I. Channel-Facilitated Solute Transport Studied at the Single-Channel Level: Molecular Coulter-Counting. Movement of solutes (mono- and di-nucleotides, sugars, antibiotics) through bacterial and mitochondrial porins induces current fluctuations that are readable in terms of the molecular dynamics of solute transport. Large channels seem to be designed by nature to serve a different purpose from that of the small channels of neurophysiology, i.e., to regulate transport of molecular species that are larger than halide or metal ions. These large channels are pathways for metabolites and macromolecules such as proteins and nucleic acids. At the same time, they can pass small ions. By measuring the current carried by the small ions one can access not only the structural changes in the channel molecule resulting from processes such as voltage or ligand gating, but also the transport of larger molecules through their transient occlusion of small-ion current. Studies of maltoporin-facilitated sugar transport have shown that single-channel experiments offer new possibilities to reveal molecular details of the interaction between the metabolite and the channel. The maltoporin channel (LamB) is asymmetric and, added from one side of the membrane only, predominantly inserts in an oriented manner. By analyzing time-resolved transient interruptions in the maltoporin ionic current in the presence of differently sized maltodextrins, it is found that only one sugar molecule is required to completely block one of the pores in the maltoporin trimer. The probability of simultaneous blockage of different pores increases with sugar concentration in a manner that demonstrates their mutual independence. The asymmetry of the channel structure manifests itself in two ways. First, it is seen as an asymmetric response to applied voltage under otherwise symmetric conditions; second, as asymmetric rates of sugar entry into the channel with asymmetric (one-sided) sugar addition. Importantly, it is demonstrated that the sugar residence time in the pore does not depend on which side the sugar was added. This voltage-dependent time is the same for either symmetric, cis, or trans sugar addition. This observation suggests that once a sugar molecule is captured by the greasy slide of the channel structure, it spends enough time there to """"""""forget"""""""" from what entrance it was captured. This also means that the blockage events represent sugar translocation events, and not just binding at and release from the same entrance of the channel. The molecular Coulter counter technique was also applied to investigate the mechanisms of antibiotic translocation through bacterial pores. It is well known that membrane permeability barriers are among the main reasons for the bacteria antibiotic resistance. Using ion channel reconstitution and high-resolution conductance recording, it is found that penicillins and cephalosporins are designed, either by targeting organisms or by pharmaceutical laboratories, to interact strongly with bacterial porin, OmpF. This demonstrates that, in analogy to substrate-specific channels that evolved to bind certain metabolite molecules, antibiotics have """"""""evolved"""""""" to be channel-specific. The charge distribution of an efficient antibiotic complements the charge distribution at the narrowest part of the bacterial porin and, due to the resulting attraction, facilitates its translocation. II. Theory of Channel-Facilitated Solute Transport. Accumulating evidence (see above) demonstrates that substrate-specific channels display pronounced binding to the corresponding substrates. The problem of solute translocation through membrane channels has been addressed using the Smoluchowski equation for diffusion in coordinate-dependent potential and with coordinate-dependent mobility. In a one-dimensional diffusion model with radiation boundary conditions obtained in a previous work, exact analytical expressions for the particle translocation probabilities were obtained. Brownian dynamics simulations verified the model and the quantitative predictions of this theory. Therefore, this theoretical study rationalized multiple observations of substrate-channel binding by showing that a potential well inside the channel is able to greatly increase the transit probability of the particle through the channel. III. Syringomycin E Channel: A Lipidic Pore Stabilized by Lipopeptide. Highly reproducible ion channels of the lipopeptide antibiotic syringomycin E (SRE) demonstrate unprecedented involvement of the host bilayer lipids. It has been found that in addition to a pronounced influence of lipid species on the open-channel ionic conductance, the membrane lipids play a crucial role in channel gating. The effective gating charge, which characterizes sensitivity of the conformational equilibrium of the SRE channels to the transmembrane voltage, is modified by the lipid charge and lipid dipole moment. The type of host lipid determines not only the absolute value but also the sign of the gating charge. With negatively charged bilayers, the gating charge sign inverts with increased salt concentration or decreased pH. The replacement of lamellar lipid by non-lamellar inhibits channel formation. All these observations suggest that the asymmetric channel directly incorporates lipids. The charges and dipoles resulting from the structural inclusion of lipids are important determinants of the overall energetics that underlie channel gating. These findings suggest that the SRE channel may serve as a biophysical model to link studies of ion channels with those of lipidic pores in membrane fusion.
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| Rostovtseva, Tatiana K; Nestorovich, Ekaterina M; Bezrukov, Sergey M (2002) Partitioning of differently sized poly(ethylene glycol)s into OmpF porin. Biophys J 82:160-9 |
| Kullman, Lisen; Winterhalter, Mathias; Bezrukov, Sergey M (2002) Transport of maltodextrins through maltoporin: a single-channel study. Biophys J 82:803-12 |
| Malev, Valery V; Schagina, Ludmila V; Gurnev, Philip A et al. (2002) Syringomycin E channel: a lipidic pore stabilized by lipopeptide? Biophys J 82:1985-94 |
| Aguilella, V M; Bezrukov, S M (2001) Alamethicin channel conductance modified by lipid charge. Eur Biophys J 30:233-41 |
| Ruszczynski, Peter S.; Kish, Laszlo B.; Bezrukov, Sergey M. (2001) Noise-assisted traffic of spikes through neuronal junctions. Chaos 11:581-586 |
| Rostovtseva, T K; Liu, T T; Colombini, M et al. (2000) Positive cooperativity without domains or subunits in a monomeric membrane channel. Proc Natl Acad Sci U S A 97:7819-22 |
| Bezrukov, S M (2000) Ion channels as molecular coulter counters to probe metabolite transport. J Membr Biol 174:13-Jan |
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