Voltage-sensitive Domain Research Articles

Design of Ultrapotent Genetically Encoded Inhibitors ofKv4.2 for Gating Neural Plasticity.

The Kv4.2 potassium channel plays established roles in neuronal excitability, while also being implicated in plasticity. Current means to study the roles of Kv4.2 are limited, motivating us to design a genetically encoded membrane tethered Heteropodatoxin-2 (MetaPoda). We find that MetaPoda is an ultrapotent and selective gating-modifier of Kv4.2. We narrow its site of contact with the channel to two adjacent residues within the voltage sensitive domain (VSD) and, with docking simulations, suggest that the toxin binds the VSD from within the membrane. We also show that MetaPoda does not require an external linker of the channel for its activity. In neurons (obtained from female and male rat neonates), MetaPoda specifically, and potently, inhibits all Kv4 currents, leaving all other A-type currents unaffected. Inhibition of Kv4 in hippocampal neurons does not promote excessive excitability, as is expected from a simple potassium channel blocker. We do find that MetaPoda's prolonged expression (1 week) increases expression levels of the immediate early gene cFos and prevents potentiation. These findings argue for a major role of Kv4.2 in facilitating plasticity of hippocampal neurons. Lastly, we show that our engineering strategy is suitable for the swift engineering of another potent Kv4.2-selective membrane-tethered toxin, Phrixotoxin-1, denoted MetaPhix. Together, we provide two uniquely potent genetic tools to study Kv4.2 in neuronal excitability and plasticity.

Structure-Activity Relationships of Alanine Scan Mutants αO-Conotoxins GeXIVA[1,2] and GeXIVA[1,4

αO-Conotoxin GeXIVA is a selective α9α10 nicotinic acetylcholine receptor (nAChR) inhibitor displaying two disulfide bonds that can form three isomers. The bead (GeXIVA[1,2]) and ribbon (GeXIVA[1,4]) isomers possess the highest activity on rat and human α9α10 nAChRs. However, the molecular mechanism by which they inhibit the α9α10 nAChR is unknown. Here, an alanine scan of GeXIVA was used to elucidate key interactions between the peptides and the α9α10 nAChR. The majority of GeXIVA[1,2] analogues preserved affinity at α9α10 nAChR, but [R17A]GeXIVA[1,2] enhanced selectivity on the α9α10 nAChR. The I23A replacement of GeXIVA[1,4] increased activity at both rat and human α9α10 nAChRs by 10-fold. Surprisingly, these results do not support the molecular model of an interaction in the orthosteric binding site proposed previously, but rather may involve allosteric coupling with the voltage-sensitive domain of the α9α10 nAChR. These results could help to guide further development of GeXIVA analogues as analgesics.

In Vivo Two-Photon Voltage Imaging with Sulfonated Rhodamine Dyes.

Optical methods that rely on fluorescence for mapping changes in neuronal membrane potential in the brains of awake animals provide a powerful way to interrogate the activity of neurons that underlie neural computations ranging from sensation and perception to learning and memory. To achieve this goal, fluorescent indicators should be bright, highly sensitive to small changes in membrane potential, nontoxic, and excitable with infrared light. We report a new class of fluorescent, voltage-sensitive dyes: sulfonated rhodamine voltage reporters (sRhoVR), synthetic fluorophores with high voltage sensitivity, excellent two-photon performance, and compatibility in intact mouse brains. sRhoVR dyes are based on a tetramethyl rhodamine fluorophore coupled to a phenylenevinylene molecular wire/diethyl aniline voltage-sensitive domain. When applied to cells, sRhoVR dyes localize to the plasma membrane and respond to membrane depolarization with a fluorescence increase. The best of the new dyes, sRhoVR 1, displays a 44% ΔF/F increase in fluorescence per 100 mV change, emits at 570 nm, and possesses excellent two-photon absorption of approximately 200 GM at 840 nm. sRhoVR 1 can detect action potentials in cultured rat hippocampal neurons under both single- and two-photon illumination with sufficient speed and sensitivity to report on action potentials in single trials, without perturbing underlying physiology or membrane properties. The combination of speed, sensitivity, and brightness under two-photon illumination makes sRhoVR 1 a promising candidate for in vivo imaging in intact brains. We show sRhoVR powerfully complements electrode-based modes of neuronal activity recording in the mouse brain by recording neuronal transmembrane potentials from the neuropil of layer 2/3 of the mouse barrel cortex in concert with extracellularly recorded local field potentials (LFPs). sRhoVR imaging reveals robust depolarization in response to whisker stimulation; concurrent electrode recordings reveal negative deflections in the LFP recording, consistent with the canonical thalamocortical response. Importantly, sRhoVR 1 can be applied in mice with chronic optical windows, presaging its utility in dissecting and resolving voltage dynamics using two-photon functional imaging in awake, behaving animals.

Excitation wavelength optimization improves photostability of ASAP-family GEVIs

Recent interest in high-throughput recording of neuronal activity has motivated rapid improvements in genetically encoded calcium or voltage indicators (GECIs or GEVIs) for all-optical electrophysiology. Among these probes, the ASAPs, a series of voltage indicators based on a variant of circularly permuted green fluorescent protein (cpGFP) and a conjugated voltage sensitive domain (VSD), are capable of detecting both action potentials and subthreshold neuronal activities. Here we show that the ASAPs, when excited by blue light, undergo reversible photobleaching. We find that this fluorescence loss induced by excitation with 470-nm light can be substantially reversed by low-intensity 405-nm light. We demonstrate that 405-nm and 470-nm co-illumination significantly improved brightness and thereby signal-to-noise ratios during voltage imaging compared to 470-nm illumination alone. Illumination with a single wavelength of 440-nm light also produced similar improvements. We hypothesize that reversible photobleaching is related to cis-trans isomerization and protonation of the GFP chromophore of ASAP proteins. Amino acids that influence chromophore isomerization are potential targets of point mutations for future improvements.

Observing the Movement of Heterogeneous Voltage Sensing Domains via Intermolecular FRET

Fusion of a voltage sensitive domain (VSD) to the fluorescent protein (FP), Super Ecliptic pHluorin A227D, allows for the optical measurement of changes in membrane potential. However, the mechanism coupling fluorescence changes to changes in membrane potential is not fully understood. Primary studies of this mechanism suggest that dimerization via the cytoplasmic fluorescent protein domain is required. To further explore the dimerization effect of the genetically encoded voltage indicator (GEVI) via intermolecular Förster Resonance Energy Transfer (FRET), novel constructs fusing FRET donors or FRET acceptors to the VSD were constructed and co-transfected into HEK-293 cells. CFP-YFP FRET pairs showed a higher resting FRET than GFP-RFP FRET pairs presumably due to the dimerization of the CFP-YFP pairs. Robust voltage-dependent FRET signals were observed which suggested the movement of S4 transmembrane segment upon depolarization of the plasma membrane improved the FRET efficiency between the chromophore dipoles of VSDs tuned to different potentials. For instance, a FRET donor attached to a VSD tuned to extreme positive potentials can be paired with a FRET acceptor coupled to a VSD that responds to physiologically relevant potentials. This combination results in a GEVI that will only move one S4 transmembrane segment or both S4 transmembrane segments depending on the strength of the depolarization of the plasma membrane. Research reported in this publication was supported by the National Institute Of Neurological Disorders And Stroke of the National Institutes of Health under Award Number U01NS099691.

Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor.

Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).

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.

Abstract 15933: Scn5a Gating Pore Current Causes Cardiac Arrhythmias Associated With Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is a structural heart disease that causes dilatation of cardiac chambers, impairs cardiac contractility and systolic function leading to heart failure. SCN5A gene encodes the cardiac sodium channel (Nav1.5) in which mutations have been identified in patients with arrhythmic disorders associated with DCM. However, it is not understood how a dysfunction in SCN5A may cause very atypical cardiac arrhythmias associated with dilatation remodeling of the heart. We generated control and patient specific human induced pluripotent cells (hiPS) carrying the Nav1.5/R219H mutation. hiPS were differentiated to obtain cardiomyocytes derived from hiPS (hiPS-CM). Molecular dynamic simulations experiments revealed that the R219H mutation opens up a pathway directly through the voltage sensitive domain (VSD) of Nav1.5 channels. We hypothesized that such a proton leak through the VSD caused by the R219H mutation may be responsible for the clinical phenotypes observed in our index patient. We unraveled the underlying mechanism responsible for the association of SCN5A and DCM. Indeed, the data shows the presence of a gating pore or omega current that is responsible of the observed abnormal ionic homeostasis, absent in control hiPS-CM. Action potential recordings showed abnormal electrical activities in both atrial and ventricular hiPS-CM. Myocytes stemmed from the index patient exhibited dilatation. Single cardiomyocytes contractility probed using atomic force microscopy revealed a higher frequency of contractility with reduced force compared to control myocytes. This could be attributed to the observed contractile protein dysfunction uncovered using immunofluorescence. Overall, the results support the hypothesis that the Nav1.5/R219H mutation creates a proton leak. This leak then causes electrical, structural and contractile disturbances that may explain the related cardiac arrhythmias associated with myocytes dilatation. We concluded that gating pore current, a novel cardiac channelopathy is directly related to the association of SCN5A and DCM. We suggest that this aberrant leak current should be investigated to characterize similar SCN5A mutations with similar clinical phenotypes.

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.

Molecular biology and biophysical properties of ion channel gating pores.

The voltage sensitive domain (VSD) is a pivotal structure of voltage-gated ion channels (VGICs) and plays an essential role in the generation of electrochemical signals by neurons, striated muscle cells, and endocrine cells. The VSD is not unique to VGICs. Recent studies have shown that a VSD regulates a phosphatase. Similarly, Hv1, a voltage-sensitive protein that lacks an apparent pore domain, is a self-contained voltage sensor that operates as an H⁺ channel. VSDs are formed by four transmembrane helices (S1-S4). The S4 helix is positively charged due to the presence of arginine and lysine residues. It is surrounded by two water crevices that extend into the membrane from both the extracellular and intracellular milieus. A hydrophobic septum disrupts communication between these water crevices thus preventing the permeation of ions. The septum is maintained by interactions between the charged residues of the S4 segment and the gating charge transfer center. Mutating the charged residue of the S4 segment allows the water crevices to communicate and generate gating pore or omega pore. Gating pore currents have been reported to underlie several neuronal and striated muscle channelopathies. Depending on which charged residue on the S4 segment is mutated, gating pores are permeant either at depolarized or hyperpolarized voltages. Gating pores are cation selective and seem to converge toward Eisenmann's first or second selectivity sequences. Most gating pores are blocked by guanidine derivatives as well as trivalent and quadrivalent cations. Gating pores can be used to study the movement of the voltage sensor and could serve as targets for novel small therapeutic molecules.

Towards a complete structure of the hERG channel

A lot of attention has been drawn to the voltage gated potassium channel Kv11.1 during the last decades. In the past, both, ligand and structure based methods intended to predict if a small molecule could cause fatal heart arrhythmias, “torsades de pointe” and sudden death. However, despite the wide interest for hERG, still no experimental 3D structure is available and therefore homology modelling of parts of the channel (generally only the pore domain) is currently used to gain structural insights [1]. Here a novel structural model of hERG is presented encompassing the full transmembrane segment of hERG, including the conduction pore and the voltage sensitive domain. Furthermore, cytoplasmic domains like the cyclic nucleotide binding domain and the PAS domain have been positioned in the overall structure. Subsequent molecular dynamics simulations allow gaining novel structural and dynamic insights into hERG functioning and perturbation.

Calcium Transients in Muscle Fibers Expressing Voltage-Sensitive Phosphoinositide Phosphatases

Phosphoinositides play a role in a variety of cellular signaling processes; PtdIns(4,5)P2 regulates the function of several types of ion channels in the plasma membrane and is the source of important second messengers. We tested the possible role of changes in the PtdIns(4,5)P2 level in E-C coupling and Ca2+ homeostasis in mouse muscle fibers under voltage-clamp conditions. Measurements of indo-1 Ca2+ transients in fibers injected with PtdIns(4,5)P2 revealed no significant changes in the voltage-dependence and maximum value for peak Ca2+ release. We then studied the consequences of the activation of a voltage-sensitive PtdIns-phosphatase (VSP). Expression of either Ci-VSP or Dr-VSP was achieved by in vivo electroporation. Confocal images of N-terminally EGFP-tagged Dr-VSP revealed a double-banded pattern of expression consistent with the triadic region and membrane current measurements from a depolarized holding potential showed the presence of a charge movement component consistent with the voltage-sensitive domain active in the t-tubule membrane. Rhod-2 Ca2+ transients generated by single depolarizing pulses within the voltage-dependent range of activation of Ca2+ release appeared unaffected by the presence of either VSP. In order to strongly activate the VSPs, fibers were depolarized by 10 successive 200 ms-long pulses from −80 mV to +80 mV; Ca2+ transients were compared to the ones elicited by an analogous series of pulses to +10 mV: on average, in control and Ci-VSP-positive fibers, the value for peak Ca2+ transient in response to the 10th pulse to +80 mV was 94 ± 7 % (n=9) and 72 ± 6 % (n=7) the value in response to the corresponding pulse to +10 mV, respectively. The significant depression in Ci-VSP expressing fibers may be indicative that depletion of t-tubule PtdIns(4,5)P2 can affect voltage-activated Ca2+ release. The work was supported by AFM, CNRS and Universite Claude Bernard.

Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons.

Although unnatural amino acids (Uaas) have been genetically encoded in bacterial, fungal, and mammalian cells using orthogonal transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs, applications of this method to a wider range of specialized cell types, such as stem cells, still face challenges. While relatively straightforward in stem cells, transient expression lacks sufficient temporal resolution to afford reasonable levels of Uaa incorporation and to allow for the study of the longer term differentiation process of stem cells. Moreover, Uaa incorporation may perturb differentiation. Here, we describe a lentiviral-based gene delivery method to stably incorporate Uaas into proteins expressed in neural stem cells, specifically HCN-A94 cells. The transduced cells differentiated into neural progenies in the same manner as the wild-type cells. By genetically incorporating a fluorescent Uaa into a voltage-dependent membrane lipid phosphatase, we show that this Uaa optically reports the conformational change of the voltage-sensitive domain in response to membrane depolarization. The method described here should be generally applicable to other stem cells and membrane proteins.

A New Approach to the Structural Investigation of the Voltage-Sensitive Domain of Voltage-Gated Cation Channels as a Function of the Transmembrane Voltage

Two fundamentally different approaches, self-assembly from solution and directed-assembly at the water-gas interface, are effective in the vectorial immobilization of the expressed voltage-sensitive domain of the KvAP channel on a suitably alkylated surface of silicon. The solvation of the immobilized protein was subsequently exchanged to form a phospholipid bilayer by incubating in a phospholipid-detergent solution in the presence of BioBeads. The formation of the protein monolayer and the vectorial-orientation of the protein molecules therein were investigated via interferometric X-ray reflectivity. The electron density profile of the tethered protein monolayer is consistent with the profile computed from the crystal structure, irrespective of the preparation procedure. Formation of lipid bilayer will require confirmation via neutron reflectivity using deuterated phospholipid. This approach enables an investigation of the structure of the VSD itself, as well as within the intact Kv-channel, as a function of the applied transmembrane voltage via a number of time-resolved techniques.

Absorption and emission spectroscopic characterisation of the LOV2-His domain of phot from Chlamydomonas reinhardtii

The absorption and emission behaviour of flavin mononucleotide (FMN) in the wild-type light, oxygen and voltage sensitive domain LOV2 of the photoreceptor phot from the green alga Chlamydomonas reinhardtii is studied. Actually a LOV2-His protein (LOV2 domain bound at N-terminal to 15 His aminoacids via a Gly aminoacid) expressed in an Escherichia coli strain is investigated. For fresh samples stored in the dark an initial fluorescence quantum yield of ϕ F = 0.12 ± 0.01 and an effective fluorescence lifetime of τ F = 2.4 ± 0.1 ns are determined. Blue-light photo-excitation generates an intermediate photoproduct (flavin-C(4a)-cysteinyl adduct with absorption peak at 390 nm) resulting in an intensity-dependent fluorescence quenching. In the aqueous solutions at pH 8 approximately 3.8% of the FMN molecules are not bound to the protein binding pocket, whereas 96.2% are non-covalently bound. Even at high-intensity light excitation at 428 nm a fraction of about 7% of the non-covalently bound FMN remains non-converted to an FMN-Cys adduct because of photo-induced back-relaxation of the adduct to non-covalently bound FMN. Two holo-LOV2-His conformations with different adduct recovery time constants are revealed by spectrally and temporally resolved fluorescence and absorption measurements: A fraction of about 48% forms FMN-Cys adducts with a fast recovery time constant of τ Ad,f = 19 ± 2 s in the dark, and the rest forms adducts with a slow recovery time constant of τ F,s = 5.5 ± 1 min. Prolonged blue light irradiation of the flavin-C(4a)-cysteinyl adducts reduces their ability to recover back in the dark to non-covalently bound FMN (photo-induced permanent adduct formation). Numerical simulations of the intensity-dependent absorption depletion reveals a quantum yield of intermediate photo-adduct formation of ϕ Ad = 0.9 ± 0.1. Simulation of the adduct absorption dynamics gives a quantum yield of photo-induced adduct back-relaxation of ϕ Ad,b = 0.15 ± 0.01 and a quantum yield of photo-induced permanent adduct formation of ϕ Ad,p = (2.6 ± 0.5) × 10 −4 .