Large ion channels are key structural elements of metabolite exchange between cellular compartments and between cells. To study the channels under precisely controlled conditions, we reconstitute channel-forming proteins into planar lipid bilayers. Our goal is to investigate the physical principles of channel-facilitated transport of metabolites and other large solutes across cell and organelle membranes. The proteins and peptides we work with include VDAC (voltage-dependent anionic channel from the outer membrane of mitochondria), OmpF (general bacterial porin), LamB (sugar-specific bacterial porin), alpha-hemolysin (toxin from Staphylococcus aureus), alamethicin (amphiphilic peptide toxin from Trichoderma viride), syringomycin E (lipopeptide toxin from Pseudomonas syringae), and anthrax protective antigen. These proteins and peptides form channels with aqueous pores that are 1 nm or larger in diameter. We approach these large channels by complementing traditional electrophysiological methods with an original concept of ion channels as molecular Coulter counters, which distinguishes us from the main body of contemporary ion channel researchers. Specifically, we focus on studying metabolite transport at the level of single molecules, elucidating the molecular mechanisms responsible for metabolite flux regulation under normal conditions and in pathology.
Involvement of VDAC in apoptosis
Rostovtseva, Bezrukov; in collaboration with Colombini, Tan
Research on VDAC, the major channel from mitochondria outer membrane (MOM), has accelerated as evidence grows of its importance in mitochondrial function and apoptosis. This small, ancient, highly conserved protein appears to be involved in many cellular processes. During the past year, we attempted to identify its role in apoptosis and to separate reliable information from more questionable claims. VDAC channels can exist in a variety of functional states that differ in their ability to pass non-electrolytes and to conduct ions. VDAC is permeable to small ions, including Ca2+. It is a well-established fact that cytosolic Ca2+ triggers opening of the permeability transition pore (PTP) in the mitochondria inner membrane, which consequently allows passage of water and solutes up to approximately 1.5 kDa. Opening of the PTP is one of the mechanisms responsible for MOM permeabilization, cytochrome C release, and, consequently, apoptotic cell death. VDAC is thought to be one of the major components of the PTP. If VDAC is part of the PTP, then it seems logical that closure of VDAC would close the PTP and protect mitochondria from cytosolic PTP activators, such as Ca2+ ions. In experiments with VDAC channel reconstituted unto the planar lipid membranes, we have shown that Ca2+ permeates through both the open and “closed” states of VDAC. The double-positive charge does not exclude Ca2+ cations from the open state because the anion selectivity is moderate. The closed state favors cations. The presence or absence of Ca2+ does not change the conductance of the open state of VDAC. Moreover, we have shown that Ca2+ presence does not affect voltage gating. Closure of VDAC does not prevent Ca2+ flux, and the closed state of VDAC has a similar molecular size cut-off as the PTP. We concluded that the closure of VDAC cannot protect against PTP opening. Thus, Ca2+ cannot regulate the PTP by opening or closing VDAC. The notion of a supramolecular PTP complex is fashionable but seems unnecessary given the large number of VDAC channels in the outer membrane and the higher permeability of VDAC than that measured for the PTP. Correspondingly, the influx of metabolites that leads to the swelling of the matrix must flow through VDAC whether or not the supramolecular complex exists. These data and the analysis of the results obtained by other groups confirm our previously suggested model wherein closure of VDAC, not VDAC opening, leads to MOM permeabilization and apoptosis.
Rostovtseva TK, Antonsson B, Suzuki M, Youle RJ, Colombini M, Bezrukov SM. Bid but not Bax regulates VDAC channels. J Biol Chem 2004;279:13575-13583.
Rostovtseva TK, Tan W, Colombini M. On the role of VDAC in apoptosis: fact and fiction. J Bioenerg Biomembr 2005;37:129-144.
Water-soluble polymers as molecular probes
Bezrukov; in collaboration with Berezhkovskii, Krasilnikov, Zitserman
The past year’s progress in quantitative understanding of polymer probing of ion channels resulted in the development of a theoretical model explaining the observed polymer-concentration dependence of the partition coefficient. We developed the model by recognizing that non-ideality of polymer solution in the pore is weaker than in the bulk because the overlap volume fraction of the polymer in the pore is higher than that in the bulk. The reason is that polymer molecules in the pore form cigars with high intramolecular monomer density. Therefore, the observed concentration dependence of the partition coefficient cannot be explained by using the standard approach, which assumes that non-ideality of the polymer solution in the pore is identical to non-ideality in the bathing solution; measured partitioning is a much sharper function of polymer concentration than predicted by identical non-ideality. In a separate study, we also analyzed the data on the electrical conductivity and viscosity of aqueous solutions of polyethylene glycol in order to use the data as reference information in studying channels. Over wide ranges of concentration and polymer molecular weight, conductivity is independent of the molecular weight for long chains and weakly dependent on the molecular weight for short chains. To explain the weak conductivity sensitivity of the length of the polymer chains, we qualitatively analyzed the processes responsible for a decrease in the mobility of ions. We demonstrated that experiments can be interpreted by using the microviscosity concept. Microviscosity increases with the addition of a polymer much less rapidly than usual (macroscopic) viscosity. We suggested a simple empirical formula describing the dependence of conductivity on the polymer concentration.
Bezrukov SM, Krasilnikov OV, Yuldasheva LN, Berezhkovskii AM, Rodrigues CG. Field-dependent effect of crown ether (18-crown-6) on ionic conductance of alpha-hemolysin channels. Biophys J 2004;87:3162-3171.
Krasilnikov OV, Bezrukov SM. Polymer partitioning from non-ideal solutions into protein voids. Macromolecules 2004;37:2650-2657.
Zitserman VY, Berezhkovskii AM, Parsegian VA, Bezrukov SM. Non-ideality of polymer solutions in the pore and concentration-dependent partitioning. J Chem Phys 2005;123:146101-146102.
Zitserman VY, Stojilkovic KS, Berezhkovskii AM, Bezrukov SM. Electrical conductivity of aqueous solutions of polyethylene glycol. Russ J Phys Chem 2005;79:1083-1089.
Theory of channel-facilitated metabolite transport
Bezrukov; in collaboration with Berezhkovskii
Membrane channels with large aqueous pores are traditionally regarded as “molecular sieves” that discriminate between molecules based on their size. This simplified view, however, contradicts emerging experimental evidence that permeation through these channels involves intimate molecular interactions. Metabolite-specific channels exhibit affinity for their metabolites; permeating molecules do not just slip through the pore but instead feel strong attraction to the pore-lining residues. We have rationalized these observations in a theoretical model. Using the language of the Smoluchowski equation for single-particle diffusion, we show that, counter-intuitively, a particle “sticking” to the channel is able to increase overall flux through the membrane. Our analysis is based on the diffusion model, which describes the solute motion in the channel as one-dimensional diffusion of a point particle along the channel axis, incorporating interaction with the channel into the potential of mean force and the position-dependent diffusion coefficient. The model is used to study how the interaction affects translocation of a single molecule entering the channel. Earlier, we had shown that the particle translocation probability reaches its upper limit when the potential well occupies the entire channel and its depth tends to infinity. However, the translocation probability is not the only factor that determines flux through the channel. The second important factor is the mean residence time of the molecule in the channel, given that a molecule sitting in the channel blocks the passage of other molecules. The deeper the well, the longer the molecule stays in the channel. We show an optimum well depth that makes channel functioning most efficient: a deep well holds molecules for too long while a shallow well does not substantially increase the translocation probability. We also analyzed a model in which the translocating solute jumps between neighboring binding sites and showed that the results predicted by the diffusion model can be recovered from the analysis of the binding-site model in a special limiting case. The binding-site model also describes flux through a narrow channel where molecules cannot jump one over the other, thus realizing the regime of single-file diffusion.
Berezhkovskii AM, Bezrukov SM. Channel-facilitated membrane transport: constructive role of particle attraction to the channel pore. Chem Phys 2005;319:342-349.
Berezhkovskii AM, Bezrukov SM. Optimizing transport of metabolites through large channels: molecular sieves with and without binding. Biophys J 2005;88:L17-L19.
Publication Related to Other Work
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 2005:102:15075-15080.
COLLABORATORS
Bruno Antonsson, PhD, Serono Pharmaceutical, Geneva, Switzerland
Alexander M. Berezhkovskii, PhD, Center for Information Technology, NIH, Bethesda, MD
Marco Colombini, PhD, University of Maryland, College Park, MD
Oleg V. Krasilnikov, PhD, Universidad Federal de Pernambuco, Recife, Brazil
Motoshi Suzuki, PhD, Surgical Neurobiology Branch, NINDS, Bethesda, MD
Wenzhi Tan, BS, University of Maryland, College Park, MD
Richard Youle, PhD, Surgical Neurobiology Branch, NINDS, Bethesda, MD
Vladimir Yu Zitserman, PhD, Institute for High Temperature, Moscow, Russia
For further information, contact bezrukos@mail.nih.gov.