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Insights into the transport side of the human SLC38A9 transceptor. Pizzo, A. The membrane raft protein Flotillin-1 is essential in dopamine neurons for amphetamine-induced behavior in Drosophila.
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Psychiatry 18 , — Karam, C. Phosphorylation of the amino terminus of the dopamine transporter: regulatory mechanisms and implications for amphetamine action. Psychiatry 19 , — Wang, R. Aptamer-based fluorescent biosensors. Marvin, J. An optimized fluorescent probe for visualizing glutamate neurotransmission. Methods 10 , — Marcos, E. Tinberg, C. Computational design of ligand-binding proteins with high affinity and selectivity. Download references. We thank R. Altman for the preparation of microscope slides and other reagents for single-molecule experiments, and M. All authors contributed to overall experimental design and the writing of the manuscript.
Correspondence to Matthias Quick or Jonathan A. Javitch or Scott C. For each concentration, FRET efficiencies were summed across time to generate a histogram symbols and fit to Gaussian functions lines to obtain estimates of the mean FRET value. Data were collected at least three times with similar results. Analogous data with mean FRET values are shown for each concentration for isoleucine red , valine blue and leucine black were fit with equation 1 to determine the K d.
This indicates that the binding activity of LIV-BP is unaffected by encapsulation within the liposome lumen. FRET values from all selected traces count at top right of each panel summed into time-dependent population FRET histograms, represented as contour plots. Scale bar is shown at the right. All experiments were performed at least three times with similar results.
As expected for a bimolecular interaction, the ligand-binding rate increased linearly with ligand concentrations, whereas the ligand-dissociation rate remained constant. Together, these results are consistent with the observed fold difference in binding affinity in the two sensor variants Fig.
The mean background fluorescence is shown, from a representative experiment in which LIV-BP WT -containing liposomes that lack MhsT were imaged before and after vertical dashed line the injection of Cy5-labelled DNA tracer, with the exact time of injection determined by the midpoint of the step-like increase in fluorescence. Inset, zoomed-in view of the period immediately before and after injection. Experiments were performed at least three times with similar results.
Traces shown are representative of experiments performed at least three times. This indicates that, while in ideal circumstances we should be able to monitor individual transport events in this assay, in practice such an analysis would be difficult.
Funcionament del sistema tarifari integrat (STI)
Leucine uptake by wild-type MhsT exhibited a K m of 0. Transport is observed only in the presence of MhsT in the outside-out orientation. Both preparations show robust activity, indicating that both vesicle preparations are intact and contain functional transporters. Lactic acid was added arrow to vesicles during the glycine-uptake time course to establish a proton gradient relative to the vesicle orientation.
This creates an inwardly directed proton gradient in outside-out vesicles, and the opposite in inside-out vesicles. We observe an increase in the rate of glycine transport in outside-out vesicles and a marked decrease in inside-out vesicles, as expected, which demonstrates that we have prepared the vesicles in the indicated orientation.
The partially filled square indicates the virtual overlap of data points. The specific radioactivity—decays per minute correlation was verified using known amounts of 3 H-leucine. Shorter sampling times yielded higher turnover rates and lower K m values the highest V max of 1. Traces are representatives of experiments performed at least three times.
The right panel shows transport data from proteoliposomes that contain a single mixed orientation MhsT transporter immobilized by His-tagged lipids. Occupancy of the high-FRET state following injection of leucine grey dashed line represents the proportion of vesicles in which transport has occurred. The N in top right corner indicates the total number of traces recorded over three separate experiments.