I. Inhalational anesthetic halothane changes the domain structure of a binary lipid membrane The molecular mechanisms of volatile anesthetic action remain obscure despite a long history of research and clinical use for over a century and a half. The remarkable correlation between anesthetic solubility in oil and anesthetic potency, the so-called Meyer-Overton rule, strongly suggests a lipid membrane mediated mechanism. Paradoxically, structural studies of lipid bilayer membranes in the presence of anesthetics yielded negligible effects. In classic experiments 30 years ago employing X-ray and neutron diffraction from dimyristoylphosphatidylcholine (DMPC)/cholesterol membranes, Franks and Lieb found that for inhalation anesthetics ...at surgical concentrations, however, there are no significant changes in bilayer structure. Since then, the conceptual view of cell membranes has shifted from relatively homogeneous lipid bilayers with interspersed proteins to complex lipid mixtures, with laterally separated membrane domains formed as a result of lipid demixing. Accumulating evidence indicates that certain membrane proteins are clustered in domains such as cholesterol-rich lipid rafts. Guided by this conceptual shift, jointly with NIST scientists we performed X-ray and neutron diffraction experiments on a binary lipid membrane. Specifically, we studied a 1:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dilauroylphaphatidylcholine (DLPC) to demonstrate that halothane, but not dichlorohexafluorocyclobutane, a halogenated nonanesthetic of close properties, produces a pronounced redistribution of lipids between different domains at physiologically relevant concentrations. The domains of different lipid types were identified through their different lamellar d-spacings and isotope composition. These results have demonstrated a specific effect of inhalational anesthetics on mixing phase equilibria. Combined with a growing body of data revealing that conformational dynamics of transmembrane proteins are very sensitive to the parameters of the lipid bilayer within which they reside, our findings suggest that halothane and other volatile anesthetics may act through the cell membrane by changing its domain structure. We hope that these results are of ultimate help in the search for more potent and safe anesthetizing agents. II. VDAC inhibition by tubulin and its physiological implications Recently we have identified an important missing player in the regulation of the Voltage-Dependent Anion Channel (VDAC) of the outer mitochondrial membrane as the abundant cytoskeletal protein tubulin. We have now extended our study of tubulin-VDAC interaction in three directions. First, we studied the structural features of the blocked state as revealed by its sensitivity to the charge of the membrane surface. We found that the relative residual conductance of the tubulin-blocked state of VDAC is sensitive to the surface charge of the membrane thus confirming the tail-in-the-pore model of the blockage suggested by us earlier. Second, we demonstrated that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3beta or cAMP-dependent protein kinase A, increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude. Experiments on human hepatoma cells HepG2 supported our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevented microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this led to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Third, we have demonstrated 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. Thus, in addition to revealing an important step in tubulin-VDAC interaction, our results give a new 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. Immediate physiological implications of these findings include new insights into cell signaling pathways and cytoskeleton/microtubule activity in health and disease, especially in the case of the highly dynamic microtubule network which is characteristic of carcinogenesis and cell proliferation. These findings may help to identify new mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs, and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect). III. Physical theory of channel-facilitated transport Further development of the physical theory of channel-facilitated transport is required for deeper understanding of its regulation mechanisms. This year we have concentrated on two topics: the effects of entropy barriers and clustering. Transport in systems of varying geometry that creates entropy barriers in one-dimensional description has become the subject of growing interest among researchers in recent years. We investigated transport of point Brownian particles in a tube formed by identical periodic compartments of varying diameter, focusing on the effects that are due to the compartment asymmetry. In particular, we studied the force-dependent mobility of the particle and found that the mobility is a symmetric non-monotonic function of the driving force when the compartment is symmetric. However, the compartment asymmetry gives rise to an asymmetric force-dependent mobility, which remains non-monotonic when the compartment asymmetry is not too high and becomes monotonic in tubes formed by highly asymmetric compartments. The transition of the dependence from non-monotonic to monotonic behavior results in important consequences for the particle motion under the action of a time-periodic force with zero mean: In a tube formed by moderately asymmetric compartments, the particle under the action of such a force moves with an effective drift velocity that vanishes at small and large values of the force amplitude having a maximum in between. In a tube formed by highly asymmetric compartments, the effective drift velocity monotonically increases with the amplitude of the driving force and becomes unboundedly large as the amplitude tends to infinity. Clustering of receptors, transporters, and ion channels, which seems to be the rule rather than the exception, complicates the qualitative description of transport in biological systems. We have analyzed the effects of clustering by considering an aggregate of absorbing disks on the otherwise reflecting wall. Trapping of diffusing particles by such an aggregate is a manifestly many-body problem because of the disk competition for the particles. By replacing the cluster with an effective uniformly absorbing spot, we derived a simple algebraic expression for the rate constant that characterizes the trapping. The formula shows how the rate constant depends on the size, shape, and the density of packing in the cluster. The obtained analytical results may provide a rationale for surface clustering on the plasma membranes in addition to the usual explanation of enhancing two-dimensional macromolecular interaction.

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Bezrukov, Sergey M; Nestorovich, Ekaterina M (2016) Inhibiting bacterial toxins by channel blockage. Pathog Dis 74:
Aksoyoglu, M Alphan; Podgornik, Rudolf; Bezrukov, Sergey M et al. (2016) Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin. Proc Natl Acad Sci U S A 113:9003-8
Noskov, Sergei Yu; Rostovtseva, Tatiana K; Chamberlin, Adam C et al. (2016) Current state of theoretical and experimental studies of the voltage-dependent anion channel (VDAC). Biochim Biophys Acta 1858:1778-90
Kasianowicz, John J; Bezrukov, Sergey M (2016) On 'three decades of nanopore sequencing'. Nat Biotechnol 34:481-2
Sheldon, Kely L; Gurnev, Philip A; Bezrukov, Sergey M et al. (2015) Tubulin Tail Sequences and Posttranslational Modifications Regulate Closure of Mitochondrial Voltage-Dependent Anion Channel (VDAC). J Biol Chem :
Berezhkovskii, Alexander M; Dagdug, Leonardo; Bezrukov, Sergey M (2015) Biased diffusion in three-dimensional comb-like structures. J Chem Phys 142:134101
Rostovtseva, Tatiana K; Gurnev, Philip A; Protchenko, Olga et al. (2015) α-Synuclein Shows High Affinity Interaction with Voltage-dependent Anion Channel, Suggesting Mechanisms of Mitochondrial Regulation and Toxicity in Parkinson Disease. J Biol Chem 290:18467-77
Nestorovich, Ekaterina M; Bezrukov, Sergey M (2014) Designing inhibitors of anthrax toxin. Expert Opin Drug Discov 9:299-318
Teijido, Oscar; Rappaport, Shay M; Chamberlin, Adam et al. (2014) Acidification Asymmetrically Affects Voltage-dependent Anion Channel Implicating the Involvement of Salt Bridges. J Biol Chem 289:23670-82
Gurnev, Philip A; Yap, Thai Leong; Pfefferkorn, Candace M et al. (2014) Alpha-synuclein lipid-dependent membrane binding and translocation through the *-hemolysin channel. Biophys J 106:556-65

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