Biophysics of Large Membrane Channels

Sergey M. Bezrukov, PhD, Head, Section on Molecular Transport
Tatiana K. Rostovtseva, PhD, Staff Scientist
Ekaterina M. Nestorovich, PhD, Research Fellow
Philip A. Gurnev, PhD, Postdoctoral Fellow
Kely L. Sheldon, MS, Postbaccalaureate Fellow

To address fundamental questions of membrane transport, we apply physical theory to experiments on membrane channels reconstituted in planar lipid bilayers, focusing on channel-facilitated transport of metabolites and other large solutes across cell and organelle membranes. The corresponding large ion channels are not only the gateways of metabolite exchange between different cellular compartments and cells but are also recognized as multifunctional membrane receptors and components of many toxins. We explore channel-forming proteins such as 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). To study these channels under precisely controlled conditions, we purify and then reconstitute the channel-forming proteins in planar lipid bilayer membranes. Our main goal is to elucidate the physical principles and molecular mechanisms responsible for metabolite flux regulation under normal and pathological conditions. Our research will provide the basis for the development of new approaches to treat various diseases for which regulation of transport through ion channels plays the key role.

Role of cytosolic proteins in the regulation of mitochondrial respiration

Rostovtseva, Sheldon, Bezrukov; in collaboration with Sackett, Saks

Positioned at the interface between mitochondria and the cytosol, the VDAC is at the control point of mitochondrial life and death. This large channel functions as a “switch” that defines whether mitochondria will engage in suppression of mitochondrial metabolism, which leads to apoptosis and cell death, or in normal respiration. VDAC, the most abundant protein in the mitochondrial outer membrane (MOM), is responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM. VDAC controls exchange and fluxes by switching between “closed” states, which are virtually impermeable to ATP and ADP, and open states. This control has dual importance in maintaining normal mitochondrial respiration and in triggering apoptotic signals that lead to release of cytochrome c and other apoptogenic factors from the intermembrane space into the cytosol. Emerging evidence indicates that VDAC closure promotes apoptotic signals, although VDAC itself is not directly involved in the permeability transition pore or hypothetical Bax-containing, cytochrome c–permeable pores. We analyzed closure of VDAC induced by dissimilar cytosolic proteins such as pro-apoptotic tBid and dimeric tubulin to show that the involved mechanisms are distinct. While tBid largely modulates VDAC voltage gating, tubulin blocks the channel with the efficiency of voltage-controlled blockage. With high-affinity binding of tubulin to isolated mitochondria reported long ago, we are now able to demonstrate the mechanism of VDAC regulation by tubulin in vitro—by reconstituting the channel in planar lipid membranes in the presence of dimeric tubulin—and to relate the regulation to the action of this protein in vivo. We found that blockage of VDAC by tubulin is highly voltage-dependent and may be described by a first-order reaction. Interpolated to zero applied voltage, the reaction is characterized by an equilibrium binding constant as high as inverse nanomoles. We confirmed our findings in experiments with isolated mitochondria from mouse heart and brain. Accordingly, we believe that we have identified a natural cytoplasmic regulator of mitochondrial respiration in permeabilized cells—the potent but evasive “Factor-X.” With such control, tubulin may selectively regulate metabolic fluxes between mitochondria and the cytoplasm. Thus, our results not only reveal a novel mechanism of mitochondrial respiration regulation but also uncover a new functional role for the cytoskeleton protein dimeric tubulin.

We also investigated the long-standing question of how VDAC voltage gating observed in planar lipid bilayers at relatively high potentials (greater than 30 mV) could be relevant to the situation in vivo and in isolated mitochondria wherein the presence of the VDAC should make the potential difference across MOM very low. This potential is most likely just the Donan potential stemming from the high concentration of charged polyions (such as cytochrome c) in the intermembrane space, which was estimated at close to 10 mV. As now follows from our findings, cytosolic concentrations of tubulin could markedly increase VDAC sensitivity to voltage and induce significant VDAC closure at transmembrane potentials even smaller than 10 mV. To conclude, we note that tubulin, as a newly discovered and potentially active player, adds another level of complexity to the VDAC’s regulation of mitochondrial signals, suggesting possible competition between tubulin and hexokinase for VDAC binding. Interestingly, others found that paclitaxel, a well-known antitumor drug that inhibits the dynamics of microtubules and subsequently induces apoptosis, induced cytochrome c release from mitochondria in intact human neuroblastoma cells and isolated mitochondria. It is likely, however, that paclitaxel and other microtubule-active antitumor drugs might modify interactions of microtubules and/or tubulin with VDAC and thus deliver a signal for mitochondria permeabilization and apoptosis induction.

Monge C, Beraud N, Kuznetsov AV, Rostovtseva T, Sackett D, Schlattner U, Vendelin M, Saks VA. Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase. Mol Cell Biochem 2008;318:147-165.
Rostovtseva TK, Bezrukov SM. VDAC regulation: role of cytosolic proteins and mitochondrial lipids. J Bioenerg Biomembr 2008;40:163-170.

Peptide-lipid interactions revealed by kinetics of a model channel

Rostovtseva, Bezrukov; in collaboration with Petrache, Weinrich

Critical to biological processes such as secretion and transport, protein-lipid interactions within the membrane and at the membrane-water interface still raise many questions. Residing within the inner oily part of the membrane, transmembrane proteins are significantly affected by non-specific, hydrophobic interactions. To quantify tractable energetic contributions of these interactions within an ingenuous physical model, others introduced the concept of hydrophobic mismatching. In essence, in this model, any mismatch in hydrophobic dimensions between the protein and the lipid incurs an energetic penalty that causes lipid and protein deformations or structural adaptations. Many studies, including ours, support this model. We used the benchmark antibiotic gramicidin A to show that the polar part (the “other part”) of the lipid bilayer claims its own role in lipid-channel interactions. We compared the dissociation rate of single gramicidin channels in “solvent-free” planar bilayers made of dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE ), and diether-DOPC (DEPC ) lipids (or mixtures); we also evaluated the effect of monovalent salt concentration. While headgroup demethylation from DOPC to DOPE decreases the life time of gramicidin channels by an order of magnitude in accordance with the currently accepted hydrophobic mismatch mechanism, our results for diether-DOPC suggest the importance of the headgroup-peptide interactions. According to our X-ray diffraction measurements, this lipid has the same hydrophobic thickness as DOPC but increases gramicidin channel life time by a factor of 2. Thus, using gramicidin channels as a probe, we showed that peptide-headgroup interactions may dominate over the effect of hydrophobic mismatch in regulating protein function. To conclude, our findings demonstrate that, even in this simple case, the channel regulation involves both nonspecific (hydrophobic mismatch) and specific (headgroup-peptide) interactions, thus highlighting the importance of the latter in the function of membrane proteins.

Rostovtseva TK, Petrache HI, Kazemi N, Hassanzadeh E, Bezrukov SM. Interfacial polar interactions affect gramicidin channel kinetics. Biophys J 2008;94:L23-5.

Physics of channel-facilitated transport

Bezrukov; in collaboration with Berezhkovskii, Pustovoit, Szabo, Zitserman

Our effort in physical theory of channel-facilitated membrane transport concentrated on further development of the continuum diffusion model of solute dynamics in a membrane channel. The first important advance of the past year was the development of an analytical approach that permits calculation of the optimal intrachannel potential of mean force that maximizes the channel-facilitated flux driven by the solute concentration gradient. Paradoxically, it would be preferable for a solute to “bind” more strongly near the exit rather than near the entrance of the channel. Another interesting observation is that the optimum value of the interaction potential depends on the concentrations of the solute outside the channel, suggesting that, in a given organism and depending on solute concentration, channel proteins designed to transport the same molecule might have different amino acid sequences. One gene might encode a channel protein that functions at high solute concentrations while another might encode a channel protein that works at low concentrations.

Second, we were able to formulate a fluctuation theorem for membrane transport. By counting translocations of single solutes through an arbitrarily biased channel, we established the statistical relation between particle transport and particle dynamics in the channel and reservoirs. We showed that the same fluctuation theorem is true for both single- and multichannel transport of non-interacting particles and particles that strongly repel each other. The theory provides the fine statistics of solute translocation through the channel and, in the limit of increasing time, leads to the expression for the steady-state flux derived earlier. The results of our work are helpful in interpretation of singlemolecule translocation experiments.

Third, we analyzed relaxation of the particle number in a membrane channel at arbitrary particlechannel interactions and derived general expressions for the relaxation time and low-frequency limit of the power spectral density. The expressions simplify significantly when the channel is symmetric. For a square-well potential of mean force that occupies the entire channel, we verified the accuracy of the analytical predictions by Brownian dynamics simulations. Interestingly, we showed that, for such a channel, as the depth of the well increases, the familiar scaling of the relaxation time with the channel length squared is transformed into a linear dependence on the length.

Berezhkovskii AM, Bezrukov SM. Counting translocations of strongly repelling particles through single channels: fluctuation theorem for membrane transport. Phys Rev Lett 2008;100:038104.
Berezhkovskii AM, Bezrukov SM. Fluctuation theorem for channel-facilitated membrane transport of interacting and non-interacting solutes. J Phys Chem B 2008;112:6228-6232.
Bezrukov SM, Berezhkovskii AM, Szabo A. Diffusion model of solute dynamics in a membrane channel: mapping onto the two-site model and optimizing the flux. J Chem Phys 2007;127:115101.
Zitserman VY, Berezhkovskii AM, Pustovoit MA, Bezrukov SM. Relaxation and fluctuations of the number of particles in a membrane channel at arbitrary particle-channel interaction. J Chem Phys 2008;129:095101.

Collaborators

Alexander M. Berezhkovskii, PhD, Division of Computational Bioscience, CIT, NIH, Bethesda, MD
Horia I. Petrache, PhD, Department of Physics, Indiana University, Indianapolis, IN
Mark A. Pustovoit, PhD, St. Petersburg Nuclear Physics Institute, Gatchina, Russia
Dan Sackett, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Valdur Saks, PhD, Université Joseph Fourier, Grenoble, France
Attila Szabo, PhD, Laboratory of Chemical Physics, NIDDK, Bethesda, MD
Michael Weinrich, MD, National Center for Medical Rehabilitation Research, NICHD, Bethesda, MD
Vladimir Zitserman, PhD, Joint Institute for High Temperatures, Moscow, Russia

For further information, contact bezrukovs@mail.nih.gov or visit http://smt.nichd.nih.gov.