Abstract
We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (τ1-on ~10 ms, τ2-on ~ 50 ms) are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (τ) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the Ciona-based ArcLight (~35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals.
Highlights
Genetically-encoded fluorescence protein (FP) voltage probes convert voltage changes across biological membranes into optical signals [1]
Unlike Ciona-based probes the “on” and “off” kinetics of chicken ArcLight-Q175 were well fit with single exponential curves; there was no improvement in fit with a double exponential
The action potential fractional changes, ΔF/F, with chicken ArcLight were the same as those found with the Ciona-based ArcLight [14], even though the changes in response to a 300 ms depolarizing step of 100 mV was ~4 times smaller for the chicken ArcLight (Figure 1A and B)
Summary
Genetically-encoded fluorescence protein (FP) voltage probes convert voltage changes across biological membranes into optical signals [1]. In principle, these probes allow for electrode-free electrophysiology of genetically-targeted cell types. Several design concepts have been used in engineering FP-based voltage probes. These include conjugating the voltage-sensing domains of membrane proteins with single FPs [1,2,3], Förster resonance energy transfer (FRET) pairs [4,5,6,7], split FPs [8] or circularly permuted FPs [9,10]. The above probes have not found practical use because of one or more limitations, including small response amplitude, slow dynamics, low brightness, unwanted physiological activity and/or poor membrane localization e.g. [13]
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