Membrane permeability barriers are among the factors contributing to the intrinsic resistance of bacteria to antibiotics. We have been able to resolve single ampicillin molecules moving through a channel of the general bacterial porin, OmpF (outer membrane protein F), believed to be the principal pathway for the ␤-lactam antibiotics. With ion channel reconstitution and high-resolution conductance recording, we find that ampicillin and several other efficient penicillins and cephalosporins strongly interact with the residues of the constriction zone of the OmpF channel. Therefore, we hypothesize that, in analogy to substrate-specific channels that evolved to bind certain metabolite molecules, antibiotics have ''evolved'' to be channel-specific. Molecular modeling suggests that the charge distribution of the ampicillin molecule complements the charge distribution at the narrowest part of the bacterial porin. Interaction of these charges creates a region of attraction inside the channel that facilitates drug translocation through the constriction zone and results in higher permeability rates.

Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation, dependent on the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. A long-standing puzzle is that in permeabilized cells, adenine nucleotide translocase (ANT) is less accessible to cytosolic ADP than in isolated mitochondria. We solve this puzzle by finding a missing player in the regulation of MOM permeability: the cytoskeletal protein tubulin. We show that nanomolar concentrations of dimeric tubulin induce voltage-sensitive reversible closure of VDAC reconstituted into planar phospholipid membranes. Tubulin strikingly increases VDAC voltage sensitivity and at physiological salt conditions could induce VDAC closure at <10 mV transmembrane potentials. Experiments with isolated mitochondria confirm these findings. Tubulin added to isolated mitochondria decreases ADP availability to ANT, partially restoring the low MOM permeability (high apparent K m for ADP) found in permeabilized cells. Our findings suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondriacytosol interface. This tubulin-VDAC interaction requires tubulin anionic C-terminal tail (CTT) peptides. The significance of this interaction may be reflected in the evolutionary conservation of length and anionic charge in CTT throughout eukaryotes, despite wide changes in the exact sequence. Additionally, tubulins that have lost significant length or anionic character are only found in cells that do not have mitochondria.xidative phosphorylation requires transport of metabolites, including cytosolic ADP, ATP, and inorganic phosphate, across both mitochondrial membranes for F 1 F 0 -ATPase to generate ATP in the matrix. Voltage-dependent anion channel (VDAC, also called mitochondrial porin) is the most abundant protein in mitochondrial outer membrane (MOM) and is known to be primarily responsible for ATP/ADP flux across the outer membrane (1, 2). Until recently, VDAC was generally viewed as a part of the pathway for release of cytochrome c and other apoptogenic factors from the mitochondrial intermembrane space into the cytosol at the early stage of apoptosis. The recent genetic studies undermined this view (3) but still left open a lot of questions concerning the role of VDAC in MOM permeabilization in apoptosis (4-6). A conserved property of VDACs in vitro is the ability to adopt a unique fully open state and multiple states with significantly smaller conductance (7). It was demonstrated that the latter, so called ''closed states'' are impermeable to ATP but still permeable to small ions (8), including Ca 2ϩ (9). In isolated mitochondria, respiration is characterized by an apparent K m for exogenous ADP that is Ϸ10-fold lower than in permeabilize...

Membrane-bound proteinaceous nanoscale pores allow us to simultaneously observe the thermodynamic and kinetic properties of differently sized polymers within their confines. We determine the dynamic partitioning of poly(ethylene glycol) (PEG) into the pore formed by Staphylococcus aureus α-toxin and evaluate the free energy of polymer confinement by measuring polymer-induced changes to the pore's ionic conductance. The free energy deduced from the partition coefficient has a sharper dependence on polymer length (or weight) than scaling theory predicts. Moreover, the polymer-induced conductance fluctuations show a striking nonmonotonic dependence on the polymer molecular weight. The movement of polymer inside the pore is characterized by a diffusion coefficient that is orders of magnitude smaller than that for polymer in the bulk aqueous solution, which suggests that PEG has an attractive interaction with the pore. Using an ad-hoc approach, we show that a simple molecular weight-dependent modification of the polymer's diffusion coefficient accounts for these results, but only qualitatively. Given that PEG associates with hydrophobic regions in proteins, we also conclude that, contrary to the conventional view of ion channels, the aqueous cavity of the α-toxin pore's interior is, to some extent, hydrophobic.

Growth of the malaria parasite in human red blood cells (RBCs) is accompanied by an increased uptake of many solutes including anions, sugars, purines, amino acids and organic cations. Although the pharmacological properties and selectivity of this uptake suggest that a chloride channel is involved, the precise mechanism has not been identified. Moreover, the location of this uptake in the infected RBC is unknown because tracer studies are complicated by possible uptake through fluid-phase pinocytosis or membranous ducts. Here we have studied the permeability of infected RBCs using the whole-cell voltage-clamp method. With this method, uninfected RBCs had ohmic whole-cell conductances of less than 100 pS, consistent with their low tracer permeabilities. In contrast, trophozoite-infected RBCs exhibited voltage-dependent, non-saturating currents that were 150-fold larger, predominantly carried by anions and abruptly abolished by channel blockers. Patch-clamp measurements and spectral analysis confirmed that a small (< 10 pS) ion channel on the infected RBC surface, present at about 1,000 copies per cell, is responsible for these currents. Because its pharmacological properties and substrate selectivities match those seen with tracer studies, this channel accounts for the increased uptake of small solutes in infected RBCs. The surface location of this new channel and its permeability to organic solutes needed for parasite growth indicate that it may have a primary role in a sequential diffusive pathway for parasite nutrient acquisition.