Voltage-sensitive Phosphatase Research Articles

Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence.

Voltage-sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization.

Controlling the Activity of a Phosphatase and Tensin Homolog (PTEN) by Membrane Potential

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor and catalytic domains. Our findings reveal that the main domains of VSPs and related phosphoinositide phosphatases are intrinsically modular and define structural requirements for coupling of enzymatic activity to a voltage sensor domain. A key feature of this prototype of novel engineered voltage-sensitive enzymes, termed Ci-VSPTEN, is the novel ability to switch enzymatic activity of PTEN rapidly and reversibly. We demonstrate that experimental control of Ci-VSPTEN can be obtained either by electrophysiological techniques or more general techniques, using potassium-induced depolarization of intact cells. Thus, Ci-VSPTEN provides a novel approach for studying the complex mechanism of activation, cellular control, and pharmacology of this important tumor suppressor. Moreover, by inducing temporally precise perturbation of phosphoinositide concentrations, Ci-VSPTEN will be useful for probing the role and specificity of these messengers in many cellular processes and to analyze the timing of phosphoinositide signaling.

A ci-VSP/Tpip Chimera Exhibiting Voltage-Dependent Pi(4,5)P2/PIP3-5’-Phosphatase Activity

Phosphoinositide (PI) concentrations in cell membranes play an important role in many cellular processes. PI phosphatases are crucial for the regulation of these concentrations. Among these phosphatases are the voltage-sensitive phosphatases (VSPs) like the prototypical Ci-VSP, which provide a new paradigm for the control of enzymatic activity. Recently, we showed that voltage sensitivity can be conferred to the cytoplasmic phosphatase PTEN: by fusing the voltage sensor domain (VSD) of Ci-VSP to the catalytic domain (CD) of PTEN, we generated an engineered chimeric VSP, Ci-VSPTEN. Here we extend this work and report on a chimera created by fusing Ci-VSP's VSD to the CD of the putative PI-phosphatase TPIPα. We demonstrate that this Ci-VSP/TPIP chimera exhibits voltage dependent enzymatic activity. The substrate and position specificity of Ci-VSP/TPIP is analyzed using genetically encoded PI-specific fluorescence labeled probes, the membrane binding of which is assessed using total internal reflection fluorescence (TIRF) microscopy. Control over the membrane voltage is achieved by whole-cell patch clamping. We find that upon depolarization, the membrane binding of the PIP3 specific probe, Btk-PH, and the PI(4,5)P2 specific probe, PLCδ1-PH, decreases, when co-expressed with Ci-VSP/TPIP. In contrast, membrane binding of the PI(3,4)P2 specific TAPP1-PH and the PI(4)P specific OSBP-PH probes increases upon depolarization. These findings identify the Ci-VSP/TPIP chimera as a voltage-sensitive PI(4,5)P2/PIP3-5’-phosphatase. We conclude that TPIPα is a 5’-phosphatase in-vivo, in contrast to PIP3-3’-phosphatase activity in-vitro reported previously (Walker et al., Biochem. J. (2001) 360, 277 −283). These data will help to further the understanding of the mechanism by which voltage control is exerted in VSPs. Additionally, our experiments demonstrate the usefulness of engineered VSPs as a novel tool for the analysis of PI-phosphatases in-vivo, an obligatory complement to in-vitro characterization. Supported by Deutsche Forschungsgemeinschaft (SFB593 TPA12 to D.O.)

Dynamic Modulation of Voltage-Dependent Kv7.2-7.3 Channel Opening by Voltage-Sensor Relaxation

S4-type voltage-sensor (VS) domains are common to voltage-gated ion channels (VGCs) and voltage-sensitive phosphatases (VSPs). In ciVSP, depolarization to +80 mV initially causes VS activation, generating rapid sensing charge (Q) movement. Sustained depolarization subsequently elicits a voltage-independent conformational rearrangement termed relaxation. Because the relaxed state is thermodynamically more stable than the activated state, relaxation causes a negative shift in the Q-V relation. Relaxation may be an intrinsic property of the VS domain because it is observed in the absence of the ciVSP catalytic domain. Since the VS and pore domains are coupled in VGCs, we wondered whether VS relaxation might alter voltage-dependent opening of an ion channel conductance (G). By analogy to ciVSP, we hypothesized that relaxation would shift the G-V relation toward negative potentials. In order to test this hypothesis, we used two-electrode voltage clamp to measure K+ currents in Xenopus oocytes expressing Kv7.2 and Kv7.3 mRNAs. As expected, prolonged depolarizations to +80 mV caused the steady-state G-V relation to shift toward negative potentials, indicating that VS relaxation alters the voltage-dependence of channel opening in heteromeric Kv7.2-7.3 channels. Furthermore, the magnitude of the shift in the midpoint of the G-V relation was found to depend on the duration of the +80 mV prepulse. Similar to ciVSP, the progressive shift in the G-V relation may be interpreted as an index of relaxation and used to measure the rates of entry into and recovery from the relaxed state. Our data therefore imply that the voltage dependence of VGC opening is likely to be dynamically modulated by conformational transitions that are intrinsic to the VS domain in a voltage-gated channel complex. VS relaxation may represent a widespread mechanism for regulating VGC function altering the relative positions of the Q-V and G-V relations.

Voltage-Sensor Relaxation in Hv1 Proton Channels

Hv1 is a voltage-gated and proton-selective channel composed of four helical transmembrane segments (S1-S4) that adopt a voltage-sensor (VS) domain fold that is common to voltage-dependent ion channels and voltage-sensitive phosphatases (VSPs). Unlike other voltage-gated channels, the Hv1 VS also functions as a sensor for the transmembrane pH gradient and contains a water-wire for H+ conduction. However, despite exhibiting biophysical properties that are specific to its proton channel function, the Hv1 VS appears to share significant structural and functional homology with other VS domains. VS relaxation is manifested by a negative shift in the voltage dependence of sensing charge (Q) movement in response to prolonged depolarization. Previous studies on ciVSP led to the hypothesis that depolarization-induced movement of S4 leads to a rapid distortion of the VS structure. A compensatory voltage-independent conformational transition termed relaxation allows the VS to adopt a more thermodynamically stable state, and is revealed by a negative shift in the Q-V relation. We postulated that because relaxation is likely to represent an intrinsic feature of VS domain function, Hv1 VS would also enter a relaxed state after sustained depolarization. In order to test this hypothesis, we elicited Hv1 opening by applying voltage steps to positive potentials for varying durations and then measured the voltage dependence of channel closure (V0.5). As predicted, depolarizing pre-pulses caused V0.5 to shift toward negative potentials. The magnitude of the shift in V0.5 was observed to depend on the duration of the prepulse over the range of 0.1-5.0 s. These observations demonstrate that Hv1 manifests VS relaxation and suggest that relaxation occurs concomitantly with proton channel opening.

The Nature of the Energy Barrier for the Charge Movement in Voltage-Sensors

Voltage-sensors (VS) are protein domains which transduce changes in membrane potential into conformational changes thereby controlling the opening of a pore or the activity of a phosphatase domain. This operation creates measurable gating currents (Ig) by a local rearrangement of charged groups in the VS. Although there is high conservation of these gating charges among VS, the kinetics of Ig varies greatly, spreading over several orders of magnitudes. These kinetic differences explain the temporal separation of Na+ and K+ conductances in excitable cells, critical for the generation and propagation of the action potential. Despite its fundamental importance, the molecular mechanism underlying this phenomenon is not well understood. Here, we identified in the Shaker K+ channel two hydrophobic residues Ile241 and Ile287 located in S1 and S2 respectively which directly interact with S4 arginines during gating charge movement. We show that Shaker gating kinetics can be speeded up ∼ 3 fold by mutating Ile287 to more hydrophilic residues threonine or serine. Interestingly, voltage-dependent sodium channel (Nav) possesses threonines in domains I-III at positions homologous to Ile287 and exhibit Ig ∼ 10 times faster than that of Shaker. Mutating these threonines to isoleucines in Nav1.4 slows down Ig 2-3 times. Moreover, the sensing currents of the voltage-sensitive phosphatase Ci-VSP, which are ∼ 8 times slower than that of Shaker, can be speeded 3-4 fold by substituting Ile126, (homologous to Ile241 in Shaker) to threonine. Taken together, our data indicate that the side chains of these two key positions form a hydrophobic plug separating the external and internal media. This plug constitutes the main energy barrier for charge crossing and its amplitude is modulated by its degree of hydrophobicity, thus controlling the kinetics of the sensor. Support: NIH-GM030376.

Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP

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.

Kinetics of PIP2metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells

Kinetics of PIP₂metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells

Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells

The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) is synthesized in two steps from phosphatidylinositol by lipid kinases. It then interacts with KCNQ channels and with pleckstrin homology (PH) domains among many other physiological protein targets. We measured and developed a quantitative description of these metabolic and protein interaction steps by perturbing the PIP2 pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP2 with a time constant of τ <300 ms and fully inhibits KCNQ currents in a similar time. PIP2 was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, τ = 11 s. In contrast, resynthesis of PIP2 after activation of phospholipase C by muscarinic receptors took ∼130 s. These kinetic experiments showed that (1) PIP2 activation of KCNQ channels obeys a cooperative square law, (2) the PIP2 residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP2 by PIP 5-kinase is fast and limited primarily by a step(s) that replenishes the pool of plasma membrane PI(4)P. We extend the kinetic model for signaling from M1 muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910344), with this new information on PIP2 synthesis and KCNQ interaction.

Modular Nature of the Main Domains in Voltage Sensitive Phosphatases

Voltage Sensitive Phosphatases (VSPs) constitute a family of enzymes controlled by membrane potential. Its members have a Voltage Sensing Domain (VSD) in the N-terminus and a Phosphatase Domain (PD) in their C-terminus, both connected by a Phospholipid Binding Motif (PBM). Remarkably, N- and C-terminal domains display high homology to two different types of proteins. The VSD of VSPs resembles the VSD of voltage gated channels; while their PD shares a striking homology to the tumor suppressor phosphoinositide phosphatase PTEN. This feature of VSPs makes them look like natural chimeras. Recently, it has been shown that the catalytic activity of Ci-VSP, a member of the VSP family, depends on the binding of the PBM onto the membrane, which is, in turn, controlled by the membrane potential-driven movement of the VSD. For PTEN, it is known that the binding of PTEN onto the plasma membrane mediated by its N-terminus is sine qua non for enzymatic activity.Based on this similarity, we built a chimera by replacing the PD of Ci-VSP by PTEN. This chimera, Ci-VSPTEN, was successfully expressed in Xenopus oocytes and displayed sensing currents resembling those observed in Ci-VSP. As for its enzymatic characteristics, Ci-VSPTEN was expressed in CHO cells and its activity tested by measuring membrane association of GFP-tagged phosphoinositide sensors by TIRF microscopy. Depolarization-induced decline in membrane fluorescence indicated that Ci-VSPTEN has voltage dependent PI(3,4,5)P3 3’-phosphatase activity. Because the binding of the PBM induces an allosteric activation of PTEN, these observations strongly support the idea that the binding of the PBM onto the membrane is a key step in the activation of Ci-VSP. In a broader view, these results show that the VSD and the PD of Ci-VSP, and presumably other VSPs, are naturally modular. (Support: GM030376, DFG OL240/2)

Sensing Charges of Ci-VSP

The Ciona intestinalis Voltage Sensor-containing Phosphatase (Ci-VSP) is a member of the Voltage Sensitive Phosphatase family that exhibits membrane potential-controlled enzymatic activity. Alignments of the amino acid sequence of Ci-VSP against Voltage Gated Channels (VGC) indicate that its Voltage Sensing Domain (VSD) is formed by four transmembrane segments. The putative fourth segment (S4) of Ci-VSP extends between the arginine (R) in position 217 and the glutamine (Q) in position 239, containing five arginines, which might be the voltage sensing charges. Although it has been shown that R229 and R232 are critical for voltage sensing in Ci-VSP (Murata et al., 2005), the role of the remaining charges is still unclear. To address this issue, we have performed a partial Histidine Scanning of the S4 of Ci-VSP, following the paradigm established for the VGC Shaker (Starace and Bezanilla, 2004). The voltage dependence of the sensing current of the R217H mutant was modulated by pH. Decreasing the external pH shifted the Q-V curve towards positive potentials, while a pH increase had the converse effect, consistent with the finding that neutralizing R217 (R217Q) produces a negative shift of the voltage dependence of Ci-VSP (Kohout et al., 2008). However, the total net charge of R217H did not change with pH, indicating that R217 does not participate in sensing the membrane potential. When the second arginine is replaced by histidine, the resultant mutant (R223H) exhibits a voltage dependent proton current which closes at positive potentials, resembling the current recorded from Shaker-W434F with its first gating charge replaced by histidine (R362H). This result strongly suggests that R223 has access to both the intra- and the extracellular media depending on voltage. Taken together, our results indicate that R223 is the most extracellularly located sensing charge of the Ci-VSP S4 segment. (Support: NIHGM030376)

Optimizing Protein-Based Optical Voltage Sensitive Probes: A Systematic Study

Replacement of the phosphatase domain of the Ciona intestinalis voltage sensitive phosphatase (CiVSP) with fluorescence protein (FP) FRET pairs generates chimeric proteins that respond to voltage fluctuations with changes in FRET efficiency. VSFP 2.1 and Mermaid represent two different CiVSP-based voltage probes. We have developed a single FP-based, CiVSP-based probe (pKF215). These probes have different FPs and linker domains. As these constructs represent the most promising probes developed to date, we have undertaken a structure/function analysis of these constructs by: i) altering the inserted FPs and ii) replacing the VSD with species orthologs. The goal is to produce probes with a larger and faster ΔF/F response. We have created a matrix of 33 different constructs replacing the FPs of the original constructs with a range of red-shifted FPs (mCherry, mStrawberry, mTangerine, mOrange, tdTomato and TagRFP). We have also replaced the inserts with the two currently most viable FRET pairs (CFP/YFP and the Mermaid FRET pair). Finally we have chosen three other FPs which we have previously identified as ‘modulatable’ (mCerulean, PHluorin and eGFP). As the S1 to S4 domain of CiVSP has produced the most successful fluorescent voltage sensing constructs, we sought orthologs of Ciona VSD that might perform better. We identified VSPs orthologs from a variety of different species. We cloned the VSP orthologs from chicken, mouse, human, Xenopus laevis, Xenopus tropicalis, and zebra fish. To test these VSDs, we have created VSFP 2.1-like constructs from these cDNAs by inserting the CFP/YFP FRET pairs at homologous positions in the orthologous sequence. The constructs are being tested for membrane expression and voltage-dependent fluorescence changes in HEK293 cells.

Two Structurally Distinct Pathways for the Voltage-Sensing S4 Helices

In voltage-dependent ion channels, the movement of the voltage-sensing S4 helices produces gating currents. The charge displaced as a function of the membrane potential (Q-V) is well described by a sequential two-state Boltzmann relation, indicating that there are at least two steps of gating charge movement from their Resting state to the Active state. In addition, it has been shown that at a maintained positive potential, the S4 helices of voltage-gated Na, Ca and K channels and the voltage sensitive phosphatase Ci-VSP, undergo a slower secondary conformational transition stabilizing the sensor in a Relaxed (inactivated) state. From the Relaxed state, the Q-V relation exhibits a strong shift towards negative potentials when compared to the Q-V relation measured from the resting state. We engineered gating perturbations in the Shaker potassium channel, by substituting specific aromatic residues in positions spatially close to the S4. One of these mutants, in position I241 of S1, part of the hydrophobic plug of the voltage sensor, when mutated to tryptophan (I241W), produces a strong split in the Q-V when measured from the resting state. By labeling M356C with TMRM we also find the same split in the fluorescence-voltage curve. We propose that the presence of the tryptophan in the 241 position favors an interaction with one of the positively-charged arginines along the S4, thus stabilizing a fleeting intermediate state in the gating pathway. However, in the I241W mutant, the split in the Q-V almost disappears when the gating currents are measured from the relaxed state and the same result is seen with the fluorescence-voltage curve. This result and the effect of other tryptophan perturbations near the S4 segment strongly support the existence of two structurally distinct gating pathways for the movement of the S4 helices. Supported: NIHGM030376.

Optical Recording of Electrical Activity of Cortical Layer 2/3 Pyramidal Neurons Using A Genetically-Encoded Voltage Probe

Voltage-Sensitive Fluorescent Protein 2.3, VSFP2.3, is a genetically-encoded probe of membrane voltage using fluorescence resonance energy transfer (FRET) between a pair of cyan (CFP) and yellow (YFP) fluorescent proteins to convert voltage-activated motions of a voltage sensor domain from Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) into a differential voltage dependent fluorescence signal. To evaluate the utility of VSFP2.3 as a probe of electrical activity of neurons in intact brain tissue, we performed targeted whole cell current clamp and simultaneous optical recordings from L2/3 pyramidal cells of the mouse cerebral cortex in acute brain slices. VSFP2.3 expression in neocortical cells was achieved by in-utero electroporation into the cortical ventricular zone at embryonic age 15.5 of a plasmid vector containing VSFP2.3 under the CAG hybrid promoter. This procedure resulted in strong VSFP2.3 fluorescence at postnatal age (up to day 30 tested) from a restricted cortical area, mostly within somato-sensory cortex, with the fluorescence originating from a clustered population of pyramidal neurons with cell bodies in layer 2/3. Electric current injection into VSFP2.3-positive cells (postnatal day 16-22) revealed an optical response signal to sub-threshold slow depolarization of the somatic membrane that could be resolved in single trials. While the optical signal in response to fast action potentials was noisy in single trials, S/N above two was obtained by event-triggered averaging over a few (5-10) action potentials. We also tested for the optical response to synaptically evoked EPSPs which were reliably detected at near threshold amplitudes in single trials. Our results provide the first demonstration of an optical readout of neuronal activity at cellular resolution using a genetically-targetable voltage probe in intact brain tissue in-vitro.

A Voltage Sensitive Phosphatase from Xenopus Laevis Testis

Voltage sensitive phosphatases (VSPs) are transmembrane proteins comprised of a voltage sensor domain characteristic of an ion channel, coupled to a phosphatidylinositol phosphatase; their phosphatase activity is activated by membrane depolarization (Murata and Okamura, 2007, J. Physiol. 583:875-889). Because VSPs are expressed predominantly in sperm, it has been proposed that they might function in the voltage-dependent regulation of sperm-egg membrane fusion, which in many species provides a fast block to polyspermy. To characterize the properties of a VSP from a species amenable to transgenesis and fertilization studies, we identified a VSP homolog from Xenopus laevis testis and expressed it in Xenopus oocytes, together with the fluorescent PIP2 sensor PLCδPH-GFP. Using a photodiode to measure PLCδPH-GFP fluorescence from the pigmented surface of voltage clamped oocytes, we showed that XlVSP enzymatic activity is regulated over a range of membrane potentials (50% activation at ∼ +10 mV) similar to those that regulate sperm-egg fusion. In agreement with previous studies of ascidian and zebrafish VSPs (Hossain et al., 2008, J. Biol. Chem. 283: 18248-59), an R152Q point mutation shifted the XlVSP activation curve towards more hyperpolarized membrane potentials (50% activation at ∼ −12mV). Future studies of the voltage dependence of fertilization using sperm from transgenic frogs expressing XlVSPR152Q should allow investigation of the functional significance of voltage sensitive phosphatases in sperm-egg fusion.

Probing Phosphoinositide Kinetics With A Voltage-sensitive Phosphatase

Voltage-sensitive phosphatases (VSPs) have a voltage sensor linked to a phosphoinositide (PI) 5-phosphatase, which hydrolyzes plasma membrane PI-(4,5)-bisphosphate (PIP2) to PI(4)P (Iwasaki, PNAS 105, 7970). We used PIP2 hydrolysis by VSP from Ciona intestinalis (ci-VSP) and zebrafish (dr-VSP) to better understand PIP2 binding and resynthesis. PIP2 was monitored using the PIP2-sensitive M-current (KCNQ2/3) and FRET between a pair of PIP2-binding probes (PH-PLCδ1-CFP & PH-PLCδ1-YFP). Depolarizations to +100 mV lasting >50 ms reduced M-current and PH-probe FRET. PIP2 depletion was saturated by depolarizations lasting 500-1000 ms. Evidently PH-probe FRET and M-current respond quickly to changes in plasma membrane PIP2.After repolarization, PH-probe FRET and M-current relaxed to baseline values with time constants of ~10 s in a wortmannin-insensitive manner. This reflects endogenous PIP 5-kinase converting PI(4)P back to PI(4,5)P2. Overexpression of PIP 5-kinase increased the length of depolarization required to deplete PIP2, and speeded PIP2 recovery after repolarization.Recovery of PIP2 after VSP activation is ~10x faster than after PLC activation. However, it only requires PIP 5-kinase, whereas recovery after PLC activation requires PI 4-kinase and PIP 5-kinase in series. Thus PI 4-kinase must be the slower enzyme. To estimate the rate of PI 4-kinase, we compared translocation of a fluorescent probe that reports plasma membrane PI(4)P (PH-OSH2, T.Balla) to translocation of the PIP2-binding probe in confocal time-lapse imaging. Upon PLC activation through M1 receptors, the plasma membrane PI(4)P signal decreased 20 s later than the PIP2 signal. Both probes recovered with similar time courses. FRET photometry between either PIP2-probes or PI(4)P-probes showed comparable results. The simultaneous recovery of both probes is consistent with the hypothesis that recovery of PIP2 is governed by rate-limiting synthesis of PI(4)P by the PI 4-kinase, followed by rapid conversion of PI(4)P into PI(4,5)P2 by the PIP 5-kinase.NIH-NS008174&HFSP

Improved FRET Sensing Of Membrane Voltage With Voltage Sensitive Phosphatase And New Coral Fluorescence Proteins

Ciona voltage sensitive phosphatase (Ci-VSP) is an unique enzyme discovered first in asicdian genome, in which phosphatase activity is regulated by transmembrane potential. Ci-VSP consists of a phosphatase domain and a preceding voltage sensing domain (VSD) which is homologous to the S1-S4 transmembrane domain found in conventional voltage-gated ionic channels. Possible mechanism of Ci-VSP should be that changes in transmembrane potential elicit conformational changes in the VSD, which then induce conformational changes in the phosphatase domain, regulating enzymatic activity. Analogously, by replacing the phosphatase domain with two fluorescent proteins that act as fluorescence resonance energy transfer (FRET) donor and acceptor, it is expected that transmembrane potential can be optically probed as FRET readout. Using two new coral fluorescent proteins, we developed such a membrane potential reporter, named Mermaid, that displays 40% changes in emission ratio per 100 mV change, allowing for visualization of spatiotemporal dynamics in electrical activities of excitable cells. Notably, Mermaid has fast on-off kinetics at warm temperatures and can report voltage spikes comparable to action potentials.

2P-147 電位感受性ホスファターゼを用いた細胞内イノシトールリン脂質シグナル経路の電気的操作(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

2P-147 電位感受性ホスファターゼを用いた細胞内イノシトールリン脂質シグナル経路の電気的操作(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

Sensing membrane voltage with voltage sensitive phosphatase and new coral fluorescence proteins

Sensing membrane voltage with voltage sensitive phosphatase and new coral fluorescence proteins

Structural Dynamics of an Isolated Voltage-Sensor Domain in a Lipid Bilayer

A strong interplay between the voltage-sensor domain (VSD) and the pore domain (PD) underlies voltage-gated channel functions. In a few voltage-sensitive proteins, the VSD has been shown to function without a canonical PD, although its structure and oligomeric state remain unknown. Here, using EPR spectroscopy, we show that the isolated VSD of KvAP can remain monomeric in a reconstituted bilayer and retain a transmembrane conformation. We find that water-filled crevices extending deep into the membrane around S3, a scaffold conducive to transport of protons/cations, are intrinsic to the VSD. Differences in solvent accessibility in comparison to the full-length KvAP allowed us to define an interacting footprint of the PD on the VSD. This interaction is centered around S1 and S2 and suggests a rotation of 70 degrees -100 degrees relative to Kv1.2-Kv2.1 chimera. Sequence-conservation patterns in Kv channels, Hv channels, and voltage-sensitive phosphatases reveal several near-universal features suggesting a common molecular architecture for all VSDs.