α-Toxin permeabilized rat pheochromocytoma cells

The channel forming α-toxin of Staphylococcus aureus (about 50 μg/ml) markedly reduces the Ca2+ requirement for dopamine release by the rat pheochromocytoma cell line (PC 12). Maximal secretion by intact cells requires approximately 1 mM Ca2+, whereas release by α-toxin-permeabilized cells can already be triggered with μM concentrations of Ca2+. The latter process reaches a plateau at about 1 μM free Ca2+ and increases again with 10–20 μM free Ca2+. The sensitivity to low concentrations of Ca2+ indicates that the toxin, as a selective cell membrane permeabilizing agent, can be used as a powerful instrument to study stimulus-secretion coupling

Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization

Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules

Location of a Constriction in the Lumen of a Transmembrane Pore by Targeted Covalent Attachment of Polymer Molecules

Few methods exist for obtaining the internal dimensions of transmembrane pores for which 3-D structures are lacking or for showing that structures determined by crystallography reflect the internal dimensions of pores in lipid bilayers. Several approaches, involving polymer penetration and transport, have revealed limiting diameters for various pores. But, in general, these approaches do not indicate the locations of constrictions in the channel lumen. Here, we combine cysteine mutagenesis and chemical modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of the staphylococcal α-hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric reagents (MePEG-OPSS) of different masses towards individual single cysteine mutants, comprising a set with cysteines distributed over the length of the lumen of the pore, were determined by macroscopic current recording. The rates for the three larger polymers (1.8, 2.5, and 5.0 kD) were normalized with respect to the rates of reaction with a 1.0-kD polymer for each of the seven positions in the lumen. The rate of reaction of the 5.0-kD polymer dropped dramatically at the centrally located Cys-111 residue and positions distal to Cys-111, whether the reagent was applied from the trans or the cis side of the bilayer. This semi-quantitative analysis sufficed to demonstrate that a constriction is located at the midpoint of the pore lumen, as predicted by the crystal structure, and although the constriction allows a 2.5-kD polymer to pass, transport of a 5.0-kD molecule is greatly restricted. In addition, PEG chains gave greater reductions in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interior of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels

Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼10(4)-fold more avidly than the remaining αHL pores, including WT-αHL. The lower K (d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors

Nanopore Detector based analysis of single-molecule conformational kinetics and binding interactions

BACKGROUND: A Nanopore Detector provides a means to transduce single molecule events into observable channel current changes. Nanopore-based detection can report directly, or indirectly, on single molecule kinetics. The nanopore-based detector can directly measure molecular characteristics in terms of the blockade properties of individual molecules – this is possible due to the kinetic information that is embedded in the blockade measurements, where the adsorption-desorption history of the molecule to the surrounding channel, and the configurational changes in the molecule itself, imprint on the ionic flow through the channel. This rich source of information offers prospects for DNA sequencing and single nucleotide polymorphism (SNP) analysis. A nanopore-based detector can also measure molecular characteristics indirectly, by using a reporter molecule that binds to certain molecules, with subsequent distinctive blockade by the bound-molecule complex. RESULTS: It is hypothesized that reaction histories of individual molecules can be observed on model DNA/DNA, DNA/Protein, and Protein/Protein systems. Preliminary results are all consistent with this hypothesis. Nanopore detection capabilities are also described for highly discriminatory biosensing, binding strength characterization, and rapid immunological screening. CONCLUSION: In essence, the heart of chemistry is now accessible to a new, single-molecule, observation method that can track both external molecular binding states, and internal conformation states