Engineering and Characterization of an Enhanced Fluorescent Protein Voltage Sensor

Background Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells. Results Here we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells. Conclusions and Significance This biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously.

[1]  C. Deutsch The Birth of a Channel , 2003, Neuron.

[2]  J. Timmermans,et al.  Domain analysis of Kv6.3, an electrically silent channel , 2005, The Journal of physiology.

[3]  C. Deutsch,et al.  Structure Acquisition of the T1 Domain of Kv1.3 during Biogenesis , 2004, Neuron.

[4]  U. Baumann,et al.  An efficient one-step site-directed and site-saturation mutagenesis protocol. , 2004, Nucleic acids research.

[5]  T. Knöpfel,et al.  Olfactory nerve stimulation-evoked mGluR1 slow potentials, oscillations, and calcium signaling in mouse olfactory bulb mitral cells. , 2006, Journal of neurophysiology.

[6]  Thomas Knöpfel,et al.  Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins. , 2003, Methods.

[7]  Yasushi Okamura,et al.  Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor , 2005, Nature.

[8]  J. M. Robinson,et al.  Coupled Tertiary Folding and Oligomerization of the T1 Domain of Kv Channels , 2005, Neuron.

[9]  E. K. Kosmidis,et al.  Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells , 2007, Journal of Neuroscience Methods.

[10]  M. Ohkura,et al.  Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein , 2005, The European journal of neuroscience.

[11]  Mark A Rizzo,et al.  An improved cyan fluorescent protein variant useful for FRET , 2004, Nature Biotechnology.

[12]  Javier Díez-García,et al.  Optical probing of neuronal circuit dynamics: genetically encoded versus classical fluorescent sensors , 2006, Trends in Neurosciences.

[13]  Vincent A Pieribone,et al.  A genetically targetable fluorescent probe of channel gating with rapid kinetics. , 2002, Biophysical journal.

[14]  R. Tsien,et al.  Reducing the Environmental Sensitivity of Yellow Fluorescent Protein , 2001, The Journal of Biological Chemistry.

[15]  T. Knöpfel,et al.  Olfactory nerve stimulation-induced calcium signaling in the mitral cell distal dendritic tuft. , 2006, Journal of neurophysiology.

[16]  Thomas Knöpfel,et al.  In vivo calcium imaging from genetically specified target cells in mouse cerebellum , 2007, NeuroImage.

[17]  T. Knöpfel,et al.  Design and characterization of a DNA‐encoded, voltage‐sensitive fluorescent protein , 2001, The European journal of neuroscience.

[18]  Ehud Y Isacoff,et al.  A Genetically Encoded Optical Probe of Membrane Voltage , 1997, Neuron.

[19]  H. Mutoh,et al.  Long-Term Depression at Olfactory Nerve Synapses , 2005, The Journal of Neuroscience.