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Monthly Archives: October 2012
There has been almost two decades of research in the field of genetically-encoded voltage indicators (GEVIs). Early probes carried channel-based voltage sensors (e.g. derived from potassium and sodium channels) but showed poor membrane localization. Membrane localization of GEVIs was greatly improved by replacing channel-based sensors with the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSD). Despite this major improvement, the goal to record single action potentials and subthreshold electrical events in mammalian neurons with adequate temporal and spatial resolution still presents a challenge. The latest step toward this goal was taken by the Pieribone lab which came up with two Ci-VSD-based monochromatic GEVIs.
The first one, called ArcLight and published in Neuron, was obtained by combining the Ci-VSD and a super ecliptic pHluorin that carries a critical point mutation (A227D). Based on ArcLight, five probes were engineered with super ecliptic pHluorin A227D relocated closer to the S4 domain of the Ci-VSD, after amino acids Q239, M240, K241, A242, or S243. Out of these 5 variants, ArcLight A242 emerged as the best, exhibiting -35% change in ΔF/F in response to 100mV depolarizing steps in HEK293 cells and up to -5% change in ΔF/F for single action potentials in neurons. ArcLight A242 … Continue reading
In a recent paper published in PLoS One, Ohkura et al. introduce R-CaMP1.07, an improved variant of a recently developed red fluorescent Ca2+ indicator protein called R-GECO1. While the sensitivity of R-CaMP1.07 is similar to that of R-GECO1 (Kd for Ca2+ is around 200 nM), R-CaMP1.07 shows 1.5–2-fold greater signals than R-GECO1 due to its enhanced dynamic range (Fmax/Fmin is near 30). The greatest advantage of R-CaMP1.07 is that its excitation wavelength ranges from 500 to 580 nm, which rarely overlaps with the photo-stimulation range of channelrhodopsin-2 (ChR2). Taking this merit of R-CaMP1.07, the authors demonstrate an application example of this indicator in hippocampal pyramidal neurons expressing ChR2: the successful detection of Ca2+ signals in response to action potentials evoked by the photo-stimulation of ChR2. Needless to say, this red fluorescent R-CaMP1.07 can be used for Ca2+ imaging of cells expressing blue, cyan or green fluorescent proteins.