Large ion channels 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. The channel-forming proteins we explore 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). To study these channels under precisely controlled conditions, we purify and then reconstitute the channel-forming proteins in planar lipid bilayer membranes. ? ? I. Role of cytosolic proteins in regulation of mitochondria respiration. Positioned on the interface between mitochondria and the cytosol, the voltage-dependent anion channel, VDAC, is at the control point of mitochondria life and death. This large channel plays the role of a switch that defines in which direction mitochondria will go: to normal respiration or to suppression of mitochondria metabolism that leads to apoptosis and cell death. The most abundant protein in the mitochondrial outer membrane (MOM), VDAC is known to be responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM. This control has dual importance: in maintaining normal mitochondria respiration and in triggering apoptotic signal leading 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, but 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 such dissimilar cytosolic proteins as pro-apoptotic tBid and dimeric tubulin to show that the involved mechanisms are rather distinct. While tBid mostly modulates VDAC voltage gating, tubulin blocks the channel with the efficiency of blockage controlled by voltage. The high-affinity binding of tubulin to isolated mitochondria was shown long ago. Now we are 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 it to the action of this protein in vivo. We find that blockage of VDAC by tubulin is highly voltage-dependent and can be described by the first-order reaction. Interpolated to zero applied voltage the reaction is characterized by an equilibrium binding constant as high as inverse nanomoles. These findings were confirmed in the experiments with isolated mitochondria from mouse heart and brain. Based on this, we believe that we have identified a natural cytoplasmic regulator of mitochondria respiration in permeabilized cells the potent but evasive Factor-X. By this type of control, tubulin may selectively regulate metabolic fluxes between mitochondria and the cytoplasm. Thus our results not only reveal a novel mechanism of mitochondria respiration regulation, but also discover a new functional role for the cytoskeleton protein, dimeric tubulin. To conclude, we note that tubulin, as a newly discovered and potentially active player, adds another level of complexity to the VDAC regulation of mitochondrial signals, suggesting a possible competition between tubulin and hexokinase for VDAC binding. Interestingly, a well-known anti-tumor drug, paclitaxel, which inhibits the dynamics of microtubules and subsequently induces apoptosis, was found to induce cytochrome c release from mitochondria in intact human neuroblastoma cells and isolated mitochondria. It is likely, however, that paclitaxel and other microtubule-active anti-tumor drugs might modify interactions of microtubules and/or tubulin with VDAC and thus deliver a signal for mitochondria permeabilization and apoptosis induction.? ? II. Peptide-lipid interactions revealed by kinetics of a model channel. 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, the concept of hydrophobic mismatching was introduced. In essence, in this model, any mismatch in hydrophobic dimensions between the protein and the lipid incurs an energetic penalty causing lipid and protein deformations or structural adaptations. Many studies, including ours, support this insightful model. Now we use 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 compare the dissociation rate of single gramicidin channels in solvent-free planar bilayers made of dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), diether-DOPC (DEPC) lipids (or mixtures) and, in addition, we evaluate the effect of monovalent salt concentration. We find that while headgroup demethylation from DOPC to DOPE decreases the lifetime 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 lifetime by a factor of 2. To conclude, our findings demonstrate that even in this simple case the channel regulation involves both non-specific (hydrophobic mismatch) and specific (headgroup-peptide) interactions, thus highlighting the importance of the latter in functioning of membrane proteins.? ? III. Physics of channel-facilitated metabolite transport. 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 most important advance of this year was the development of an analytical approach that allows one to calculate the optimal intra-channel potential of mean force that maximizes the channel-facilitated flux driven by the solute concentration gradient. Surprisingly, we find that it is 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. This may suggest that in a given organism, depending on solute concentration, channel proteins designed to transport the same molecule might have different amino-acid sequences. One gene might code for a channel protein that functions at high solute concentrations, while another for the one that works at low concentrations.

Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2008
Total Cost
$858,248
Indirect Cost
City
State
Country
United States
Zip Code
Khattab, Ahmed; Yuen, Tony; Al-Malki, Sultan et al. (2016) A rare CYP21A2 mutation in a congenital adrenal hyperplasia kindred displaying genotype-phenotype nonconcordance. Ann N Y Acad Sci 1364:5-10
Meyer-Bahlburg, Heino F L; Dolezal, Curtis; Haggerty, Rita et al. (2012) Cognitive outcome of offspring from dexamethasone-treated pregnancies at risk for congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Eur J Endocrinol 167:103-10
Lin-Su, Karen; Harbison, Madeleine D; Lekarev, Oksana et al. (2011) Final adult height in children with congenital adrenal hyperplasia treated with growth hormone. J Clin Endocrinol Metab 96:1710-7
Berezhkovskii, Alexander M; Pustovoit, Mark A; Bezrukov, Sergey M (2010) Fluxes of non-interacting and strongly repelling particles through a single conical channel: Analytical results and their numerical tests. Chem Phys 375:523-528
Alcaraz, Antonio; Nestorovich, Ekaterina M; Lopez, M Lidon et al. (2009) Diffusion, exclusion, and specific binding in a large channel: a study of ompf selectivity inversion. Biophys J 96:56-66
Ostroumova, Olga S; Gurnev, Philip A; Schagina, Ludmila V et al. (2007) Asymmetry of syringomycin E channel studied by polymer partitioning. FEBS Lett 581:804-8
Karginov, Vladimir A; Nestorovich, Ekaterina M; Schmidtmann, Frank et al. (2007) Inhibition of S. aureus alpha-hemolysin and B. anthracis lethal toxin by beta-cyclodextrin derivatives. Bioorg Med Chem 15:5424-31
Bezrukov, Sergey M; Berezhkovskii, Alexander M; Szabo, Attila (2007) Diffusion model of solute dynamics in a membrane channel: mapping onto the two-site model and optimizing the flux. J Chem Phys 127:115101
Berezhkovskii, A M; Pustovoit, M A; Bezrukov, S M (2007) Diffusion in a tube of varying cross section: numerical study of reduction to effective one-dimensional description. J Chem Phys 126:134706
Pustovoit, M A; Berezhkovskii, A M; Bezrukov, S M (2006) Analytical theory of hysteresis in ion channels: two-state model. J Chem Phys 125:194907

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