Filamentous cells of Escherichia coli can be produced by treatment with the antibiotic cephalexin, which blocks cell division but allows cell growth. To explore the effect of cell size on chemotactic activity, we studied the motility and chemotaxis of filamentous cells. The filaments, up to 50 times the length of normal E. coli organisms, were motile and had flagella along their entire lengths. Despite their increased size, the motility and chemotaxis of filaments were very similar to those properties of normal-sized cells. Unstimulated filaments of chemotactically normal bacteria ran and stopped repeatedly (while normal-sized bacteria run and tumble repeatedly). Filaments responded to attractants by prolonged running (like normal-sized bacteria) and to repellents by prolonged stopping (unlike normal-sized bacteria, which tumble), until adaptation restored unstimulated behavior (as occurs with normal-sized cells). Chemotaxis mutants that always ran when they were normal sized always ran when they were filament sized, and those mutants that always tumbled when they were normal sized always stopped when they were filament sized. Chemoreceptors in filaments were localized to regions both at the poles and at intervals along the filament. We suggest that the location of the chemoreceptors enables the chemotactic responses observed in filaments. The implications of this work with regard to the cytoplasmic diffusion of chemotaxis components in normal-sized and filamentous E. coli are discussed.Escherichia coli organisms have about six flagella, located randomly around their surfaces, that serve to propel the bacterium. When flagellar rotation is counterclockwise, the flagella form a bundle at one end to push the bacterium forward; this is known as a "run" and typically lasts about 1 to 2 s. When the rotation is clockwise, the flagella pull in opposing directions, causing the bacterium to "tumble" for less than a second. The bacteria alternately run and tumble in the absence of stimuli. When an attractant is encountered, running is promoted and tumbling is suppressed, which causes the bacteria to swim towards the attractant. When a repellent is encountered, the bacteria tumble, which prevents them from swimming into repellent. Adaptation to the attractant or repellent returns the bacteria to unstimulated behavior despite the continued presence of attractant or repellent.Detection of attractants and repellents is mediated by a series of chemoreceptors in the cytoplasmic membrane, the methyl-accepting chemotaxis proteins (MCPs). Binding of repellents induces phosphorylation of the soluble cytoplasmic protein CheY, whereas binding of attractants results in CheY dephosphorylation. CheY interacts with the flagellar motor components and controls the direction of flagellar spin; phosphorylated CheY (CheY-P) stimulates clockwise rotation (tumbling), while unphosphorylated CheY results in counterclockwise rotation (running). For recent reviews of motility and chemotaxis see references 7 and 30.The MCPs are located primarily at t...

The human multidrug transporter P-glycoprotein (Pgp, ABCB1) contributes to the poor bioavailability of many anticancer and antimicrobial agents as well as to drug resistance at the cellular level. For rational design of effective Pgp inhibitors, a clear understanding of its mechanism of action and functional regulation is essential. In this study, we demonstrate that inhibition of Pgp-mediated drug transport by cis-(Z) Cellular expression of human P-glycoprotein (Pgp), 1 the product of the MDR1 gene, confers resistance to a broad variety of structurally unrelated chemotherapeutic agents and restricts bioavailability of many therapeutic drugs in experimental models (1, 2). Pgp is a 1280-amino acid plasma membrane protein that has two homologous halves separated by a linker region of about 80 amino acids (3). Each half of the protein contains a hydrophobic region with six putative transmembrane (TM) helices, followed by a cytoplasmic consensus ATPbinding/hydrolysis site (3). The TM regions are presumed to form the drug-translocating pathway (4), whereas the ATP sites, through ATP hydrolysis, provide the necessary driving force for transport (5, 6).-Pgp-mediated drug transport is inhibited by a number of structurally unrelated compounds known as reversing agents or modulators (for review see Ref.2). Although some of the modulators are currently being tested for their clinical effectiveness, there remains a growing need for molecules with higher efficacy (7-9). To develop such compounds, a clear knowledge of the mechanisms of action of the existing repertoire is essential. Some of the Pgp modulators themselves, such as verapamil (10) and cyclosporin A (11), are substrates of the pump and inhibit drug transport in a competitive manner without interrupting the catalytic turnover (catalytic cycle) of Pgp (12-15). However, for many others, the inhibitory mechanisms are yet to be fully understood.Recent studies on the mechanism of action of Pgp modulators indicated an allosteric mode of action for several compounds. Martin et al. (16) demonstrated that inhibition of vinblastine transport by the anthranilic acid derivative XR9576 is not through direct physical competition for the drug translocating pathway, indicating an allosteric effect on substrate recognition or ATP hydrolysis (17). A similar study suggested that the indolizin sulfone SR33557 affected vinblastine binding to Pgp through interaction with a site distinct from the site of substrate recognition (17). Boer et al. (18) and Ferry et al. (19) have shown that modulators like dexniguldipine and prenylamine inhibited vinblastine interaction with Pgp by a non-competitive mechanism. Based on these and other similar studies, drug interaction sites of Pgp have been categorized into the following two discrete types: 1) transport sites, where translocation of drug across the lipid bilayer can occur, and 2) regulatory sites, which modulate Pgp function (20,21). Most of these studies investigated the effect of the modulators on substrate binding in isolated membranes, ...

The human P-glycoprotein (Pgp) 3 functions as an ATP-driven drug transporter conferring multidrug resistance in cancer cells and restricting bioavailability of many antimicrobial and anticancer agents (1, 2). It is a 1280-amino acid integral membrane protein of the ATP-binding cassette (ABC) transporter family (3, 4), with two highly homologous halves, each containing a hydrophobic transmembrane domain and a relatively hydrophilic cytosolic domain. Each hydrophobic domain contains six putative transmembrane helices that, in conjunction with the transmembrane regions of the other half, form the drug-translocating pathway (substrate site) across the lipid bilayer (1, 5). The cytosolic domains each contain three consensus sequences (3) that together contribute to the formation of two ATP binding/hydrolysis sites (ATP sites) (6).Both ATP sites are essential for the drug transport function of Pgp (7-10) but are believed to hydrolyze ATP in an alternate sequence (11-13). Pgp possesses a basal rate of ATP hydrolysis that is stimulated upon interaction with many transport substrates as well as with several Pgp modulators (14). Binding and hydrolysis of ATP induce a conformational change in that is coupled to substrate translocation across the lipid bilayer and its subsequent dissociation (18 -24). The two ATP sites of Pgp were believed to be functionally equivalent with drug translocation and regeneration of the transporter directly coupled to a single round of ATP binding or/and hydrolysis (12,25,26). However, evidence suggests that although the two ATP sites assume similar structural conformation, they have distinct functional roles within a single catalytic turnover of (18,19), whereas hydrolysis by the other resets the transporter for the subsequent round of transport activity (27,28). Vanadate (Vi), a phosphate analog, replaces phosphate at the catalytic site (30) when present during ATP hydrolysis. Due to its low rate of dissociation, vanadate stabilizes Pgp in a catalytic intermediate (mimicking a conformational state) that immediately follows the first ATP hydrolytic event (11,18,19,22,23 (18,19). This is accompanied by an experimentally detectable conformational change (15,17). Chemical cross-linking with thiol reagents and cysteine-scanning mutagenesis revealed increased accessibility of the drug-translocating pathway from the extracellular side of the lipid bilayer due to vanadate trapping (22).Interestingly, the [ 125 I]IAAP-binding site once transformed to its low affinity state remains unaltered even after dissociation of vanadate and

The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.

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