BIOPHYSICS OF LARGE MEMBRANE CHANNELS
, Head, Section on Molecular Transport
Philip A. Gurnev, PhD, Postdoctoral Fellow
Ekaterina M. Nestorovich, PhD, Research Fellow
Tatiana K. Rostovtseva, PhD, Staff Scientist
Large ion channels of cell and organelle membranes are not only the gateways of metabolite exchange between different cellular compartments and cells; they are also recognized as multifunctional membrane receptors and components of many toxins. To study these channels under precisely controlled conditions, we purify and then reconstitute channel-forming proteins into planar lipid bilayers. The proteins we work with include anthrax protective antigen (from Bacillus anthracis), VDAC (voltage-dependent anionic channel from the outer membrane of mitochondria), OmpF (general bacterial porin from Escherichia coli), LamB (sugar-specific bacterial porin from Escherichia coli), alpha-hemolysin (toxin from Staphylococcus aureus), OprF (porin from Pseudomonas aeruginosa), alamethicin (amphiphilic peptide toxin from Trichoderma viride), and syringomycin E (lipopeptide toxin from Pseudomonas syringae). Channels formed by these proteins and peptides have aqueous pores of 1 to 2 nm diameter. We study these large channels by complementing traditional electrophysiological methods with an original concept of ion channels as molecular Coulter counters. Specifically, we focus on metabolite and other large solute transport at the level of single molecules. Using this strategy, we elucidate the physical principles and molecular mechanisms responsible for metabolite flux regulation under normal and pathological conditions.
High-affinity blocking of anthrax and other toxin channels
Many pathogens use transmembrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered important virulence factors, making them attractive targets for the discovery of new therapeutic agents. Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. Now, as a next step, we are evaluating a series of new beta-cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. We tested newly synthesized hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl derivatives of beta-cyclodextrin with alkyl spacers of various lengths for their ability both to inhibit cytotoxicity of LeTx in cells and to block ion conductance through PA channels reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax LeTx action at low or sub-micromolar concentrations. They also blocked ion conductance through PA channels at concentrations as low as 0.1 nM. We found that the activity of the derivatives in both cell protection and channel blocking depended on the length and chemical nature of the substituent groups. One of the compounds also blocked the edema toxin activity. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcus aureus alpha-hemolysin (HL), which is regarded as a major virulence factor with an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of HL and LeTx in cell-based assays at low micromolar concentrations. We tested one of the compounds for its ability to block ion conductance through the pores formed by HL and PA in artificial lipid membranes. We expect that this approach will provide the basis for a structure-directed drug discovery program to find new and effective therapies against various pathogens that use pore-forming proteins as virulence factors.
Karginov VA, Nestorovich EM, Moayeri M, Leppla SH, Bezrukov SM. Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design. Proc Natl Acad Sci USA 2005;102:15075-80.
Karginov VA, Nestorovich EM, Schmidtmann F, Robinson TM, Yohannes A, Fahmi NE, Bezrukov SM, Hecht SM. Inhibition of S. aureus alpha-Hemolysin and B. anthracis lethal toxin by beta-cyclodextrin derivatives. Bioorg Med Chem 2007;15:5424-31.
Karginov VA, Nestorovich EM, Yohannes A, Robinson TM, Fahmi NE, Schmidtmann F, Hecht SM, Bezrukov SM. Search for cyclodextrin-based inhibitors of anthrax toxins: synthesis, structural features, and relative activities. Antimicrob Agents Chemother 2006;50:3740-53.
Regulation of VDAC by non-lamellar lipids of mitochondrial membranes
Evidence is accumulating to suggest that lipids play important roles in permeabilization of the mitochondria outer membrane (MOM) at an early stage of apoptosis. Lamellar phosphatidylcholine (PC) and non-lamellar phosphatidylethanolamine (PE) lipids are the major membrane components of the MOM. Cardiolipin (CL), the characteristic lipid from the mitochondrial inner membrane, is another non-lamellar lipid recently shown to play a role in MOM permeabilization. We investigate the effect of these three key lipids on the gating properties of VDAC, the major channel in MOM. We found that PE induces voltage asymmetry in VDAC current-voltage characteristics by promoting channel closure at cis-negative applied potentials. CL also induces significant asymmetry. The observed differences in VDAC behavior in PC and PE membranes cannot be explained by differences in the insertion orientation of VDAC in these membranes. Rather, it is clear that the two nonlamellar lipids affect VDAC gating. Using gramicidin A channels as a tool to probe bilayer mechanics, we show that VDAC channels are much more sensitive to the presence of CL than would be expected from experiments with gramicidin channels. We suggest that this sensitivity is attributable to the preferential insertion of VDAC into CL-rich domains. We propose that the specific lipid composition of the mitochondria outer membrane and/or of contact sites might influence MOM permeability by regulating VDAC gating.
Rostovtseva TK, Kazemi N, Weinrich M, Bezrukov SM. Voltage gating of VDAC is regulated by non-lamellar lipids of mitochondrial membranes. J Biol Chem 2006;281:37496-506.
Physics of channel-facilitated metabolite transport
The past year’s progress in quantitative understanding of channel-facilitated transport led to three major findings. First, to probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide syringomycin E, we used penetrating polyethylene glycols’ partial blockage of ion current. Earlier experiments with symmetric application of these polymers yielded a radius estimate of about 1 nm. Motivated by the asymmetric non-ohmic current-voltage curves reported for this channel, we explored the channel’s structural asymmetry. We gauged the asymmetry by studying the channel conductance after the one-sided addition of differently sized polyethylene glycols. We found 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 the results to suggest that the water-filled pore of the channel is conical with cis- and trans-radii differing by a factor of 2 to 3 and that the smaller cis-radius is in the range of 0.25 to 0.35 nm. In symmetric two-sided addition, polymers entering the pore from the larger opening dominate blockage.
Second, we studied the distribution of direct translocation times for particles passing through membrane channels between two reservoirs. The direct translocation time is a conditional first-passage time defined as the residence time of the particle in the channel while passing through the membrane directly, that is, without returning to the reservoir from which it entered. We showed that the distributions of direct translocation times are identical for translocation in both directions, independent of any asymmetry in the potential across the channel and, hence, the translocation probabilities.
Third, channel-forming proteins in a lipid bilayer of a biological membrane usually respond to variation of external voltage by changing their conformations. Periodic voltages with frequency comparable to the inverse relaxation time of the protein produce hysteresis in the occupancies of the protein conformations. If the channel conductance changes when the protein jumps between these conformations, hysteresis in occupancies is observed as hysteresis in ion current through the channel. We developed an analytical theory of this phenomenon that is based on the assumption that the channel conformational dynamics can be described in terms of a two-state model. The theory describes transient behavior of the channel after the periodic voltage is switched on as well as the shape and area of the stationary hysteretic loop as functions of the frequency and amplitude of the applied voltage.
Berezhkovskii AM, Bezrukov SM. Site model for channel-facilitated membrane transport: invariance of translocation time distribution with respect to direction of passage. J Phys Condens Matter 2007;19:065148.
Berezhkovskii AM, Hummer G, Bezrukov SM. Identity of distributions of direct uphill and downhill translocation times for particles traversing membrane channels. Phys Rev Lett 2006;97:020601.
Berezhkovskii AM, Pustovoit MA, Bezrukov SM. Diffusion in a tube of varying cross section: numerical study of reduction to effective one-dimensional description. J Chem Phys 2007;126:134706.
Ostroumova OS, Gurnev PA, Schagina LV, Bezrukov SM. Asymmetry of syringomycin E channel studied by polymer partitioning. FEBS Lett 2007;581:804-8.
Pustovoit MA, Berezhkovskii AM, Bezrukov SM. Analytical theory of hysteresis in ion channels: two-state model. J Chem Phys 2006;125:194907.
COLLABORATORS
Alexander M. Berezhkovskii, PhD, Division of Computational Bioscience, CIT, NIH, Bethesda, MD
Gerhard Hummer, PhD, Theoretical Biophysics Section, NIDDK, Bethesda, MD
Vladimir A. Karginov, PhD, Innovative Biologics, Inc., Manassas, VA
Mark A. Pustovoit, PhD, St. Petersburg Nuclear Physics Institute, Gatchina, Russia
Ludmila V. Schagina, PhD, Institute of Cytology, St. Petersburg, Russia
Michael Weinrich, MD, National Center for Medical Rehabilitation Research, NICHD, Bethesda, MD
For further information, contact bezrukos@mail.nih.gov.
