ABSTRACT
Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.
Subject(s)
Bacterial Proteins/chemistry , Norepinephrine/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Sodium/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Catalytic Domain , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Liposomes/chemistry , Liposomes/metabolism , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Norepinephrine/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodamines/chemistry , Sodium/metabolism , Spectrometry, Fluorescence/methodsABSTRACT
Here, we bind the sodium dependent amino acid transporter on nitrilotriacetic acid/polyethylene glycol functionalized gold sensors in detergents and perform a detergent-lipid exchange with phosphatidylcholine. We characterize the LeuT structure in the adsorbed film by magnetic contrast neutron reflection using the predicted model from molecular dynamic simulations.
Subject(s)
Amino Acid Transport Systems/metabolism , Molecular Dynamics Simulation , Amino Acid Transport Systems/chemistry , Detergents/chemistry , Gold/chemistry , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Nitrilotriacetic Acid/chemistry , Phosphatidylcholines/chemistry , Polyethylene Glycols/chemistry , Quartz Crystal Microbalance Techniques , Sodium/chemistryABSTRACT
Calcium electroporation may offer a simple general tool for anticancer therapy. Transient permeabilization of cancer cell membranes created by applying short, high-voltage pulses in tumors enables high calcium influxes that trigger cell death. In this study, we compared the relative sensitivity of different human tumor models and normal tissues to calcium electroporation. Plasma membrane Ca2+-ATPase (PMCA) protein expression was confirmed in vitro in all cancer cell lines and normal primary dermal fibroblasts studied. In all tumor types tested in vivo, calcium electroporation effectively induced necrosis, with a range of sensitivities observed (36%-88%) 2 days after treatment. Necrosis was induced using calcium concentrations of 100-500 mmol/L and injection volumes 20%-80% of tumor volume. Notably, only limited effects were seen in normal tissue. Calcium content increased >7-fold in tumor and skin tissue after calcium electroporation but decreased in skin tissue 4 hours after treatment to levels comparable with untreated controls, whereas calcium content endured at high levels in tumor tissue. Mechanistic experiments in vitro indicated that calcium influx was similar in fibroblasts and cancer cells. However, we observed decreased PMCA expression in cancer cells compared with fibroblasts, offering a potential explanation for the different calcium content in tumor cells versus normal tissues. Overall, our results suggest that calcium electroporation can elicit a rapid and selective necrosis of solid tumors, with limited deleterious effects on surrounding normal tissues. Cancer Res; 77(16); 4389-401. ©2017 AACR.