Intrinsic Lipid Curvature and Bilayer Elasticity as Regulators of Channel Function: A Comparative Single-Molecule Study.

Perturbations in bilayer material properties (thickness, lipid intrinsic curvature and elastic moduli) modulate the free energy difference between different membrane protein conformations, thereby leading to changes in the conformational preferences of bilayer-spanning proteins. To further explore the relative importance of curvature and elasticity in determining the changes in bilayer properties that underlie the modulation of channel function, we investigated how the micelle-forming amphiphiles Triton X-100, reduced Triton X-100 and the HII lipid phase promoter capsaicin modulate the function of alamethicin and gramicidin channels. Whether the amphiphile-induced changes in intrinsic curvature were negative or positive, amphiphile addition increased gramicidin channel appearance rates and lifetimes and stabilized the higher conductance states in alamethicin channels. When the intrinsic curvature was modulated by altering phospholipid head group interactions, however, maneuvers that promote a negative-going curvature stabilized the higher conductance states in alamethicin channels but destabilized gramicidin channels. Using gramicidin channels of different lengths to probe for changes in bilayer elasticity, we found that amphiphile adsorption increases bilayer elasticity, whereas altering head group interactions does not. We draw the following conclusions: first, confirming previous studies, both alamethicin and gramicidin channels are modulated by changes in lipid bilayer material properties, the changes occurring in parallel yet differing dependent on the property that is being changed; second, isolated, negative-going changes in curvature stabilize the higher current levels in alamethicin channels and destabilize gramicidin channels; third, increases in bilayer elasticity stabilize the higher current levels in alamethicin channels and stabilize gramicidin channels; and fourth, the energetic consequences of changes in elasticity tend to dominate over changes in curvature.

Probucol is anti-hyperalgesic in a mouse peripheral nerve injury model of neuropathic pain.

2,6-di-tert-butylphenol (2,6-DTBP) ameliorates mechanical allodynia and thermal hyperalgesia produced by partial sciatic nerve ligation in mice, and selectively inhibits HCN1 channel gating. We hypothesized that the clinically utilized non-anesthetic dimerized congener of 2,6-DTBP, probucol (2,6-di-tert-butyl-4-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)sulfanylpropan-2-ylsulfanyl]phenol), would relieve the neuropathic phenotype that results from peripheral nerve damage, and that the anti-hyperalgesic efficacy in vivo would correlate with HCN1 channel inhibition in vitro. A single oral dose of probucol (800 mg/kg) relieved mechanical allodynia and thermal hyperalgesia in a mouse spared-nerve injury neuropathic pain model. While the low aqueous solubility of probucol precluded assessment of its possible interaction with HCN1 channels, our results, in conjunction with recent data demonstrating that probucol reduces lipopolysaccharide-induced mechanical allodynia and thermal hyperalgesia, support the testing/development of probucol as a non-opioid, oral antihyperalgesic albeit one of unknown mechanistic action.

Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity.

Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five- to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates.

Screening for bilayer-active and likely cytotoxic molecules reveals bilayer-mediated regulation of cell function.

A perennial problem encountered when using small molecules (drugs) to manipulate cell or protein function is to assess whether observed changes in function result from specific interactions with a desired target or from less specific off-target mechanisms. This is important in laboratory research as well as in drug development, where the goal is to identify molecules that are unlikely to be successful therapeutics early in the process, thereby avoiding costly mistakes. We pursued this challenge from the perspective that many bioactive molecules (drugs) are amphiphiles that alter lipid bilayer elastic properties, which may cause indiscriminate changes in membrane protein (and cell) function and, in turn, cytotoxicity. Such drug-induced changes in bilayer properties can be quantified as changes in the monomer↔dimer equilibrium for bilayer-spanning gramicidin channels. Using this approach, we tested whether molecules in the Pathogen Box (a library of 400 drugs and drug-like molecules with confirmed activity against tropical diseases released by Medicines for Malaria Venture to encourage the development of therapies for neglected tropical diseases) are bilayer modifiers. 32% of the molecules in the Pathogen Box were bilayer modifiers, defined as molecules that at 10 µM shifted the monomer↔dimer equilibrium toward the conducting dimers by at least 50%. Correlation analysis of the molecules' reported HepG2 cell cytotoxicity to bilayer-modifying potency, quantified as the shift in the gramicidin monomer↔dimer equilibrium, revealed that molecules producing <25% change in the equilibrium had significantly lower probability of being cytotoxic than molecules producing >50% change. Neither cytotoxicity nor bilayer-modifying potency (quantified as the shift in the gramicidin monomer↔dimer equilibrium) was well predicted by conventional physico-chemical descriptors (hydrophobicity, polar surface area, etc.). We conclude that drug-induced changes in lipid bilayer properties are robust predictors of the likelihood of membrane-mediated off-target effects, including cytotoxicity.

Capsaicin as an amphipathic modulator of NaV1.5 mechanosensitivity.

SCN5A-encoded NaV1.5 is a voltage-gated Na+ channel that drives the electrical excitability of cardiac myocytes and contributes to slow waves of the human gastrointestinal smooth muscle cells. NaV1.5 is mechanosensitive: mechanical force modulates several facets of NaV1.5's voltage-gated function, and some NaV1.5 channelopathies are associated with abnormal NaV1.5 mechanosensitivity (MS). A class of membrane-active drugs, known as amphiphiles, therapeutically target NaV1.5's voltage-gated function and produce off-target effects including alteration of MS. Amphiphiles may provide a novel option for therapeutic modulation of NaV1.5's mechanosensitive operation. To more selectively target NaV1.5 MS, we searched for a membrane-partitioning amphipathic agent that would inhibit MS with minimal closed-state inhibition of voltage-gated currents. Among the amphiphiles tested, we selected capsaicin for further study. We used two methods to assess the effects of capsaicin on NaV1.5 MS: (1) membrane suction in cell-attached macroscopic patches and (2) fluid shear stress on whole cells. We tested the effect of capsaicin on NaV1.5 MS by examining macro-patch and whole-cell Na+ current parameters with and without force. Capsaicin abolished the pressure- and shear-mediated peak current increase and acceleration; and the mechanosensitive shifts in the voltage-dependence of activation (shear) and inactivation (pressure and shear). Exploring the recovery from inactivation and use-dependent entry into inactivation, we found divergent stimulus-dependent effects that could potentiate or mitigate the effect of capsaicin, suggesting that mechanical stimuli may differentially modulate NaV1.5 MS. We conclude that selective modulation of NaV1.5 MS makes capsaicin a promising candidate for therapeutic interventions targeting MS.

Regulation of Gramicidin Channel Function Solely by Changes in Lipid Intrinsic Curvature.

Membrane protein function is regulated by the lipid bilayer composition. In many cases the changes in function correlate with changes in the lipid intrinsic curvature (c 0), and c 0 is considered a determinant of protein function. Yet, water-soluble amphiphiles that cause either negative or positive changes in curvature have similar effects on membrane protein function, showing that changes in lipid bilayer properties other than c 0 are important-and may be dominant. To further investigate the mechanisms underlying the bilayer regulation of protein function, we examined how maneuvers that alter phospholipid head groups effective "size"-and thereby c 0-alter gramicidin (gA) channel function. Using dioleoylphospholipids and planar bilayers, we varied the head groups' physical volume and the electrostatic repulsion among head groups (and thus their effective size). When 1,2-dioleyol-sn-glycero-3-phosphocholine (DOPC), was replaced by 1,2-dioleyol-sn-glycero-3-phosphoethanolamine (DOPE) with a smaller head group (causing a more negative c 0), the channel lifetime (τ) is decreased. When the pH of the solution bathing a 1,2-dioleyol-sn-glycero-3-phosphoserine (DOPS) bilayer is decreased from 7 to 3 (causing decreased head group repulsion and a more negative c 0), τ is decreased. When some DOPS head groups are replaced by zwitterionic head groups, τ is similarly decreased. These effects do not depend on the sign of the change in surface charge. In DOPE:DOPC (3:1) bilayers, pH changes from 5→9 to 5→0 (both increasing head group electrostatic repulsion, thereby causing a less negative c 0) both increase τ. Nor do the effects depend on the use of planar, hydrocarbon-containing bilayers, as similar changes were observed in hydrocarbon-free lipid vesicles. Altering the interactions among phospholipid head groups may alter also other bilayer properties such as thickness or elastic moduli. Such changes could be excluded using capacitance measurements and single channel measurements on gA channels of different lengths. We conclude that changes gA channel function caused by changes in head group effective size can be predicted from the expected changes in c 0.

Mechanisms underlying drug-mediated regulation of membrane protein function.

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. Drug regulation of membrane protein function thus may be mediated by both direct interactions with the protein and drug-induced alterations of bilayer properties, in which the latter will alter the energetics of protein conformational changes. To tease apart these mechanisms, we examine how the prototypical, proton-gated bacterial potassium channel KcsA is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). All tested drugs inhibited KcsA activity, and the changes in the different gating steps varied with bilayer thickness, suggesting a coupling to the bilayer. Examining the correlations between changes in KcsA gating steps and bilayer properties reveals that drug-induced regulation of membrane protein function indeed involves bilayer-mediated mechanisms. Both direct, either specific or nonspecific, binding and bilayer-mediated mechanisms therefore are likely to be important whenever there is overlap between the concentration ranges at which a drug alters membrane protein function and bilayer properties. Because changes in bilayer properties will impact many diverse membrane proteins, they may cause indiscriminate changes in protein function.

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity.

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD's localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.

Atomistic Characterization of Gramicidin Channel Formation.

We investigated gramicidin A (gA) subunit dimerization in lipid bilayers using microsecond-long replica-exchange umbrella sampling simulations, millisecond-long unbiased molecular dynamics simulations, and machine learning. Our simulations led to a dimer structure that is indistinguishable from the experimentally determined gA channel structures, with the two gA subunits joined by six hydrogen bonds (6HB). The simulations also uncovered two additional dimer structures, with different gA-gA stacking orientations that were stabilized by four or two hydrogen bonds (4HB or 2HB). When examining the temporal evolution of the dimerization, we found that two bilayer-inserted gA subunits can form the 6HB dimer directly, with no discernible intermediate states, as well as through paths that involve the 2HB and 4HB dimers.

Assessing the Perturbing Effects of Drugs on Lipid Bilayers Using Gramicidin Channel-Based In Silico and In Vitro Assays.

Partitioning of bioactive molecules, including drugs, into cell membranes may produce indiscriminate changes in membrane protein function. As a guide to safe drug development, it therefore becomes important to be able to predict the bilayer-perturbing potency of hydrophobic/amphiphilic drugs candidates. Toward this end, we exploited gramicidin channels as molecular force probes and developed in silico and in vitro assays to measure drugs' bilayer-modifying potency. We examined eight drug-like molecules that were found to enhance or suppress gramicidin channel function in a thick 1,2-dierucoyl-sn-glycero-3-phosphocholine (DC22:1PC) but not in thin 1,2-dioleoyl-sn-glycero-3-phosphocholine (DC18:1PC) lipid bilayer. The mechanism underlying this difference was attributable to the changes in gramicidin dimerization free energy by drug-induced perturbations of lipid bilayer physical properties and bilayer-gramicidin interactions. The combined in silico and in vitro approaches, which allow for predicting the perturbing effects of drug candidates on membrane protein function, have implications for preclinical drug safety assessment.

How perceptions of a successful physician-scientist varies with gender and academic rank: toward defining physician-scientist's success.

BACKGROUND: Physician-scientists (the physician-scientist workforce) are aging, and there are too few physician-scientists in the pipeline to replace those who retire. Moreover, the pipeline is leaky because some trainees and junior physician-scientists choose other career paths. Significant attention has been directed toward patching the leaking pipeline, thereby increasing the quantity of physician-scientists. Less attention has been devoted to identifying and training more successful physician-scientists, thereby increasing the quality of the pool and making up for the attrition. Though all training programs strive to develop more successful graduates, there is no clear understanding of what constitutes predictors of future success. Identifying characteristics of success would enable those who recruit trainees-and later hire and fund physician-scientists-to make more informed decisions. It also could impact on the training, as it would be possible to focus on competencies that foster success. Predictors of success are therefore important. Prior to taking on this task, however, we must first define success for physician-scientists. METHODS: To identify likely characteristics of success, we undertook a qualitative case study where 21 physician-scientists were interviewed to determine their perceptions of what constitutes a successful physician-scientist. Sixteen interviewees were selected based on convenience sampling, while the remaining five were selected based on the snowball effect. Interviews were transcribed and coded in Dedoose® and a qualitative analysis was conducted using an inductive approach to content analysis. RESULTS: There was considerable variation in their perceptions based on seniority and gender. Junior physician-scientists focused on metrics on which their promotion is based, e.g., publications and grants; senior physician-scientists focused on their legacy, e.g., contribution to the field and mentoring. Women were more likely to emphasize objective measures of success, like publications, while concurrently concentrating on relational skills, like networking, collaboration and public recognition. Men emphasized the impact of science and subjective characteristics like boldness, confidence and critical thinking. CONCLUSION: Clearly, physician-scientists are not working off of a uniform metric of success, thereby making their evaluation and remuneration a convoluted process, especially if those evaluating the physician-scientists are not of the same mind as to the definition of success.

Molecular Mechanism for Gramicidin Dimerization and Dissociation in Bilayers of Different Thickness.

Membrane protein functions can be altered by subtle changes in the host lipid bilayer physical properties. Gramicidin channels have emerged as a powerful system for elucidating the underlying mechanisms of membrane protein function regulation through changes in bilayer properties, which are reflected in the thermodynamic equilibrium distribution between nonconducting gramicidin monomers and conducting bilayer-spanning dimers. To improve our understanding of how subtle changes in bilayer thickness alter the gramicidin monomer and dimer distributions, we performed extensive atomistic molecular dynamics simulations and fluorescence-quenching experiments on gramicidin A (gA). The free-energy calculations predicted a nonlinear coupling between the bilayer thickness and channel formation. The energetic barrier inhibiting gA channel formation was sharply increased in the thickest bilayer (1,2-dierucoyl-sn-glycero-3-phosphocholine). This prediction was corroborated by experimental results on gramicidin channel activity in bilayers of different thickness. To further explore the mechanism of channel formation, we performed extensive unbiased molecular dynamics simulations, which allowed us to observe spontaneous gA dimer formation in lipid bilayers. The simulations revealed structural rearrangements in the gA subunits and changes in lipid packing, as well as water reorganization, that occur during the dimerization process. Together, the simulations and experiments provide new, to our knowledge, insights into the process and mechanism of gramicidin channel formation, as a prototypical example of the bilayer regulation of membrane protein function.

Quantitative Characterization of Protein-Lipid Interactions by Free Energy Simulation between Binary Bilayers.

Using a recently developed binary bilayer system (BBS) consisting of two patches of laterally contacting bilayers, umbrella sampling molecular dynamics (MD) simulations were performed for quantitative characterization of protein-lipid interactions. The BBS is composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with an embedded model membrane protein, a gramicidin A (gA) channel. The calculated free energy difference for the transfer of a gA channel from DLPC (hydrophobic thickness ≈ 21.5 Å) to DMPC (hydrophobic thickness ≈ 25.5 Å) bilayers, ΔG(DLPC → DMPC), is -2.2 ± 0.7 kcal/mol. This value appears at odds with the traditional view that the hydrophobic length of the gA channel is ∼22 Å. To understand this discrepancy, we first note that recent MD simulations by different groups have shown that lipid bilayer thickness profiles in the vicinity of a gA channel differ qualitatively from the deformation profile predicted from continuum elastic bilayer models. Our MD simulations at low and high gA:lipid molar ratios and different membrane compositions indicate that the gA channel's effective hydrophobic length is ∼26 Å. Using this effective hydrophobic length, ΔG(DLPC → DMPC) determined here is in excellent agreement with predictions based on continuum elastic models (-3.0 to -2.2 kcal/mol) where the bilayer deformation energy is approximated as a harmonic function of the mismatch between the channel's effective hydrophobic length and the hydrophobic thickness of the bilayer. The free energy profile for gA in the BBS includes a barrier at the interface between the two bilayers which can be attributed to the line tension at the interface between two bilayers with different hydrophobic thicknesses. This observation implies that translation of a peptide between two different regions of a cell membrane (such as between the liquid ordered and disordered phases) may include effects of a barrier at the interface in addition to the relative free energies of the species far from the interface. The BBS allows for direct transfer free energy calculations between bilayers without a need of a reference medium, such as bulk water, and thus provides an efficient simulation protocol for the quantitative characterization of protein-lipid interactions at all-atom resolution.

Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors (mGluRs) are class C, synaptic G-protein-coupled receptors (GPCRs) that contain large extracellular ligand binding domains (LBDs) and form constitutive dimers. Despite the existence of a detailed picture of inter-LBD conformational dynamics and structural snapshots of both isolated domains and full-length receptors, it remains unclear how mGluR activation proceeds at the level of the transmembrane domains (TMDs) and how TMD-targeting allosteric drugs exert their effects. Here, we use time-resolved functional and conformational assays to dissect the mechanisms by which allosteric drugs activate and modulate mGluR2. Single-molecule subunit counting and inter-TMD fluorescence resonance energy transfer measurements in living cells reveal LBD-independent conformational rearrangements between TMD dimers during receptor modulation. Using these assays along with functional readouts, we uncover heterogeneity in the magnitude, direction, and the timing of the action of both positive and negative allosteric drugs. Together our experiments lead to a three-state model of TMD activation, which provides a framework for understanding how inter-subunit rearrangements drive class C GPCR activation.

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

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.

Antidepressants are modifiers of lipid bilayer properties.

The two major classes of antidepressants, tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), inhibit neurotransmitter reuptake at synapses. They also have off-target effects on proteins other than neurotransmitter transporters, which may contribute to both desired changes in brain function and the development of side effects. Many proteins modulated by antidepressants are bilayer spanning and coupled to the bilayer through hydrophobic interactions such that the conformational changes underlying their function will perturb the surrounding lipid bilayer, with an energetic cost (ΔG def) that varies with changes in bilayer properties. Here, we test whether changes in ΔG def caused by amphiphilic antidepressants partitioning into the bilayer are sufficient to alter membrane protein function. Using gramicidin A (gA) channels to probe whether TCAs and SSRIs alter the bilayer contribution to the free energy difference for the gramicidin monomer⇔dimer equilibrium (representing a well-defined conformational transition), we find that antidepressants alter gA channel activity with varying potency and no stereospecificity but with different effects on bilayer elasticity and intrinsic curvature. Measuring the antidepressant partition coefficients using isothermal titration calorimetry (ITC) or cLogP shows that the bilayer-modifying potency is predicted quite well by the ITC-determined partition coefficients, and channel activity is doubled at an antidepressant/lipid mole ratio of 0.02-0.07. These results suggest a mechanism by which antidepressants could alter the function of diverse membrane proteins by partitioning into cell membranes and thereby altering the bilayer contribution to the energetics of membrane protein conformational changes.

Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase.

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca2+-activated scramblases, but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.

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 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.