Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP.
A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.
Lundby
Alicia
A
Laboratory for Neuronal Circuit Dynamics, RIKEN Brain Science Institute, Wako-City, Saitama, 351-0198, Japan alicia.lundby@cpr.ku.dk
Akemann
Walther
W
Knöpfel
Thomas
T
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004553:Electric Conductivity; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D008564:Membrane Potentials; D016716:PC12 Cells; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D013696:Temperature
D007473:Ion Channels; D008164:Luminescent Proteins; D001120:Arginine; C509671:voltage-sensor-containing phosphatase, Ciona intestinalis; D010744:Phosphoric Monoester Hydrolases