The Unit on Molecular Transport examines the channel-facilitated transport of metabolites and other large solutes across cell and organelle membranes. Channel structural equilibrium is extremely sensitive to its immediate environment, including, for example, surrounding lipids and local acidity. We reconstitute channels of different origin and biological function into planar lipid bilayers in order to study functional states under precisely controlled conditions. Our channels include VDAC (Voltage-Dependent Anionic Channel from the outer membrane of mitochondria), alpha-Hemolysin (formed in the target cell membranes by a toxin from Staphylococcus aureus), OmpF (general bacterial porin), LamB (sugar-specific bacterial porin), and Alamethicin (oligomeric peptide channel). All these channels have large-diameter aqueous pores (1 nm or more). By analyzing the statistical properties of channel-mediated ion currents, we address structure and function using modern physico-chemical approaches. Our laboratory initiated several of the following approaches: probing channel aqueous pore geometry and channel hydration related to its "gating" by using water-soluble polymers; examining channel structure by analyzing current noise from reversible ionization of channel amino acid residues; and studying channel transport properties using the "molecular Coulter counter" concept.

Ion Channels as Molecular Coulter Counters that Probe Metabolite Transport
Kullman, Nestorovich, Rostovtseva, Komarov, Winterhalter, Bezrukov
Despite the obvious clue that the main function of large channels is to pass large metabolites, peptides, and other high molecular weight organic solutes through biological membranes, most studies concentrate on small ion conduction through large channels. To address the actual functional properties of these channels, that is, their ability to transfer solutes other than water and small ions, we have introduced "molecular Coulter counting." Its underlying idea is similar to the resistive pulse principle used since 1953 in Coulter counters. To count and size particles suspended in electrolyte solution, Coulter counters use the transient changes in resistance of a small electrolyte-filled capillary caused by the passage of micron-sized particles driven by solution flow. In our molecular version, we use the fluctuations in ion currents through single protein channels. The basic difference is that Coulter counters employ a macroscopic capillary as a sensing device that can detect particles of several tenths of a micron or larger while, with a protein channel, we can detect the passage of neutral molecules as small as 10 to 15 ?ngstroms.

Antibiotic Translocation through Bacterial Walls Studied at the Single-Molecule Level
Nestorovich, Winterhalter, Bezrukov
We have watched the transport of the betalactam antibiotic ampicillin at the single-molecule level. Using the three-barreled bacterial porin, Ompf, reconstituted into planar lipid bilayer membranes, we found that addition of ampicillin to the membrane-bathing solution introduces transient interruptions in small-ion current through the channel. Time-resolved measurements show that one ampicillin molecule reduces channel conductance by one third, completely blocking one of the monomers in the Ompf trimer for characteristic times in the range of hundreds of microseconds. Asymmetric addition of ampicillin reveals that, during translocation, the molecules interact with the same binding site independently of the side of antibiotic addition. The frequency of current transients shows a pronounced nonmonotonic dependence on the bulk solution pH. Taken together with the data on ampicillin ionic equilibria in solution and the results of computer modeling, the result suggests that the zwitterionic form of ampicillin interacts favorably with the constriction zone of the Ompf pore. The transient binding facilitates antibiotic translocation and thereby enhances its efficiency.

Probing OmpF Channel with Water-Soluble Soft Polymers
Rostovtseva, Nestorovich, Bezrukov
To understand the physics of polymer equilibrium and dynamics in the confines of ion channel pores, we studied the partitioning of polyethyleneglycols (PEGs) of different molecular weights into the bacterial porin, OmpF. Thermodynamic and kinetic parameters of partitioning are deduced from the effects of polymer addition on ion currents through single OmpF channels reconstituted into planar lipid bilayer membranes. The equilibrium partition coefficient is inferred from the average reduction of channel conductance in the presence of PEG; rates of polymer exchange between the pore and the bulk are estimated from PEG-induced conductance noise. Observed PEG partitioning into the OmpF pore depends more sharply on polymer molecular weight than predicted by hard-sphere, random-flight, or scaling models. A 1360 Da polymer separates regimes of partitioning and exclusion {for small polymers, which partition into the channel easily, there is a regime of partitioning (or "equi-partitioning" between the bulk solution and the pore), whereas, for the range of molecular weights that are larger than a certain "characteristic molecular weight," there is a regime of exclusion}. Comparison of its characteristic size with the size of a 2200 Da polymer previously found to separate these regimes for the a-toxin shows good agreement with the x-ray structural data for these channels. The PEG-induced conductance noise is compatible with a reduced polymer mobility inside the OmpF pore, an order of magnitude smaller than its value in bulk solution.

Role of Membrane Surface Charge in Ion Channel Functioning
Aguilella, Bezrukov
Membrane surface charge modifies conductance of ion channels by changing electric potential and redistributing ionic composition in the ions' vicinity. We have studied the effects of lipid charge on conductance of a multistate channel formed in planar lipid bilayers by a peptide antibiotic, alamethicin. The channel conductance was measured in two lipids: in a neutral dioleoylphosphatidylethanolamine (DOPE) and in a negatively charged dioleoylphosphatidylserine (DOPS). The charge state of DOPS was manipulated by the pH of the membrane-bathing solution. We found that at high salt concentrations (e.g., 2M NaCl) the effect of the lipid charge is below the accuracy of our measurements. However, when the salt concentration in the membrane-bathing solution is decreased, the surface charge manifests itself as an increase in conductance of the first two channel levels that correspond to the smallest conductive alamethicin aggregates. Our theoretical analysis shows that both the salt and pH dependence of the surface charge effect can be rationalized within the nonlinear Poisson-Boltzmann approach. Given channel conductance in neutral lipids, we use different procedures to account for the surface charge (e.g., introduction of averaging over the channel aperture and taking into account Na? adsorption to DOPS heads) but only one adjustable parameter: an effective distance from the nearest lipid charge to the channel mouth center. We show that the distance varies by 3 to 4 Ångstroms upon channel transition from the minimal conducting aggregate (Level 0) to the next larger aggregate (Level 1). This conclusion is in accord with a simple geometrical model of alamethicin aggregation.

FIGURE 39

Maltoporin channel in the presence of penetrating sugars illustrates the "molecular Coulter counter" principle. Sugar molecules passing through one of the water-filled channels in the maltoporin trimer induce transient blockage of small ion current. For large sugars, the single-molecular events are long enough to be time-resolved (Kullman et al. 2002).