Journal: eLife
Article Title: Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid
doi: 10.7554/eLife.85836
Figure Lengend Snippet: ( A ) Confocal images of HEK 293 cells in bright-field (top panel), mCherry fluorescence (middle row, magenta), and Anap fluorescence (bottom row, green). Each column shows the same field, and the bottom labels indicate transfection of the H V 1-A197TAG plasmid in the presence or absence pAnap plasmid and L-Anap in the culture media. The images were obtained with a Leica TCS SP5 inverted confocal microscope (RRID: SCR_020233 Leica Microsystems, Germany). Fluorescence of mCherry was excited with a He-Ne laser at 543 nm, and the emission was filtered at 580–610 nm, while Anap was excited with a 405 nm laser diode, and the emission was filtered at 415–458 nm. Scale bar = 10 µm. ( B ) Summary of current density quantification at 60 mV test pulse (ΔpH = 1) in HEK293 cells cotransfected with the hH V 1-TAG plasmid in different positions (Q191, blue circles; A197, red triangles; L201, orange squares; F150A-A197, green diamonds) and the pAnap plasmid with (+, filled markers) or without (−, empty markers) L-Anap added to incubation media. Note that the cells that were not incubated with L-Anap presented similar current density as non-transfected cells (gray marks), showing a low probability of non-specific amino acid incorporation. Current density values: non-transfected cells: 2.6±0.98 pA/pF, n=7. H V 1-Q191TAG +pAnap: 3.4±1.04 pA/pF (n=7). H V 1-Q191TAG + pAnap + L-Anap: 89.7±17.53 pA/pF (n=7). H V 1-A197TAG +pAnap: 2.1±1.2 pA/pF (n=4). H V 1-A197TAG +pAnap + L-Anap: 149.9±27.42 pA/pF (n=11). H V 1-L201TAG +pAnap: 2.6±1.47 pA/pF (n=6). H V 1-L201TAG +pAnap + L-Anap: 151.1±5.4 pA/pF (n=4). H V 1-F150A-A197TAG +pAnap: 4.6±2.4 pA/pF (n=6). H V 1-F150A-A197TAG +pAnap + L-Anap: 95.7±16.98 pA/pF (n=4). Horizontal bars and error bars represent these mean ± SEM values, and markers are individual experiments.
Article Snippet: These cells were cotransfected with 0.1–1 μg of mutant hH V 1-TAG plasmid and 0.7 μg of pAnap plasmid (a gift from Dr. Sharona Gordon, University of Washington, Seattle, WA, USA; RRID: Addgene_48696 ) using the transfection reagent JetPei (Polyplus-transfection).
Techniques: Fluorescence, Transfection, Plasmid Preparation, Microscopy, Incubation
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