Ciona Intestinalis Voltage Sensitive Phosphatase Research Articles

Tracking the movement of discrete gating charges in a voltage-gated potassium channel.

Positively charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. We optically tracked the movement of the two most extracellular charged residues (R1 and R2) in the Shaker potassium channel voltage sensor using a fluorescent positively charged bimane derivative (qBBr) that is strongly quenched by tryptophan. By individually mutating residues to tryptophan within the putative pathway of gating charges, we observed that the charge motion during activation is a rotation and a tilted translation that differs between R1 and R2. Tryptophan-induced quenching of qBBr also indicates that a crucial residue of the hydrophobic plug is linked to the Cole-Moore shift through its interaction with R1. Finally, we show that this approach extends to additional voltage-sensing membrane proteins using the Ciona intestinalis voltage-sensitive phosphatase (CiVSP).

Biophysical Characterization of Genetically Encoded Voltage Sensor ASAP1: Dynamic Range Improvement

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1. Gating charge movement and fluorescence kinetics track closely with each other, although ASAP1 gating currents are significantly faster than those of Ciona intestinalis voltage-sensitive phosphatase. Altering the residue before the first gating charge removes a split in the ASAP1 QV curve, but preserves the accelerated kinetics that allow for the faithful tracking of action potentials in neurons.

Gating Charge Calculations by Computational Electrophysiology Simulations

Electrical cell signaling requires adjustment of ion channel, receptor, or transporter function in response to changes in membrane potential. For the majority of such membrane proteins, the molecular details of voltage sensing remain insufficiently understood. Here, we present a molecular dynamics simulation-based method to determine the underlying charge movement across the membrane—the gating charge—by measuring electrical capacitor properties of membrane-embedded proteins. We illustrate the approach by calculating the charge transfer upon membrane insertion of the HIV gp41 fusion peptide, and validate the method on two prototypical voltage-dependent proteins, the Kv1.2 K+ channel and the voltage sensor of the Ciona intestinalis voltage-sensitive phosphatase, against experimental data. We then use the gating charge analysis to study how the T1 domain modifies voltage sensing in Kv1.2 channels and to investigate the voltage dependence of the initial binding of two Na+ ions in Na+-coupled glutamate transporters. Our simulation approach quantifies various mechanisms of voltage sensing, enables direct comparison with experiments, and supports mechanistic interpretation of voltage sensitivity by fractional amino acid contributions.

A Microscopic Capacitor Model of Voltage Coupling in Membrane Proteins: Gating Charge Fluctuations in Ci-VSD

The voltage sensitivity of membrane proteins is reflected in the response of the voltage sensing domains (VSDs) to the changes in membrane potential. This response is implicated in the displacement of positively charged residues, associated with the conformational changes of VSDs. The displaced charges generate nonlinear (i.e., voltage-dependent) capacitance current called the gating current (and its corresponding gating charge), which is a key experimental quantity that characterizes voltage activation in VSMP. However, the relevant theoretical/computational approaches, aimed to correlate the structural information on VSMP to electrophysiological measurements, have been rather limited, posing a broad challenge in computer simulations of VSMP. Concomitant with the development of our coarse-graining (CG) model of voltage coupling, we apply our theoretical framework for the treatments of voltage effects in membrane proteins to modeling the VSMP activation, taking the VSDs (Ci-VSD) derived from the Ciona intestinalis voltage sensitive phosphatase (Ci-VSP) as a model system. Our CG model reproduces the observed gating charge of Ci-VSD activation in several different perspectives. In particular, a new closed-form expression of the gating charge is evaluated in both nonequilibrium and equilibrium ways, while considering the fluctuation-dissipation relation that connects a nonequilibrium measurement of the gating charge to an equilibrium measurement of charge fluctuations (i.e., the voltage-independent linear component of membrane capacitance). In turn, the expression uncovers a novel link that connects an equilibrium measurement of the voltage-independent linear capacitance to a nonequilibrium measurement of the voltage-dependent nonlinear capacitance (whose integral over voltage is equal to the gating charge). In addition, our CG model yields capacitor-like voltage dependent free energy parabolas, resulting in the free energy difference and the free energy barrier for the Ci-VSD activation at "zero" (depolarization) membrane potential. Significantly, the resultant voltage dependent energetics enables a direct evaluation of capacitance-voltage relationship (C-V curve) as well as charge-voltage relationship (Q-V curve) that is in a good agreement with the observed measurement of Ci-VSD voltage activation. Importantly, an extension of our kinetic/thermodynamic model of voltage dependent activation in VSMP allows for novel derivations of voltage-dependent rate constants, whose parameters are expressed by the intrinsic properties of VSMP. These novel closed-form expressions offer a physicochemical foundation for the semiempirical Eyring-type voltage dependent rate equations that have been the cornerstone for the phenomenological (kinetic) descriptions of gating and membrane currents in the mechanistic study of ion channels and transporters. Our extended theoretical framework developed in the present study has potential implications on the roles played by gating charge fluctuations for the spike generations in nerve cells within the framework of the Hodgkin-Huxley-type model.

Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different “Nabi1” constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz.

Development of Novel FRET-Based Fluorescent Voltage Sensor Proteins

Recently, we developed a series of FRET (Fluorescence Resonance Energy Transfer)basedvoltage sensors, Nabi,utilizing the voltage sensing domain of CiVSP (Ciona intestinalis voltage sensitive phosphatase). The Nabi series of probes contained FPs inserted at different locations in the Ciona voltage sensitive domain.Nabi2 probes with Clover (green) and mRuby2 (red) as donor and acceptor showed large signal size (9% ΔF/F for a 100 mv depolarization) and fast taus (<3 msec). In order to improve further FRET efficiency of voltage responses, we investigated the behaviors of individual FPs in the probes.

Improving Signal Dynamics of Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions

FRET (Förster Resonance Energy Transfer)-based voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair removes common sources of noise such as heart beat artifacts. We improved the performance of genetically encoded fluorescent voltage sensors by optimizing FRET interactions between donor and acceptor fluorescent proteins (FPs) flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. The donor FP, UKG, was positioned at 8 different locations downstream of the voltage-sensing domain while the acceptor FP, mKO, was placed at 6 positions upstream of the voltage-sensing domain resulting in 39 different “Nabi1” constructs. Several of these combinations resulted in large signals and fast kinetics. Nabi1 probes responded to changes in membrane potential with improved signal size (up to 11% ΔF/F for a 100 mv depolarization) and fast response time constants both for signal activation (∼2 ms) and signal decay (∼3 ms). Replacement of the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) improved expression in cultured neurons enabling the optical resolution of individual action potentials. We are analyzing a series of Nabi2 probes with different insertion sites of FPs. This effort may identify novel probes that may be useful for optical recording of neuronal activities. Supported by US NIH Grants DC005259 and NS054270 and grant WCI 2009-003 from the National Research Foundation of Korea.

Investigating the Regulatory Interaction of Linker Region of Ciona intestinalis Voltage-sensitive Phosphatase with Lipid Membrane

E-mail: ykim@kku.ac.krReceived July 15, 2014, Accepted July 31, 2014Key Words : Ci-VSP, Membrane Interaction, PTEN, Phosphoinositides, Lipid phosphataseCiona intestinalis voltage-sensitive phosphatase (Ci-VSP),which possesses the voltage-sensing domain (VSD) andphosphatase domain (PD), was discovered in sea squirt.

Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain.

Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviors. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs) has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP) prototypical design or on the voltage-dependent state transitions of microbial opsins. We recently introduced a new VSFP design in which the voltage-sensing domain (VSD) is sandwiched between a fluorescence resonance energy transfer pair of fluorescent proteins (termed VSFP-Butterflies) and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians.

Probing α-310 Transitions in a Voltage-Sensing S4 Helix

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 310 conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 310 conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 310 conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-310 helical conversions of the S4 in Ci-VSP associated with voltage activation.

Interactions of Phosphatase and Tensin Homologue (PTEN) Proteins with Phosphatidylinositol Phosphates: Insights from Molecular Dynamics Simulations of PTEN and Voltage Sensitive Phosphatase

The phosphatase and tensin homologue (PTEN) and the Ciona intestinalis voltage sensitive phosphatase (Ci-VSP) are both phosphatidylinositol phosphate (PIP) phosphatases that contain a C2 domain. PTEN is a tumor suppressor protein that acts as a phosphatase on PIP3 in mammalian cell membranes. It contains two principal domains: a phosphatase domain (PD) and a C2 domain. Despite detailed structural and functional characterization, less is known about its mechanism of interaction with PIP-containing lipid bilayers. Ci-VSP consists of an N-terminal transmembrane voltage sensor domain and a C-terminal PTEN domain, which in turn contains a PD and a C2 domain. The nature of the interaction of the PTEN domain of Ci-VSP with membranes has not been well established. We have used multiscale molecular dynamics simulations to define the interaction mechanisms of PTEN and of the Ci-VSP PTEN domains with PIP-containing lipid bilayers. Our results suggest a novel mechanism of association of the PTEN with such bilayers, in which an initial electrostatics-driven encounter of the protein and bilayer is followed by reorientation of the protein to optimize its interactions with PIP molecules in the membrane. Although a PIP3 molecule binds close to the active site of PTEN, our simulations suggest a further conformational change of the protein may be required for catalytically productive binding to occur. Ci-VSP interacted with membranes in an orientation comparable to that of PTEN but bound directly to PIP-containing membranes without a subsequent reorientation step. Again, PIP3 bound close to the active site of the Ci-VSP PD, but not in a catalytically productive manner. Interactions of Ci-VSP with the bilayer induced clustering of PIP molecules around the protein.

Nabi, a Novel FRET-Based Voltage Sensor Protein

FRET (fluorescence resonance energy transfer) has been used to develop genetically encoded biosensors. FRET-based voltage sensors can be useful to monitor change of membrane potential and neuronal activity because the ratio signal removes noise from a common source such as heart beat artifacts. Positions of fluorescent donor and acceptor in voltage sensor proteins may affect plasma membrane expression, signal intensity, and response time constant of FRET. We developed “Nabi” voltage sensors by using the voltage-sensing domain of CiVSP (Ciona intestinalis voltage sensitive phosphatase) as a backbone. We aimed at improving FRET performance of voltage sensors by optimizing interactions between fluorescent donor and acceptor. For this effort, we designed a series of constructs with insertion of two fluorescent proteins (FPs) at different locations in the CiVSP backbone. We engineered a total of 40 different combinatorial constructs to evaluate 6 different locations for one FP and 10 different locations for the other FP of FRET pairs. By examining FRET signals of these voltage sensors in response to changes in membrane potential in HEK293 cells, we identified amino acid residues of CiVSP at which insertion of FP pairs can generate efficient FRET signals. Nabi1 series of probes contained UKG and mKO (Tsutsui H et al., 2008) as FRET donor and acceptor. Nabi2 series contained Clover and mRuby2 (Lam AJ et al., 2012) as FRET pairs. Nabi voltage sensors had improved signal size (up to 15% ΔF/F) and time constant compared to the previously reported FRET based voltage probes. Nabi2 was able to respond to single action potentials in primary cultured neurons. Supported by US NIH Grants DC005259 and grant WCI 2009-003 from the National Research Foundation of Korea.

Development of a Red Genetically-Encoded Voltage Indicator and its use with Channelrhodopsin for All-Optical Electrophysiology

The need for effective genetically-encoded voltage indicators is widely recognized. Several prototypes have been made to date by fusing one or more fluorescent proteins to a voltage sensitive membrane protein (1). However, one factor that has limited the utility of these indicators is poor voltage sensitivity, making it a challenge to express the indicators in neurons and still observe detectable responses (2). In addition, none of the genetically-encoded voltage indicators reported can be combined with channelrhodopsin for optical readout of electrical activity in an all-optical electrophysiology setup. To address these limitations, we set up a directed evolution strategy to screen for both voltage indicator brightness and voltage sensitivity. First, bacterial expression enables us to screen thousands of clones in colonies of E. coli to improve the brightness of the indicator. Second, a medium throughput mammalian electric field stimulation system allows us to test for function of the voltage indicators. This screening has resulted in a family of red voltage indicators (VSDRs). They consist of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase linked to red fluorescent protein mApple. The fluorescence intensity of the VSDRs increases in response to depolarization. We show that the combination of signal size and response speed of VSDRs allows the reliable detection of spontaneous action potentials in cultured mammalian neurons in single trials with widefield fluorescent light microscopy. Critically, we also demonstrate that VSDRs faithfully reports signals from all-optical electrophysiology experiments using channelrhodopsin to depolarize mammalian cells.1. B. J. Baker et al., J. Neurosci. Methods. 2007, 161, 32-38.2. W. Akemann et al., Nat Methods. 7, 2010, 643-649.

Fluorescent Protein Voltage Probes Derived from ArcLight that Respond to Membrane Voltage Changes with Fast Kinetics

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.

Probing S4 Re-Arrangement during Gating using Optical Tools

Voltage Sensor Domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes in response to changes in the membrane potential. Voltage sensitivity relies on the transfer of gating charges located in the fourth transmembrane helix (S4) across the electric field. The gating charge transfer has been proposed to be associated with changes in the secondary structure of the S4 between alpha and 3-10 helix, yet this conformational transition has not been observed to date. Because such transitions are expected to extend the distance between the two ends of the S4, we sought to identify changes in the distance between genetically-encoded spectroscopic probes (a Lanthanide-Binding-Tag and a fluorescent mCherry) placed at the two ends of the S4 of the Ciona intestinalis Voltage-Sensitive Phosphatase (Ci-VSP) using LRET. We previously showed that the length of the S4 of Ci-VSP tagged with LBT and mCherry does not exhibit long-lived voltage-dependent changes and that it most likely exists as an alpha helix. We report here that short-lived changes of the S4 length in this construct could not be detected using time-resolved FRET, indicating the unlikeliness of transient alpha to 3-10 transitions. Since the recent x-ray structure of the NavAb, sodium channel from Arcobactor butzlen, displays its whole S4 in a 3-10 conformation, we substituted the S4 of Ci-VSP (from G216 to S236) by the S4 of NavAb (from F95 to Q115). As for Ci-VSP, our LRET results with this chimera show that the NavAb S4 in the Ci-VSP frame does not undergo long-lived alpha-3-10 transitions, suggesting that it most likely resides in an alpha conformation. Our results show that in both constructs, the voltage-sensor operation from resting to active does not require alpha to 3-10 transitions of the whole S4. Supported by GM030376 and U54GM087519.

Activation of Voltage-Sensitive Phosphatases with Pharmacological and Optogenetic Methods

Voltage-sensitivephosphatases (VSPs) are proteins that dephosphorylate phosphoinositides (PIs) upon membrane voltage dependent activation. Particularly, Ci-VSP (Ciona intestinalis Voltage-Sensitive Phosphatase) is a 5-phosphatase of PI(4,5)P2 and PI(3,4,5)P3 and has so far been studied by electrophysiological means.Here we developed more broadly applicable methods of membrane depolarization and therefore phosphatase activation. First, depolarization was achieved by coexpression of the K+ channels TASK and KCNQ and elevation of extracellular potassium concentration. Second, we utilized the capsaicin-activated TRPV1 channel, and last the photosensitive Channel Rhodopsin, ChR2, in order to activate Ci-VSP. Cultured cell lines were transfected with the different channels, Ci-VSP and sensors for PIs, e.g. the tubby and the PLCδ1-PH domain, each tagged with a fluorescent protein. Confocal microscopy was used to visualize and quantify the translocation of the PI-sensors from the membrane to the cytoplasm after the dephosphorylation of PIs. All three methods resulted in successful activation of Ci-VSP, with TRPV1 and ChR2 mediated depolarization to give the most robust results. Specifically, the ChR2 activation provides a very rapid response without the need of solution exchanges, simplifying the experimental procedures. To conclude, we suggest a series of methods that allow the manipulation of PI levels as well as the study of VSPs in living cells without electrophysiological instrumentation.This work was supported by a grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to CRH and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to DO.

Development of New Fluorescent Voltage Sensor Proteins

Our goal is to develop genetically encoded fluorescent biosensors (FP voltage sensors) and to use them for optical recording of membrane potential in excitable cells. We created a series of FRET (fluorescence resonance energy transfer) based sensors. These constructs contained the voltage sensing domain of CiVSP (Ciona intestinalis voltage sensitive phosphatase) as voltage sensor fused with two fluorescent proteins, UKG (green-emitting fluorescent protein Umi-Kinoko) and mKOK (orange-emitting fluorescent protein Kusabira orange) as donor and acceptor. We used PCR to generate a library of constructs with insertions of the green and orange fluorescent proteins into different regions in CiVSP. Insertion of FRET donor and acceptor at different locations affects plasma membrane expression, FRET signal intensity, and response time constant. We are screening the these FP voltage sensors by examining the FRET signal in response to changes in membrane potential in HEK293 cells. Several novel probes have been identified; some with relatively large signals (>7% ΔF/F) and fast taus (<3 msec). We hope this effort will lead to voltage sensor proteins useful for in vivo recording of membrane potential in neurons.Supported by US NIH Grants DC005259 and NS054270 and grant WCI 2009-003 from the National Research Foundation of Korea.

Two-Photon Imaging of Electrical Activity in Mouse Cortex using a Genetically-Encoded Voltage Indicator

Genetically-encoded voltage indicators (GEVIs) of the VSFP (voltage-sensitive fluorescent protein) family based on the Ci-VSP (Ciona intestinalis voltage-sensitive phosphatase) voltage sensor domain were demonstrated to report membrane voltage fluctuations of genetically-targeted cells in a number of in-vitro and in-vivo preparations using single photon (1P) fluorescence excitation and wide field image detection [1, 2]. However, in-vivo 1P epi-fluorescence imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon (2P) excitation imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here, we show GEVI two-photon imaging in mouse brain slices and mouse cortex in-vivo expressing VSFP Butterfly 1.2 in cortical layer2/3 pyramidal cells. We show that 2P excitation at 920-940 nm of the mCitrine/mKate2 FRET reporter in Butterfly 1.2 yields an optical voltage signal from neuron membranes in brain slices in response to field stimulation with a time course identically revealed by 1P excitation and with a 2-3 larger ΔR/R value. Two-photon imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3 of Butterfly 1.2-expressing pyramidal cells. In somatosensory barrel cortex we recorded sensory responses to single whisker deflections in anesthetized mice at 30 frames per second in full frame view. The 2P-excited voltage signal was localized within the functional map established by 1P VSFP imaging in the same preparation. Our results demonstrate the feasibility of GEVI-based functional 2P imaging of sensory-evoked electrical activity in mouse cortex.[1] Akemann W, Mutoh H, Perron A, Rossier J, Knopfel T, Nat Methods 7, 2010[2] Akemann W, Mutoh H, Perron A, Park KY, Iwamoto Y, Knopfel T, J Neurophysiol, 2012 Jul 18

Molecular mechanism for depolarization-induced modulation of Kv channel closure

Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K+ permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K+ conduction through constriction of the K+ selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history.

A fluorescent, genetically-encoded voltage probe capable of resolving action potentials.

There is a pressing need in neuroscience for genetically-encoded, fluorescent voltage probes that can be targeted to specific neurons and circuits to allow study of neural activity using fluorescent imaging. We created 90 constructs in which the voltage sensing portion (S1–S4) of Ciona intestinalis voltage sensitive phosphatase (CiVSP) was fused to circularly permuted eGFP. This led to ElectricPk, a probe that is an order of magnitude faster (taus ∼1–2 ms) than any currently published fluorescent protein-based voltage probe. ElectricPk can follow the rise and fall of neuronal action potentials with a modest decrease in fluorescence intensity (∼0.7% ΔF/F). The probe has a nearly linear fluorescence/membrane potential response to both hyperpolarizing and depolarizing steps. This is the first probe based on CiVSP that captures the rapid movements of the voltage sensor, suggesting that voltage probes designed with circularly permuted fluorescent proteins may have some advantages.