Designs and sensing mechanisms of genetically encoded fluorescent voltage indicators
Introduction
Neurons can encode and transmit information by regulating the electrical field (voltage) across their plasma membrane. Voltage dynamics track both neural inputs and outputs: voltage can be modulated by neurotransmitters released by upstream neurons; in turn, voltage controls whether neurotransmitters will be discharged onto downstream neurons. The central role of voltage as a carrier of neural information thus motivates the development of powerful tools to image voltage transients within individual cells and across large populations. Although voltage is most commonly measured with electrodes, recent engineering efforts have substantially improved the ability of protein-based fluorescent sensors to image fast electrical activity in neural tissue. Optical detection of voltage signals with protein-based sensors presents unique opportunities over monitoring voltage with electrodes. First, voltage sensors can image subcellular regions such as dendritic spines or axonal termini that are typically too small to be accessible by standard electrodes. Second, they could enable monitoring of voltage dynamics over thousands or millions of cells. By contrast, electrode arrays have lower spatial resolution given their limited number and density of electrodes. Third, protein-based voltage sensors can restrict visualization to genetically defined cell types of interest, rather than selectively monitoring electrical activity in neurons that happen to be near the recording electrode.
Yet, imaging voltage dynamics with protein sensors also poses several challenges. First, to report on membrane voltage transients, the indicator must be in the plasma membrane, or be tightly coupled to a sensor element in the membrane. As a result, the sensor must hijack the cellular plasma membrane trafficking machinery and avoid accumulating in intermediate organelles such as the endoplasmic reticulum or the Golgi apparatus. Second, voltage transients are often rapid; for example, action potentials last less than a few milliseconds, while neurotransmitter-induced depolarizations typically have time courses of less than tens of milliseconds. Sensors must therefore have sufficiently rapid kinetics and be very sensitive to detect these voltage transients. Finally, voltage indicators must be sufficiently bright and photostable to report voltage dynamics with the required spatiotemporal resolution over the course of an entire experiment. We review different strategies for developing protein-based probes that begin to address these challenges. We focus on voltage indicators that are fully genetically encoded; voltage-sensitive dyes, and hybrid sensors combining a protein component and a synthetic dye, are reviewed elsewhere [1, 2].
Section snippets
Sensors exploiting voltage-induced conformational changes in natural voltage sensing domains
In one family of genetically encoded voltage indicators, integral membrane voltage sensing domains (VSDs) are fused to fluorescent proteins from jellyfish or coral. In their native proteins, VSDs either control the opening and closing of ion channels, or the activity of a phosphatase. In all cases, VSDs are composed of four transmembrane helices, with the fourth (S4) containing several positively-charged residues — arginines, or a mixture of arginines and lysines. These residues are sensitive to
Sensors exploiting voltage-sensitive photophysical properties of microbial rhodopsins
A surprising development came in 2011 with the demonstration that microbial rhodopsins can be repurposed as voltage indicators (Figure 1, right column). Rhodopsins are light-sensitive membrane proteins composed of 7 transmembrane α-helices; they normally function as light-driven ion pumps, channels or light sensors [38]. They have met broad adoption over the last decade for optical control of neural activity, a technology called optogenetics [39]. The light sensitivity of these proteins comes
Future directions
Given the role of voltage for encoding, transforming and propagating information in animal brains, high performance genetically encoded voltage indicators are on the wish lists of many neuroscientists [50]. Interestingly, protein engineers have leveraged not one, but two distinct voltage sensing mechanisms for developing protein-based voltage sensors: voltage-sensitive conformational states, and voltage-sensitive photophysical states. These sensors are beginning to be used to capture neural
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by National Science Foundation grant 1134416 (F.S.-P., M.Z.L.), DARPA grant W911NF-14-1-0013 (M.Z.L.) and NIH Brain Initiative Grant 1U01NS090600-01 (M.Z.L.). M.Z.L. receives funding from the Rita Allen Foundation and the Burroughs Wellcome Fund.
References (61)
- et al.
Voltage-sensitive dye imaging: technique review and models
J Physiol Paris
(2010) - et al.
A genetically targetable fluorescent probe of channel gating with rapid kinetics
Biophys J
(2002) - et al.
Tuning FlaSh: redesign of the dynamics, voltage range, and color of the genetically encoded optical sensor of membrane potential
Biophys J
(2002) - et al.
Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein
Eur J Neurosci
(2001) - et al.
Engineering and characterization of an enhanced fluorescent protein voltage sensor
PLoS One
(2007) - et al.
Improved detection of electrical activity with a voltage probe based on a voltage-sensing phosphatase
J Physiol
(2013) - et al.
S4-based voltage sensors have three major conformations
Proc Natl Acad Sci U S A
(2008) - et al.
Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe
Neuron
(2012) - et al.
Temperature dependence of intrinsic membrane properties and synaptic potentials in hippocampal CA1 neurons in vitro
J Neurosci
(1985) - et al.
Fluorescent protein voltage probes derived from ArcLight that respond to membrane voltage changes with fast kinetics
PLoS One
(2013)
Genetically engineered fluorescent voltage reporters
ACS Chem Neurosci
Mechanism of voltage gating in potassium channels
Science
Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain
Nat Struct Mol Biol
Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel
Cell
Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells
J Neurosci Methods
Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor
Nature
Subunit organization and functional transitions in Ci-VSP
Nat Struct Mol Biol
Sensing charges of the Ciona intestinalis voltage-sensing phosphatase
J Gen Physiol
Genetically encoded fluorescent voltage sensors using the voltage-sensing domain of Nematostella and Danio phosphatases exhibit fast kinetics
J Neurosci Methods
Improving FRET dynamic range with bright green and red fluorescent proteins
Nat Methods
Improving membrane voltage measurements using FRET with new fluorescent proteins
Nat Methods
Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein
J Neurophysiol
Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins
Nat Methods
Second and third generation voltage-sensitive fluorescent proteins for monitoring membrane potential
Front Mol Neurosci
Transfer of Kv3.1 voltage sensor features to the isolated Ci-VSP voltage-sensing domain
Biophys J
Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain
Front Mol Neurosci
Charge movement of a voltage-sensitive fluorescent protein
Biophys J
Red-shifted voltage-sensitive fluorescent proteins
Chem Biol
Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements
PLoS One
Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight
PLoS One
Cited by (0)
- 3
CCSR 2105, 269 Campus Dr, Stanford, USA.