lundby citrine

The authors have declared that no competing interests exist. Currents related to movement of the gating particles of the sodium channels. For each cell 3—7 current traces and 2—4 fluorescence traces were recorded and averaged. VSDs are shown in blue. Published online Jun

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A genetically encoded optical probe of membrane voltage. Author information Article notes Copyright and License information Disclaimer. To test this hypothesis we photo-bleached the YFP chromophore subsequently to the measurements illustrated in Figure 2a. Data was acquired at 50 kHz and filtered by a 5 kHz low pass Bessel filter. Removal of five amino acids originating from engineered restriction sites in VSFP2.

Optical probing of neuronal circuit dynamics: Reverse engineering of the previously reported Ccitrine. Note broken time scale. Open in a separate window. The conformational transitions of the protein upon a voltage change are initiated by displacement of charged amino acids of the VSD, which gives rise to a transient current analogous to lubdby gating currents known from ion channels [9].

Subunit organization and functional transitions in Ci-VSP. The lower panel shows the fluorescence signals citrinf in the yellow and cyan channels. Received Apr 13; Accepted May Amino acids are specified for regions containing modifications between different VSFP constructs.


A coverslip with PC12 cells was placed in a recording chamber mounted on the stage of an inverted microscope Eclipse TE, Nikonand voltage-clamp recordings in the whole-cell configuration were performed using an Axopatch B amplifier Axon Instruments. The unusual large citrinf in the kinetics observed in mammalian membranes as compared to Xenopus oocytes [11] may result from an expectedly strong dependence on the lipid environment of an isolated VSD [12].

The amplitude of fluorescence transients was measured at end of the test pulse lunbdy expressed as percentage of baseline fluorescence. Data luhdby Fluorescence signals and gating currents were analyzed using Clampfit 9.

Steady-state relations recorded from single cells were normalized and fit with two-state Boltzmann distributions. For each cell 3—7 current traces and 2—4 fluorescence traces were recorded and averaged. Cells use voltage cirtine containing proteins to control the membrane potential and for signaling processes.

For the data presented in Fig. All data points are represented by mean values and error bars indicate standard error of the mean.

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All constructs were verified by DNA sequencing. The voltage-dependency of the fluorescence read-out closely resembles the activation curve for charge displacement Figure 1b—d indicating that the fluorescence signal reports the voltage-dependent conformation of the VSD. Similarly, the capacitive transient needed to charge the cell membrane was estimated by a single-exponential fit to the current recorded at the hyperpolarizing voltage step, and a linear voltage dependent exponential was subtracted from each current trace recorded.


The fluorescence output of VSFP3. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

To our knowledge Lundy. Fluorescence traces were elicited from a holding potential of —70 mV by a sequence of 20 mV activation steps lasting ms to a final potential of mV with 10 s interpulse intervals. Recordings from transiently expressing PC12 cells were performed 48—72 h post transfection.

Genetically encodable fluorescent voltage probes hold great promise in neuroscience, where ulndby that allow recordings of electrical activity from multiple identified neurons simultaneously are needed [7][8].

John Lewis & Partners Lina Throw, Citrine

Tranfections were done one day after plating using Lipofectamine Invitrogen reagent according to the manufacturer’s instructions. The main cutrine of these VSFP variants is that their fluorescence read-out is slower than required for measurement of fast electrical signals in neurons.

Fluorescence measurements Fluorescence was induced by light nm from a computer controlled monochromator Polychrome IV, T.