Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles

© 2019 Biophysical Society Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes

Synthetic Analogues of the Snail Toxin 6-Bromo-2-mercaptotryptamine Dimer (BrMT) Reveal That Lipid Bilayer Perturbation Does Not Underlie Its Modulation of Voltage-Gated Potassium Channels

Drugs do not act solely by canonical ligand–receptor binding interactions. Amphiphilic drugs partition into membranes, thereby perturbing bulk lipid bilayer properties and possibly altering the function of membrane proteins. Distinguishing membrane perturbation from more direct protein–ligand interactions is an ongoing challenge in chemical biology. Herein, we present one strategy for doing so, using dimeric 6-bromo-2-mercaptotryptamine (BrMT) and synthetic analogues. BrMT is a chemically unstable marine snail toxin that has unique effects on voltage-gated K+ channel proteins, making it an attractive medicinal chemistry lead. BrMT is amphiphilic and perturbs lipid bilayers, raising the question of whether its action against K+ channels is merely a manifestation of membrane perturbation. To determine whether medicinal chemistry approaches to improve BrMT might be viable, we synthesized BrMT and 11 analogues and determined their activities in parallel assays measuring K+ channel activity and lipid bilayer properties. Structure–activity relationships were determined for modulation of the Kv1.4 channel, bilayer partitioning, and bilayer perturbation. Neither membrane partitioning nor bilayer perturbation correlates with K+ channel modulation. We conclude that BrMT’s membrane interactions are not critical for its inhibition of Kv1.4 activation. Further, we found that alkyl or ether linkages can replace the chemically labile disulfide bond in the BrMT pharmacophore, and we identified additional regions of the scaffold that are amenable to chemical modification. Our work demonstrates a strategy for determining if drugs act by specific interactions or bilayer-dependent mechanisms, and chemically stable modulators of Kv1 channels are reported

Fluorinated Alcohols' Effects on Lipid Bilayer Properties

Fluorinated alcohols (fluoroalcohols) have physicochemical properties that make them excellent solvents of peptides, proteins, and other compounds. Like other alcohols, fluoroalcohols also alter membrane protein function and lipid bilayer properties and stability. Thus, the questions arise: how potent are fluoroalcohols as lipid-bilayer-perturbing compounds, could small residual amounts that remain after adding compounds dissolved in fluoroalcohols alter lipid bilayer properties sufficiently to affect membranes and membrane protein function, and do they behave like other alcohols? To address these questions, we used a gramicidin-based fluorescence assay to determine the bilayer-modifying potency of selected fluoroalcohols: trifluoroethanol (TFE), HFIP, and perfluoro-tert-butanol (PFTB). These fluoroalcohols alter bilayer properties in the low (PFTB) to high (TFE) mM range. Using the same assay, we determined the bilayer partitioning of the alcohols. When referenced to the aqueous concentrations, the fluoroalcohols are more bilayer perturbing than their nonfluorinated counterparts, with the largest fluoroalcohol, PFTB, being the most potent and the smallest, TFE, the least. When referenced to the mole fractions in the membrane, however, the fluoroalcohols have equal or lesser bilayer-perturbing potency than their nonfluorinated counterparts, with TFE being more bilayer perturbing than PFTB. We compared the fluoroalcohols' molecular level bilayer interactions using atomistic molecular dynamics simulations and showed how, at higher concentrations, they can cause bilayer breakdown using absorbance measurements and 31P nuclear magnetic resonance

Synthetic Analogs of the Snail Toxin 6-Bromo-2-Mercaptotryptamine Dimer (BrMT) Reveal That Lipid Bilayer Perturbation Does Not Underlie Its Modulation of Voltage-Gated Potassium Channels

Distinguishing membrane perturbation from more direct protein-ligand interactions is an ongoing challenge in chemical biology. Herein, we present one strategy for doing so, using dimeric 6-bromo-2-mercaptotryptamine (BrMT) and synthetic analogs. BrMT is a chemically unstable marine snail toxin that has unique effects on voltage-gated K+ channel proteins, making it an attractive medicinal chemistry lead. BrMT is amphiphilic and perturbs lipid bilayers, raising the question of whether its action against K+ channels is merely a manifestation of membrane perturbation. To determine whether medicinal chemistry approaches to improve BrMT might be viable, we synthesized BrMT and 11 analogs and determined their activities in parallel assays measuring K+ channel activity and lipid bilayer properties. Our work demonstrates a strategy for determining if drugs act by specific interactions or bilayer-dependent mechanisms, and chemically stable modulators of Kv1 channels are reported

Synthetic Analogs of the Snail Toxin 6-Bromo-2-Mercaptotryptamine Dimer (BrMT) Reveal That Lipid Bilayer Perturbation Does Not Underlie Its Modulation of Voltage-Gated Potassium Channels

Distinguishing membrane perturbation from more direct protein-ligand interactions is an ongoing challenge in chemical biology. Herein, we present one strategy for doing so, using dimeric 6-bromo-2-mercaptotryptamine (BrMT) and synthetic analogs. BrMT is a chemically unstable marine snail toxin that has unique effects on voltage-gated K+ channel proteins, making it an attractive medicinal chemistry lead. BrMT is amphiphilic and perturbs lipid bilayers, raising the question of whether its action against K+ channels is merely a manifestation of membrane perturbation. To determine whether medicinal chemistry approaches to improve BrMT might be viable, we synthesized BrMT and 11 analogs and determined their activities in parallel assays measuring K+ channel activity and lipid bilayer properties. Our work demonstrates a strategy for determining if drugs act by specific interactions or bilayer-dependent mechanisms, and chemically stable modulators of Kv1 channels are reported