SummaryTo date most antibiotics are targeted at intracellular processes, and must be able to penetrate the bacterial cell envelope. In particular, the outer membrane of Gram-negative bacteria provides a formidable barrier that must be overcome. There are essentially two pathways that antibiotics can take through the outer membrane: a lipid-mediated pathway for hydrophobic antibiotics, and general diffusion porins for hydrophilic antibiotics. The lipid and protein compositions of the outer membrane have a strong impact on the sensitivity of bacteria to many types of antibiotics, and drug resistance involving modifications of these macromolecules is common. This review will describe the molecular mechanisms for permeation of antibiotics through the outer membrane, and the strategies that bacteria have deployed to resist antibiotics by modifications of these pathways.

We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherchia coli and have discovered pressure-activated ion channels. The channels have the following properties: (t) activation by slight positive or negative pressure; (it) voltage dependence; (ifi) large conductance; (iv) selectivity for anions over cations; (v) dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.Ion channels are gated protein pores found in biological membranes; these channels regulate many cellular interactions with the environment, including responses to hormonal, neuronal, and sensory stimuli (1). Ion channels have been studied in animals, plants, and microorganisms (2-4). In bacteria, in vivo channel activity has not been demonstrated, although the activity of isolated channel proteins has been measured in artificial membranes (5, 6).The patch-clamp technique allows recording of current through individual ion channels in the native membrane by sucking the membrane onto a recording pipette to form a tight (gigaohm) electrical seal (7). This method has been used to study single channels in vivo in many eukaryotic cells, and it has demonstrated that the large currents measured across the membranes of a whole cell are really composed ofmany small currents passing through individual channels.The lower limit to the diameter ofthe patch-pipette opening is about 1 ,um (1); this precludes measurement ofion channels in bacteria directly. Cells of Escherichia coli, however, can become giant spheroplasts when grown in the presence of chemicals such as mecillinam to prevent cell wall (peptidoglycan) synthesis, and membrane potential has been measured in such spheroplasts by conventional electrophysiology (8). Giant spheroplasts can also be formed by growth of cells in the presence of cephalexin to prevent cell division and form filamentous "snakes"; these snakes can then be treated with lysozyme and EDTA to dissolve the cell wall (the spheroplasts can revert to normal form when returned to growth medium in the absence of these chemicals) (9). We used this latter method to make spheroplasts with a diameter of -6 ,.m. We demonstrate here the application of in vivo patch-clamp recording to such giant spheroplasts. This method should be generally applicable to any bacterial species.We discovered that a low positive or negative pressure (tens of millimeters of mercury; 1 mm Hg = 133 Pa) applied to the spheroplast membrane activates ion channels. This pressure could be caused by an osmotic difference of as little as a few milliosmolar across the membrane. We believe that these channels may allow E. coli to detect and to respond to small osmotic changes in the surrounding medium. The preliminary work has been reported in abstract form (10). MATERIALS AND METHODSMaterials. Organic components of the growth medium were purchased from Difco. Tris was purchased from Boehringer Mannheim; other salts and chemicals for preparation of...

Recent studies have provided evidence for a role of protein phosphorylation in the regulation of the function of various potassium and calcium channels (for reviews, see refs 1, 2). As these ion channels have not yet been isolated and characterized, it has not been possible to determine whether phosphorylation of the ion channels themselves alters their properties or whether some indirect mechanism is involved. In contrast, the nicotinic acetylcholine receptor, a neurotransmitter-dependent ion channel, has been extensively characterized biochemically and has been shown to be directly phosphorylated. The phosphorylation of this receptor is catalysed by at least three different protein kinases (cyclic AMP-dependent protein kinase, protein kinase C and a tyrosine-specific protein kinase) on seven different phosphorylation sites. However, the functional significance of phosphorylation of the receptor has been unclear. We have now examined the functional effects of phosphorylation of the nicotinic acetylcholine receptor by cAMP-dependent protein kinase. We investigated the ion transport properties of the purified and reconstituted acetylcholine receptor before and after phosphorylation. We report here that phosphorylation of the nicotinic acetylcholine receptor on the gamma- and delta-subunits by cAMP-dependent protein kinase increases the rate of the rapid desensitization of the receptor, a process by which the receptor is inactivated in the presence of acetylcholine (ACh). These results provide the first direct evidence that phosphorylation of an ion channel protein modulates its function and suggest that phosphorylation of postsynaptic receptors in general may play an important role in synaptic plasticity.

We have modified the procedure of Criado and Keller (1987) to study ion channels of Escherichia coli reconstituted in liposomes. The modifications include (a) excluding the use of any detergent and (b) inducing blisters from liposomes with Mg2+. These blisters, which appear to be unilamellar, are stable for hours. They could be repeatedly sampled with different patch-clamp pipettes each achieving seal resistance greater than 10 GOhms. Activities of three types of ion channels are often observed by use of this method, including two voltage-sensitive cation channels of different conductances. Even the mechanosensitive channel, previously recorded from live E. coli cells (Martinac et al., 1987), was also detected in these blisters. Apparently the channel protein and any accessory structures, postulated to be needed for mechanotransduction, can be reconstituted together by this method.

The effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on the activity of bacterial porins OmpC and OmpF were investigated by electrophysiology. Membrane vesicles made from the outer membrane of Escherichia coli strains expressing only OmpC or OmpF were reconstituted into liposomes probed by patch clamp. The channel activity was recorded in control solutions and in the presence of increasing concentrations of a specific polyamine. In all cases, concentration-and voltage-dependent inhibitory effects were observed. They include both the suppression of channel openings and the enhancement of channel closures as well as the promotion of blocked or inactivated states. OmpF and OmpC, although highly homologous, have distinct sensitivities to modulation, especially by spermine. This compound inhibits OmpF in the nanomolar range, which is in agreement with its potency on eukaryotic channels. Putrescine was the least effective (upper millimolar range) and also had inhibitory effects qualitatively distinct from those exerted by the other polyamines. The compounds appear to bind to at least two distinct binding sites, one of which resides within the pore. The potencies to this site are lower when the polyamines are applied from the extracellular side than from the periplasmic side, suggesting an asymmetric binding site.Polyamines are a class of naturally occurring polycationic molecules produced through complex pathways involving decarboxylations of ornithine, arginine, or lysine (1, 2). The most ubiquitous are spermine, spermidine, cadaverine and putrescine. With the exception of spermine, which is associated exclusively with eukaryotes, the other three are endogenous to both eukaryotic and prokaryotic cell types. Polyamines have been implicated in a wide range of biological phenomena (1, 2). One of the most intriguing forms of polyamine action is the recently discovered modulation of ion channels of heart, muscles, and neurons (3-10).The cytoplasmic membrane of Escherichia coli cells is surrounded by an additional external membrane, the outer membrane, whose outer leaflet is made of highly negatively charged lipopolysaccharides. Polyamines are associated with the outer membrane of E. coli, possibly through their interactions with the lipopolysaccharides (11). Although polyamines have not been measured directly in the periplasmic space between the outer and cytoplasmic membranes, they are likely to accumulate in this compartment during their synthesis and transport (12)(13)(14). An arginine decarboxylase involved in the production of putrescine is located in the inner periplasmic space (12), and a lysine-cadaverine exchanger of the cytoplasmic membrane participates in the extrusion of cadaverine (13). Thus, polyamines appear to reside in the vicinity of the major poreforming proteins of the outer membrane, the porins. Porins are trimeric channels characterized extensively at the biochemical, structural, and genetic levels (15). They are the only ion channels whose structure is known at atomic re...