Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP
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Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP. / Lundby, Alicia; Akemann, Walther; Knöpfel, Thomas.
I: European Biophysics Journal, Bind 39, Nr. 12, 06.08.2010, s. 1625-1635.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › fagfællebedømt
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T1 - Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP
AU - Lundby, Alicia
AU - Akemann, Walther
AU - Knöpfel, Thomas
PY - 2010/8/6
Y1 - 2010/8/6
N2 - 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.
AB - 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.
KW - Animals
KW - Arginine
KW - Cell Membrane
KW - Electric Conductivity
KW - Electrophysiology
KW - Fluorescence Resonance Energy Transfer
KW - Ion Channel Gating
KW - Ion Channels
KW - Kinetics
KW - Luminescent Proteins
KW - Membrane Potentials
KW - PC12 Cells
KW - Phosphoric Monoester Hydrolases
KW - Rats
KW - Temperature
U2 - 10.1007/s00249-010-0620-0
DO - 10.1007/s00249-010-0620-0
M3 - Journal article
C2 - 20686764
VL - 39
SP - 1625
EP - 1635
JO - European Biophysics Journal
JF - European Biophysics Journal
SN - 0175-7571
IS - 12
ER -
ID: 45558648