Philip A. Gurnev

Syringomycin E Channel: A Lipidic Pore Stabilized by Lipopeptide?

Highly reproducible ion channels of the lipopeptide antibiotic syringomycin E demonstrate unprecedented involvement of the host bilayer lipids. We find that in addition to a pronounced influence of lipid species on the open-channel ionic conductance, the membrane lipids play a crucial role in channel gating. The effective gating charge, which characterizes sensitivity of the conformational equilibrium of the syringomycin E channels to the transmembrane voltage, is modified by the lipid charge and lipid dipolar moment. We show that the type of host lipid determines not only the absolute value but also the sign of the gating charge. With negatively charged bilayers, the gating charge sign inverts with increased salt concentration or decreased pH. We also demonstrate that the replacement of lamellar lipid by nonlamellar with the negative spontaneous curvature inhibits channel formation. These observations suggest that the asymmetric channel directly incorporates lipids. The charges and dipoles resulting from the structural inclusion of lipids are important determinants of the overall energetics that underlies channel gating. We conclude that the syringomycin E channel may serve as a biophysical model to link studies of ion channels with those of lipidic pores in membrane fusion.

α-Synuclein Shows High Affinity Interaction with Voltage-dependent Anion Channel, Suggesting Mechanisms of Mitochondrial Regulation and Toxicity in Parkinson Disease

Background: The intrinsically disordered protein α-synuclein, a hallmark of Parkinson disease, is involved in mitochondrial dysfunction in neurodegeneration and directly interacts with mitochondria. Results: α-Synuclein regulates VDAC permeability; α-synuclein toxicity in yeast depends on VDAC. Conclusion: α-Synuclein both blocks VDAC and translocates via this channel across the mitochondrial outer membrane. Significance: (Patho)physiological roles of monomeric α-synuclein may originate from its interaction with VDAC. Participation of the small, intrinsically disordered protein α-synuclein (α-syn) in Parkinson disease (PD) pathogenesis has been well documented. Although recent research demonstrates the involvement of α-syn in mitochondrial dysfunction in neurodegeneration and suggests direct interaction of α-syn with mitochondria, the molecular mechanism(s) of α-syn toxicity and its effect on neuronal mitochondria remain vague. Here we report that at nanomolar concentrations, α-syn reversibly blocks the voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane that controls most of the metabolite fluxes in and out of the mitochondria. Detailed analysis of the blockage kinetics of VDAC reconstituted into planar lipid membranes suggests that α-syn is able to translocate through the channel and thus target complexes of the mitochondrial respiratory chain in the inner mitochondrial membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. The functional interactions between VDAC and α-syn, revealed by the present study, point toward the long sought after physiological and pathophysiological roles for monomeric α-syn in PD and in other α-synucleinopathies.

Persister-promoting bacterial toxin TisB produces anion-selective pores in planar lipid bilayers

We studied membrane activity of the bacterial peptide TisB involved in persister cell formation. TisB and its analogs form multi‐state ion‐conductive pores in planar lipid bilayers with all states displaying similar anionic selectivity. TisB analogs differing by ±1 elementary charges show corresponding changes in selectivity. Probing TisB pores with poly‐(ethylene glycol)s reveals only restricted partitioning even for the smallest polymers, suggesting that the pores are characterized by a relatively small diameter. These findings allow us to suggest that TisB forms clusters of narrow pores that are essential for its mechanism of action.

Membrane Lipid Composition Regulates Tubulin Interaction with Mitochondrial Voltage-dependent Anion Channel

Background: Dimeric αβ-tubulin regulates mitochondrial respiration by blocking VDAC. Results: The on-rate of tubulin binding to VDAC varies more than 100-fold depending on the lipid type. Conclusion: VDAC blockage by tubulin involves a crucial step of tubulin interaction with the membrane. Significance: VDAC-tubulin binding is a new example of lipid-controlled protein-protein interactions that contribute to mitochondrial metabolism regulation. Elucidating molecular mechanisms by which lipids regulate protein function within biological membranes is critical for understanding the many cellular processes. Recently, we have found that dimeric αβ-tubulin, a subunit of microtubules, regulates mitochondrial respiration by blocking the voltage-dependent anion channel (VDAC) of mitochondrial outer membrane. Here, we show that the mechanism of VDAC blockage by tubulin involves tubulin interaction with the membrane as a critical step. The on-rate of the blockage varies up to 100-fold depending on the particular lipid composition used for bilayer formation in reconstitution experiments and increases with the increasing content of dioleoylphosphatidylethanolamine (DOPE) in dioleoylphosphatidylcholine (DOPC) bilayers. At physiologically low salt concentrations, the on-rate is decreased by the charged lipid. The off-rate of VDAC blockage by tubulin does not depend on the lipid composition. Using confocal fluorescence microscopy, we compared tubulin binding to the membranes of giant unilamellar vesicles (GUVs) made from DOPC and DOPC/DOPE mixtures. We found that detectable binding of the fluorescently labeled dimeric tubulin to GUV membranes requires the presence of DOPE. We propose that prior to the characteristic blockage of VDAC, tubulin first binds to the membrane in a lipid-dependent manner. We thus reveal a new potent regulatory role of the mitochondrial lipids in control of the mitochondrial outer membrane permeability and hence mitochondrial respiration through tuning VDAC sensitivity to blockage by tubulin. More generally, our findings give an example of the lipid-controlled protein-protein interaction where the choice of lipid species is able to change the equilibrium binding constant by orders of magnitude.

Tubulin-blocked state of VDAC studied by polymer and ATP partitioning

Recently reported functional interaction between voltage‐dependent anion channel of the outer mitochondrial membrane, VDAC, and dimeric tubulin is observed as a reversible channel blockage. Using partitioning of poly‐(ethylene glycol)s of different molecular weights and reversal potential measurements, we probe the size and ion selectivity of the fully open and tubulin‐blocked states of VDAC reconstituted into planar lipid bilayers. While the effective radius of the channel decreases by only a factor of 1.34 ± 0.15, the selectivity reverses from initially anionic to cationic. Directly measuring ATP partitioning we demonstrate that these changes prohibit ATP from entering the channel in its tubulin‐blocked state.

Membrane-permeabilizing activities of cyclic lipodepsipeptides, syringopeptin 22A and syringomycin E from Pseudomonas syringae pv. syringae in human red blood cells and in bilayer lipid membranes.

The pore-forming activities of cyclic lipodepsipeptides (CLPs), syringopeptin 22A (SP22A) and syringomycin E (SRE) were compared on the human red blood cell (RBC) membrane and on bilayer lipid membranes (BLMs). SP22A above a concentration of 4 x 10(5) molecules/cell significantly increased the RBC membrane permeability for 86Rb. With electric current measurements on BLM, it was proved that like SRE, the SP22A formed two types of ion channels in the membrane, small and large, the latter having six times larger conductance and longer dwell time. Both CLPs formed clusters consisting of six small channels, and the channel-forming activity of SP22A is about one order of magnitude higher than that of SRE. A Hill coefficient of 2-3 estimated from the concentration dependence of these CLPs-induced lysis gave a proof of the pore oligomerization on RBCs. Transport kinetic data also confirmed that SP22A pores were oligomers of at least three monomers. While SRE pores were inactivated in time, no pore inactivation was observed with SP22A. The 86Rb efflux through SP22A-treated RBCs approached the tracer equilibrium distribution with a constant rate; a constant integral current was measured on the BLM for as long as 2.5 h as well. The partition coefficient (Kp = 2 x 10(4) l/mol) between the RBC membrane and the extracellular space was estimated for SRE to be at least six times higher than that for SP22A. This finding suggested that the higher ion permeability of the SP22A-treated cells compared to that of SRE was the result of the higher pore-forming activity of SP22A.

The Dynamic Side of the Hofmeister Effect: A Single‐Molecule Nanopore Study of Specific Complex Formation

Beyond measurements of equilibria: An alpha-hemolysin nanopore is used as a single-molecule sensor to follow the effects of different salts on the complexation reaction of gamma-cyclodextrin and adamantane carboxylate (see picture). The kinetics underlying the dynamic equilibrium are studied to reveal qualitatively different dynamic actions of various cosolute salts.

Asymmetry of syringomycin E channel studied by polymer partitioning.

To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide syringomycin E, we use the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of ∼1 nm. Now, motivated by the asymmetric non‐ohmic current–voltage curves reported for this channel, we explore its structural asymmetry. We gauge this asymmetry by studying the channel conductance after one‐sided addition of differently sized poly(ethylene glycol)s. We find that small polymers added to the cis‐side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans‐side. We interpret our results to suggest that the water‐filled pore of the channel is conical with cis‐ and trans‐radii differing by a factor of 2–3 and that the smaller cis‐radius is in the 0.25–0.35 nm range. In symmetric, two‐sided addition, polymers entering the pore from the larger opening dominate blockage.

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications.

Alpha-Synuclein Lipid-Dependent Membrane Binding and Translocation through the α-Hemolysin Channel

Gauging the interactions of a natively unfolded Parkinson disease-related protein, alpha-synuclein (α-syn) with membranes and its pathways between and within cells is important for understanding its pathogenesis. Here, to address these questions, we use a robust β-barrel channel, α-hemolysin, reconstituted into planar lipid bilayers. Transient, ~95% blockage of the channel current by α-syn was observed when 1), α-syn was added from the membrane side where the shorter (stem) part of the channel is exposed; and 2), the applied potential was lower on the side of α-syn addition. While the on-rate of α-syn binding to the channel strongly increased with the applied field, the off-rate displayed a turnover behavior. Statistical analysis suggests that at voltages >50 mV, a significant fraction of the α-syn molecules bound to the channel undergoes subsequent translocation. The observed on-rate varied by >100 times depending on the bilayer lipid composition. Removal of the last 25 amino acids from the highly negatively charged C-terminal of α-syn resulted in a significant decrease in the binding rates. Taken together, these results demonstrate that β-barrel channels may serve as sensitive probes of α-syn interactions with membranes as well as model systems for studies of channel-assisted protein transport.