even in the complete absence of PL(-)s. The results provide a coherent molecular mechanism for the secondary anionic lipid requirement of Kir2 channel activity: PL(-) interaction with a discrete binding site results in a conformational change that pulls the cytosolic Kir domain closer to the membrane, thereby stabilizing the high-affinity PIP2 activatory site.

Perfluorocarbon-based nanoemulsions with stabilizing surface monolayers of emulsifying phospholipids are promising platforms to carry diagnostic and therapeutic agents for cancer. However, to acheive their full therapeutic potential will require investigating the microscopic mechanism of nanoemulsion interactions with biological membranes and the forces that govern cargo transfer. From such investigations and the resulting mechanistic understanding it will be possible to exploit cargo and nanoemulsion characteristics to use them more effectively in imaging and therapeutic applications. Experimental observations suggest a distinctive “contact-facilitated” nanoemulsion delivery mechanism in which cargo diffuses to the targeted cell membrane through a lipid complex formed between a nanoemulsion and the target bilayer. This complex is hypothesized to be structurally comparable to the hemifusion stalk formed during membrane fusion. We are investigating this contact-facilitated delivery mechanism at a molecular level by employing multi-scale molecular dynamics simulations. Force field parameters for the nanoemulsion perfluorocarbon molecule were developed at multiple resolutions to give good agreement to experimental data at all scales of simulation. The structural and dynamical details of the nanoemulsions were characterized at an atomic level. However, in order to access larger time and length scales, the interactions between a nanoemulsion and a target bilayer were simulated using a coarse-grained model to directly examine lipid complex formation hypothesized to precede contact-facilitated delivery. In particular, various phospholipid compositions of the surface monolayer were tested for the lipid complex formation.

Inward rectifier potassium (Kir) channels are directly regulated by phospholipids, including phosphoinositides (PIPs) and anionic glycerophospholipids (ALs) in the absence of other proteins or downstream signaling pathways. In the presence of non-specific bulk ALs Kir2.1 channels are highly selective for activation by PI(4,5)P2, and under certain lipid conditions are inhibited by other PIPs. Biochemical approaches with purified full-length Kir2.1 channels enabled us to identify specific residues that regulate binding for 6 of 7 PIP ligands, and to estimate the energetic contributions of each of these residues to the binding of each PIP. A recent PI(4,5)P2 bound Kir2.2 crystal structure (PDB entry 3SPI) suggests that PI(4,5)P2 interacts directly with residues R80, R82, R182, K185, K187, K189 in Kir2.1. While K185 contributes ∼0.89 kcal/mol to the binding of PI(4,5)P2, the other residues in this binding pocket did not alter the affinity (and thus binding energy) of ligand binding. However, when R189, R218 and R219 residues, which do not reside in this binding pocket, were mutated to glutamine, the binding affinity for PI(4,5)P2 was markedly reduced compared to WT, with calculated ΔΔG's of ∼0.44, ∼1.24 and ∼0.85 kcal/mol, respectively. Notably, the particular subsets of residues that when mutated disrupt binding are different for each PIP. We further employed ligand docking approaches on homology models of human Kir2.1 channels based on chicken Kir2.2 crystal structures to identify putative binding regions for these PIPs and ALs. These simulations suggest that different PIPs may bind in similar but non-identical locations, in multiple poses. Our combined biochemical and computational analyses provide insight to the complexities of variable specificity, competition and synergy between different phospholipids in regulation of Kir channel gating.