Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent K D values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.mass spectrometry from native state | real time substrate monitoring P -glycoprotein (P-gp) is an ATP-driven low-specificity efflux pump that plays an important role in the clearance of xenotoxins (1, 2). P-gp is also a member of the ATP-Binding Cassette (ABC) family of transporters and exports hydrophobic cytotoxic compounds as well as natural products, cyclic, and linear peptides (1, 3-5). Overexpression of P-gp in tumor cells impairs targeted drug delivery and is a major pitfall for chemotherapies. Small molecule substrates partition in the plasma membrane (6, 7), before binding in the internal hydrophobic cavity formed in the inward conformation of the pump (8). Export is then thought to proceed in an ATP-dependent way through conformational changes from the inward to the outward facing forms, evidenced by FRET spectroscopy (9). Recent highresolution structures of eukaryotic P-gp from mouse (8) and Caenorhabditis elegans (10) were obtained in inward conformations. Two prokaryotic homologs of P-gp [Sav1866 (11) and MsbA (12)] were captured crystallographically in outward-facing conformers, reflecting ATP-bound states, as well as two different inward states for MsbA. From these X-ray structures it is possible to build up a picture of P-gp, alternating between inward-and outward-facing conformations.Despite decades of careful biochemical studies (13), and recent insights from crystallography, many questions remain, however. Specifically it has not yet been possible to trap P-gp in an outwardfacing state or to show how substrate binding activates ATPase act...

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By far the most studied multidrug resistance protein is P-glycoprotein. Despite recent structural data, key questions about its function remain. P-glycoprotein (P-gp) is flexible and undergoes large conformational changes as part of its function and in this respect, details not only of the export cycle, but also the recognition stage are currently lacking. Given the flexibility, molecular dynamics (MD) simulations provide an ideal tool to examine this aspect in detail. We have performed MD simulations to examine the behaviour of P-gp. In agreement with previous reports, we found that P-gp undergoes large conformational changes which tended to result in the nucleotide-binding domains coming closer together. In all simulations, the approach of the NBDs was asymmetrical in agreement with previous observations for other ABC transporter proteins. To validate the simulations, we make extensive comparison to previous cross-linking data. Our results show very good agreement with the available data. We then went on to compare the influence of inhibitor compounds bound with simulations of a substrate (daunorubicin) bound. Our results suggest that inhibitors may work by keeping the NBDs apart, thus preventing ATP-hydrolysis. On the other hand, repeat simulations of daunorubicin (substrate) in one particular binding pose suggest that the approach of the NBDs is not impaired and that the structure would be still be competent to perform ATP hydrolysis, thus providing a model for inhibition or substrate transport. Finally we compare the latter to earlier QSAR data to provide a model for the first part of substrate transport within P-gp.

The efficacy of ␥-secretase inhibitors in vivo has, to date, been generally assessed in transgenic mouse models expressing increased levels of amyloid-␤ (A␤) peptide thereby allowing the detection of changes in A␤ production. However, it is not clear whether the in vivo potency of ␥-secretase inhibitors is independent of the level of amyloid precursor protein expression. In other words, does a ␥-secretase inhibitor have the same effect in nontransgenic physiological animals versus transgenic overexpressing animals? In the present study, an immunoassay has been developed which can detect A␤(40) in the rat brain, where concentrations are much lower than those seen in transgenic mice such as Tg2576 (c. 0.7 and 25 nM, respectively) and in cerebrospinal fluid (CSF, c. 0.3 nM). Using this immunoassay, the effects of thewere assessed and robust dose-dependent reductions in rat brain and CSF A␤(40) levels were observed with ID 50 values of 1.3 mg/kg for both brain and CSF. These values were comparable with those calculated for LY-411575 in transgenic mice.Time course experiments using LY-411575 demonstrated comparable temporal reductions in rat brain and CSF A␤(40), further suggesting these two pools of A␤ are related. Accordingly, when all the data for the dose-response curve and time course were correlated, a strong association was observed between the brain and CSF A␤ (40) levels. These data demonstrate the utility of the rat as a novel approach for assessing the effects of ␥-secretase inhibitors on central nervous system A␤(40) levels in vivo.Alzheimer's disease (AD) is one of the major neurological diseases of the elderly, characterized histopathologically by protein deposits in the brain parenchyma (plaques) or blood vessels and intracellular neurofibrillary tangles of abnormally phosphorylated protein. The plaques mainly consist of the A␤ peptides originating from cleavage of the amyloid precursor protein (APP), with the majority being the more hydrophobic A␤(42) which is particularly prone to aggregation (Selkoe 2001). Furthermore, the identification of APP gene mutations in familial Alzheimer's disease (FAD) cases gives evidence of the involvement of APP processing in AD (Goate et al., 1991;Hardy, 1997).The enzymes responsible for processing APP into A␤ are the aspartyl proteases, ␤-amyloid cleaving enzyme (BACE or 1 These authors contributed equally to this publication. Article, publication date, and citation information can be found at

Permeation of drugs across lipid bilayers is a key factor in dictating how effective they will be. In vivo, the issue is compounded by the presence of drug-exporter proteins such as P-glycoprotein. However, despite intense effort, exactly what controls permeation and susceptibility to export is still poorly understood. In this work we examine two well-studied drugs for which interaction with P-glycoprotein has been studied before: amitriptyline, a known substrate and clozapine, which is not a substrate. Extensive MD simulations, including potential of mean force (PMF) profiles of the compounds in all possible protonation states, reveal that the preferred location of the compounds in different bilayers in different protonation states is remarkably similar. For both molecules in charged states, there is a substantial barrier to crossing the bilayer. Clozapine however, shows an energetic barrier to movement across the bilayer even in a protonation state that results in an uncharged molecule. For amitriptyline there is only a very small barrier of approximately 1.3 kcal mol(-1). Further analysis revealed that the conformational and orientational behavior of the two compounds was also similar, with the sidechain interacting with the lipid headgroups. This effect was much stronger if the sidechain was charged (protonated). These interactions with lipid bilayers were confirmed by NMR ROESY experiments. The results are discussed in terms of their potential interactions with export proteins like P-glycoprotein.

Although monoamine transporters translocate substrates (neurotransmitters) across the plasma membrane, a second less studied function is their ion channel-like activity which is responsible for the passive permeation of ions through the membrane. These currents are inward at resting membrane potential and would result in cell depolarization, which may enhance presynaptic excitability, and which would help explain the elevated release of neurotransmitter associated by psychostimulants. Here we compare the ionic currents evoked by serotonin (5-HT) and S(þ)-3,4-methylenedioxy-N-methylamphetamine (S(þ)MDMA, ecstasy) in mammalian cells expressing the human serotonin transporter (hSERT). 5-HT as well as S(þ)MDMA induce inward currents in hSERT-expressing cells. Although the conductances are similar, the S(þ)MDMA induced-current reversal potential is strongly shifted towards the Na þ equilibrium potential, indicating a bigger contribution of Na þ to the overall current compared to 5-HT current. To measure changes in Na þ permeability induced by these compounds, the cells were pre-loaded with the Na þ sensor Asante NaTRIUM GREEN 2 and changes in intracellular Na þ concentration ([Na þ ]i) were monitored fluorometrically. Dose-response experiments show that 5-HT and S(þ)MDMA have similar potency (EC50 200 nM) in elevating the [Na þ ]i; however, S(þ)MDMA increases [Na þ ]i at least 3 times with respect to 5-HT. In addition, the reconstitution of hSERT on a lipid bilayer in the presence of a Na þ gradient showed channel-like activity after the application of 5-HT or S(þ)MDMA in the trans side. Taken together these results strongly suggest that S(þ)MDMA-induced current is different in composition than the 5-HT current, and moreover it must be strongly depolarizing due to its higher Na þ content. Supported by RC1DA028112 and R01DA033930.

The pathogenic Pseudomonas aeruginosa is a Gram-negative bacterium coexpressing two unique forms of lipopolysaccharides on its surface, the A and B bands. The A-band polysaccharides are thought to be translocated into the periplasm through an ATP-binding-cassette (ABC) transporter consisting of two subunits: a transmembranar protein, Wzm, and a nucleotide-binding protein, Wzt. Since Wzm and Wzt proteins possess no domains or motifs that are targeted for modifications by eukaryotic cellular machineries or exhibit any signal for organelles localization, we used Chinese hamster ovary (CHO) cells as a model system to study the protein complex structure and stoichiometry of Wzt protein in the absence or the presence of Wzm. We transfected the CHO cells with Wzt proteins fused to the green fluorescent protein (GFP2) or its variant yellow fluorescent protein (YFP) to study the protein complex structure of Wzt. A spectrally resolved two-photon microscope was used to obtain fluorescent images from transfected cells, and a novel spectral Fluorescence Resonance Energy Transfer (FRET) provided apparent FRET efficiency (Eapp) for each image pixel (Raicu et al., Nature Photonics, 2009). Distributions of FRET efficiencies were obtained from several cells, and the peak positions for the cells whose Eapp histograms showed single peak were binned to obtain a ''meta-histogram''. The meta-histograms were fitted to simulated distributions obtained from various theoretical models (Raicu et. al., Nature Photonics, 2009), to determine the stoichiometry and geometry of Wzt oligomers. Wzt formed rhombus-shaped homo-tertamers, which became square shaped upon co-expression of untagged Wzm.

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