Previous studies have shown that symmetric tetraalkylammonium ions affect, in a voltage-dependent manner, the conductance of membranes containing many channels formed by the PAn5 fragment of anthrax toxin. In this paper we analyze this phenomenon at the single-channel level for tetrabutylammonium ion (Bu4N+). We find that Bu4N + induces a flickery block of the PA65 channel when present on either side of the membrane, and this block is relieved by large positive voltages on the blocking-ion side. At high frequencies (>2 kHz) we have resolved individual blocking events and measured the dwell times in the blocked and unblocked states. These dwell times have single-exponential distributions, with time constants rb and ~'u that are voltage dependent, consistent with the two-barrier, single-well potential energy diagram that we postulated in our previous paper. The fraction of time the channel spends unblocked [Tu/(Tu + ~'b)] as a function of voltage is identical to the normalized conductance-voltage relation determined from macroscopic measurements of blocking, thus demonstrating that these single channels mirror the behavior seen with many (> 10,000) channels in the membrane. In going from large negative to large positive voltages (-100 to + 160 mV) on the c/s (PA~n-containing) side of the membrane, one sees the mean blocked time (Tb) increase to a maximum at + 60 mV and then steadily decline for voltages greater than +60 mV, thereby clearly demonstrating that Bu4N + is driven through the channel by positive voltages on the blocking-ion side. In other words, the channel is permeable to Bu4N +. An interesting finding that emerges from analysis of the voltage dependence of mean blocked and unblocked times is that the blocking rate, with Bu4N + present on the c/s side of the membrane, plateaus at large positive c/s voltages to a voltage-independent value consistent with the rate of Bu4N + entry into the blocking site being diffusion limited.Address reprint requests to Dr.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

A recently discovered antibiotic (CDA ; calcium-dependent antibiotic) of Streptomyces coelicolor A3(2) was found to be effective against a wide range of Gram-positive bacteria only in the presence of calcium ions. Producer and non-producer strains were identified and several media tested for their ability to support antibiotic production. The action of calcium was not simulated by any of the other cations tested. The antibiotic was found to induce discrete conductance fluctuations in planar lipid bilayer consistent with a channel-forming action. The electrical potential difference caused by a concentration difference of various salts across the CDAcontaining bilayer, showed the channel to be cation-selective but of a size that discriminated against tetramethyl ammonium and choline ions. The data indicate that the antibiotic activity of CDA is due to its action as a calcium-dependent ionophore.

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SummaryMycobacteria are characterized by an unusual cell wall that controls nutrient and small hydrophilic compound permeability. Porin-like proteins are necessary to ensure the transport of molecules into the cell. Here, we investigated the pore-forming properties of OmpATb, a porin from Mycobacterium tuberculosis, in lipid bilayers. Multi-channel experiments showed an asymmetric behaviour with channel closures at negative critical voltages (Vc) and a strong decrease in Vc at acidic pH. Single-channel experiments gave conductance values of about 850 ± 80 pS in 1 M KCl and displayed a weak cationic selectivity in 4-8 pH range. The production and characterization of a series of truncated OmpATb proteins, showed that the central domain (OmpATb 73-220) was sufficient to induce the ion channel properties of the native protein in lipid bilayers, i.e. asymmetric insertion, pH-dependent voltage closure, cationic selectivity and similar conductance values in 1 M KCl. Western blot analysis suggests that the presence of OmpATb is only restricted to certain pathogenic species. Therefore, the propensity of channels of native OmpATb to close at low pH may represent an intrinsic property allowing pathogenic mycobacteria to adapt and survive to mildly acidic conditions, such as those encountered within the macrophage phagosome.