Voltage-sensitive Phosphatase Research Articles

Regulation of the two-pore domain potassium channel, THIK-1 and THIK-2, by G protein coupled receptors.

A member of THIK (two pore domain halothane-inhibited K+) channels, THIK-1, was reported as a target of Gi/o-coupled receptors (Gi/o-Rs) in neurons and microglia. We confirmed that in HEK293T cells the THIK-1 channel is activated by Gi/o-Rs and found that Gq-coupled receptors (Gq-Rs) also activates the channel. The effects of Gi/o-Rs and Gq-Rs were inhibited by the Gi/o inhibitor pertussis toxin and phospholipase C (PLC) inhibitor, respectively. The effects of Gi/o-Rs were attenuated when consensus Gβγ binding motif at the C-tail of the THIK-1 channel was mutated, suggesting that Gβγ serves as a THIK-1 channel activator upon the stimulation of Gi/o-Rs. As to the effects of Gq-Rs on the THIK-1 channel, a protein kinase C inhibitor and calcium chelators failed to inhibit the effect of a Gq coupled muscarinic M1R. Neither the hydrolysis of phosphatidyl inositol bisphosphate induced by voltage sensitive phosphatase nor the application of a diacylglycerol analogue, OAG, increased the channel current. The mediator of Gq-dependent activation of the THIK-1 channel remained unsolved. The effects of Gi/o- and Gq-Rs on the THIK-2 channel were also investigated, by using a THIK-2 mutant channel whose N-terminal domain is deleted to improve the surface membrane expression. We observed that Gi/o- and Gq-Rs activate the mutated THIK-2 channel, similarly to the THIK-1 channel. Interestingly, heterodimeric channels of THIK-1 and THIK-2 responded to Gi/o-R and Gq-R stimulation. Taken together, Gi/o- or Gq-Rs activates the THIK-1 and THIK-2 channels in a Gβγ or PLC dependent manner, respectively.

Critical contributions of pre-S1 shoulder and distal TRP box in DAG-activated TRPC6 channel by PIP2 regulation

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) regulates the activities of numerous membrane proteins, including diacylglycerol(DAG)-activated TRPC3/6/7 channels. Although PIP2 binding is known to support DAG-activated TRP channel activity, its binding site remains unknown. We screened for PIP2 binding sites within TRPC6 channels through extensive mutagenesis. Using voltage-sensitive phosphatase (DrVSP), we found that Arg437 and Lys442, located in the channel’s pre-S1 domain/shoulder, are crucial for interaction with PIP2. To gain structural insights, we conducted computer protein–ligand docking simulations with the pre-S1 domain/shoulder of TRPC6 channels. Further, the functional significance of PIP2 binding to the pre-S1 shoulder was assessed for receptor-operated channel functions, cross-reactivity to DAG activation, and the kinetic model simulation. These results revealed that basic residues in the pre-S1 domain/shoulder play a central role in the regulation of PIP2-dependent gating. In addition, neutralizing mutation of K771 in the distal TRP box reversed the effect of PIP2 depletion from inhibiting to potentiating channel activity. A similar effect was seen in TRPV1 channels, which suggests that TRPC6 possesses a common but robust polarity switch mediating the PIP2-dependent effect. Overall, these mutagenesis studies reveal functional and structural insights for how basic residues and channel segments in TRP channels are controlled through phosphoinositides recognition.

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).

Differential Regulation of Ca2+-Activated Cl- Channel TMEM16A Splice Variants by Membrane PI(4,5)P2.

TMEM16A is a Ca2+-activated Cl− channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP2 in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl− currents, which are independent of the membrane potential and specific to PI(4,5)P2 depletion. The attenuation of endogenous PI(4,5)P2 levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl− currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P2, not PI4P and PI(3,4,5)P3. Finally, we also confirmed that PI(4,5)P2 resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P2 selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.

Andersen-Tawil Syndrome Is Associated With Impaired PIP2 Regulation of the Potassium Channel Kir2.1.

Andersen–Tawil syndrome (ATS) type-1 is associated with loss-of-function mutations in KCNJ2 gene. KCNJ2 encodes the tetrameric inward-rectifier potassium channel Kir2.1, important to the resting phase of the cardiac action potential. Kir-channels’ activity requires interaction with the agonist phosphatidylinositol-4,5-bisphosphate (PIP2). Two mutations were identified in ATS patients, V77E in the cytosolic N-terminal “slide helix” and M307V in the C-terminal cytoplasmic gate structure “G-loop.” Current recordings in Kir2.1-expressing HEK cells showed that each of the two mutations caused Kir2.1 loss-of-function. Biotinylation and immunostaining showed that protein expression and trafficking of Kir2.1 to the plasma membrane were not affected by the mutations. To test the functional effect of the mutants in a heterozygote set, Kir2.1 dimers were prepared. Each dimer was composed of two Kir2.1 subunits joined with a flexible linker (i.e. WT-WT, WT dimer; WT-V77E and WT-M307V, mutant dimer). A tetrameric assembly of Kir2.1 is expected to include two dimers. The protein expression and the current density of WT dimer were equally reduced to ~25% of the WT monomer. Measurements from HEK cells and Xenopus oocytes showed that the expression of either WT-V77E or WT-M307V yielded currents of only about 20% compared to the WT dimer, supporting a dominant-negative effect of the mutants. Kir2.1 sensitivity to PIP2 was examined by activating the PIP2 specific voltage-sensitive phosphatase (VSP) that induced PIP2 depletion during current recordings, in HEK cells and Xenopus oocytes. PIP2 depletion induced a stronger and faster decay in Kir2.1 mutant dimers current compared to the WT dimer. BGP-15, a drug that has been demonstrated to have an anti-arrhythmic effect in mice, stabilized the Kir2.1 current amplitude following VSP-induced PIP2 depletion in cells expressing WT or mutant dimers. This study underlines the implication of mutations in cytoplasmic regions of Kir2.1. A newly developed calibrated VSP activation protocol enabled a quantitative assessment of changes in PIP2 regulation caused by the mutations. The results suggest an impaired function and a dominant-negative effect of the Kir2.1 variants that involve an impaired regulation by PIP2. This study also demonstrates that BGP-15 may be beneficial in restoring impaired Kir2.1 function and possibly in treating ATS symptoms.

TMEM266 is a functional voltage sensor regulated by extracellular Zn2.

Voltage-activated ion channels contain S1-S4 domains that sense membrane voltage and control opening of ion-selective pores, a mechanism that is crucial for electrical signaling. Related S1-S4 domains have been identified in voltage-sensitive phosphatases and voltage-activated proton channels, both of which lack associated pore domains. hTMEM266 is a protein of unknown function that is predicted to contain an S1-S4 domain, along with partially structured cytoplasmic termini. Here we show that hTMEM266 forms oligomers, undergoes both rapid (µs) and slow (ms) structural rearrangements in response to changes in voltage, and contains a Zn2+ binding site that can regulate the slow conformational transition. Our results demonstrate that the S1-S4 domain in hTMEM266 is a functional voltage sensor, motivating future studies to identify cellular processes that may be regulated by the protein. The ability of hTMEM266 to respond to voltage on the µs timescale may be advantageous for designing new genetically encoded voltage indicators.

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates KCNQ3 K+ channels by interacting with four cytoplasmic channel domains

Phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), Kv7) K+ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP2 and performed extensive mutagenesis in regions suggested to be involved in PIP2 interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe, we found that PIP2 regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP2 interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP2 were consistent with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.

A126 in the active site and TI167/168 in the TI loop are essential determinants of the substrate specificity of PTEN

PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P2 and PI(3,4,5)P3. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTENCiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTENCiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P3, but not toward PI(4,5)P2. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P2 and PI(3,4,5)P3 of PTENCiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P3 in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN’s substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.

The oculocerebrorenal syndrome of Lowe protein (OCRL) inhibits the Na/bicarbonate cotransporter NBCe1 expressed in Xenopus laevis oocytes.

The electrogenic Na/HCO3 cotransporter NBCe1 regulates intracellular pH in many tissues and contributes to solute reabsorption/secretion in epithelia. NBCe1 is modulated by hormones, classic second messengers, and other signaling molecules/ions. We previously reported that both the phospholipid PIP2 itself and its hydrolysis to IP3 (Ca2+) stimulates the activity of NBCe1 expressed in oocytes. In this preparation, activating a co‐expressed voltage‐sensitive phosphatase (VSP) that dephosphorylates PIP2 to PIP without stimulating IP3/Ca2+ signaling decreases NBCe1 activity. In the current study, we examined the effect of the PIP2‐lowering phosphatase, inositol polyphosphate 5‐phosphatase encoded by the oculocerebrorenal syndrome of Lowe gene (OCRL) on the activity of NBCe1 variants expressed in oocytes. Mutations and variants of OCRL lead to Dent disease type 2, as well as Lowe Syndrome, which is characterized by cataracts, intellectual disability, basal ganglia calcification, and proximal renal tubular acidosis (pRTA). Using the 2‐electrode voltage‐clamp technique, we found that oocytes co‐expressing human OCRL with NBCe1‐A (both HA tagged) displayed a characteristic NBC‐mediated, HCO3–‐induced outward current (INBC) that was 59% smaller than oocytes expressing NBCe1‐A. We also observed OCRL inhibition of HA‐tagged NBCe1‐B and ‐C. We obtained similar results after replacing the HA tag on NBCe1‐A with a FLAG tag. The OCRL‐induced decrease in INBC could be explained by a decrease in NBCe1 expression at the plasma membrane. Co‐expressing OCRL caused a 44% decrease in INBC and a parallel 33% decrease in FLAG‐tagged NBCe1‐A oocyte surface expression as determined by single‐oocyte chemiluminescence. Thus, a physiological phosphatase that lowers PIP2 can regulate the activity and/or expression of NBCe1. Altered activity/expression of NBCe1 caused by mutant OCRL variants seen in Dent disease and Lowe Syndrome may contribute to observed patient symptoms, especially pRTA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Differential Regulation of PI(4,5)P2 Sensitivity of Kv7.2 and Kv7.3 Channels by Calmodulin.

HIGHLIGHTS - Calmodulin-dependent Kv7.2 current density without the need of binding calcium.- Kv7.2 current density increase is accompanied with resistance to PI(4,5)P2 depletion.- Kv7.3 current density is insensitive to calmodulin elevation.- Kv7.3 is more sensitive to PI(4,5)P2 depletion in the presence of calmodulin.- Apo-calmodulin influences PI(4,5)P2 dependence in a subunit specific manner.The identification and understanding of critical factors regulating M-current functional density, whose main components are Kv7.2 and Kv7.3 subunits, has profound pathophysiological impact given the important role of the M-current in neuronal excitability control. We report the increase in current density of Kv7.2 channels by calmodulin (CaM) and by a mutant CaM unable to bind Ca2+ (CaM1234) revealing that this potentiation is calcium independent. Furthermore, after co-expressing a CaM binding protein (CaM sponge) to reduce CaM cellular availability, Kv7.2 current density was reduced. Current inhibition after transient depletion of the essential Kv7 co-factor phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by activating Danio rerio voltage sensitive phosphatase (DrVSP) was blunted by co-expressing CaM1234 or the CaM sponge. In addition, CaM-dependent potentiation was occluded by tonic elevation of PI(4,5)P2 levels by PI(4)P5-kinase (PIP5K) expression. In contrast to the effect on homomeric Kv7.2 channels, CaM1234 failed to potentiate heteromeric Kv7.2/3 or homomeric Kv7.3 channels. Sensitivity to PI(4,5)P2 depletion of Kv7.2/3 channels was increased after expression of CaM1234 or the CaM sponge, while that of homomeric Kv7.3 was unaltered. Altogether, the data reveal that apo-CaM influences PI(4,5)P2 dependence of Kv7.2, Kv7.2/3, and of Kv7.3 channels in a subunit specific manner.

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.

Voltage Dependent Phosphatase Activity is Enhanced by Intracellular Acidification

Voltage sensitive phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4)P2/PI(3,4,5)P3-3-phosphatases. For non-mammalian VSPs, this activity is regulated by membrane voltage via a voltage sensor domain (VSD). For mammalian VSPs the VSD seems insensitive to voltage changes yet still essential for control of the phosphatase activity. Under physiological conditions, the non-mammalian VSPs strongly deplete PI(4,5)P2 in a voltage dependent manner. The physiological regulator of mammalian VSP activity remains elusive. VSPs are suggested to play a role in fertilization and development, where changes in intracellular pH are known to occur. They are found to be expressed in tissues like kidney, stomach, sperm, and ovary which are known to undergo such pH changes. Therefore, we speculated that intracellular pH might modulate VSP activity.To test this hypothesis we performed whole-cell patch-clamp experiments in CHO cells expressing diverse VSPs and fluorescent PI(4,5)P2 reported domains, the latter allowing for monitoring VSP activity by means of total internal reflection microscopy (TIRF-M). The whole-cell patch-clamp configuration allowed for control not only over membrane voltage but also intracellular pH by dialysing the cell with solutions with the desired pH.We find that acidification of the cytoplasm results in increased PI(4,5)P2 depletion, accompanied by a shift of the apparent voltage dependence towards more negative potentials. An increase in intracellular pH has the opposite effect. The voltage dependence of sensing currents was unaffected by the pH changes, suggesting that alterations of the VSD are not causal for the observed changes in voltage dependent activity. Similar effects were observed in all tested VSPs We conclude that the overall activity of the phosphatase is enhanced under acidic and diminished under alkaline conditions. Kinetic modeling predicts a shift in apparent voltage dependence under these circumstances that is in agreement with the observed shift.In conclusion, we suggest that intracellular pH can play a role in the regulation of the activity of VSPs.This work was supported by a research grant of the University Medical Center Giessen and Marburg (UKGM32/2011MR) to C.R.H and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O.

Biophysical Characterization of ASAP1

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. ASAP1 is based on the Gallus gallus voltage sensitive phosphatase (GgVSP) with 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. Expressing this molecule in Xenopus oocytes and recording fluorescence simultaneously with gating current using the cut-open voltage clamp technique, our studies show that the gating currents of ASAP1 are significantly faster than CiVSP.

Reorganization of the cytoskeleton

The effect of a modulated electromagnetic field on the cytoskeleton is possible through the action of voltage-sensitive phosphatase (VSP). Field-controlled phosphorous hydrolysis could have important roles in cytoskeleton restructuring, as well as in resonant-type behavior.

Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate.

Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K+ current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies.

PIP2 in pancreatic β-cells regulates voltage-gated calcium channels by a voltage-independent pathway.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (CaV) channels promotes insulin secretion in pancreatic β-cells. However, whether CaV channels are regulated by PIP2, as is the case for some non-insulin-secreting cells, is unknown. The purpose of this study was to investigate whether CaV channels are regulated by PIP2 depletion in pancreatic β-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). CaV channel currents were recorded by the patch-clamp technique. The CaV current amplitude was reduced by activation of the muscarinic receptor 1 (M1R) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced CaV inhibition was diminished by a high concentration of Ca2+ chelator, whereas ≥50% of this inhibition was prevented by diC8-PIP2 dialysis. Localization of PIP2 in the plasma membrane was examined by transfecting INS-1 cells with PH-PLCδ1, which revealed a close temporal association between PIP2 hydrolysis and CaV channel inhibition. Furthermore, the depletion of PIP2 by a voltage-sensitive phosphatase reduced CaV currents in a way similar to that observed following M1R activation. These results indicate that activation of the M1R pathway inhibits the CaV channel via PIP2 depletion by a Ca2+-dependent mechanism in pancreatic β- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.

Characterization of a Fast Voltage-Sensing Protein using Voltage-Clamp Fluorometry

Voltage-sensing domains in voltage-activated ion channels and other voltage-sensing proteins contain well-conserved S4 helices containing basic residues capable of sensing changes in membrane potential to trigger opening or closing of ion-selective pores or phosphatase activity in voltage-sensitive phosphatases. Here we report the discovery of a protein which contains a voltage-sensing domain capable of rapid and slow rearrangements in response to changes in voltage. In place of a pore domain, this voltage sensor has large N- and C-termini predicted to contain structured and disordered domains for interaction with intracellular proteins. Our working hypothesis is that this protein, which we name Coupled Voltage Sensor (CVS), functions as a voltage sensor that couples to intracellular signaling pathways (as yet undefined). Our goal in the present experiments was to demonstrate that CVS undergoes voltage-dependent conformation changes and to characterize those using site-specific voltage-clamp fluorometry. We identified several positions at the external end of S4 where introduced and fluorophore-labeled Cys residues produce changes in fluorescence as a function of membrane potential. Several positions give complex fluorescence responses, starting with a rapid fluorescence increase (dequenching), followed by slower fluorescence decrease (quenching). Interestingly the rapid component is as fast as the clamp speed when using the cut-open voltage clamp technique (tau ∼ 200 μs). To exclude the possibility of Stark effect we examined the effects of a series of soluble quenchers and observed enhanced quenching with membrane depolarization, consistent with an outward movement of the S4 helix. Overall, our results support a model in which CVS undergoes two temporally distinct conformation rearrangements in response to membrane depolarization.

Relaxation of the Voltage Sensing Modules of Excitation-Contraction (EC) Coupling in Mammalian Skeletal Muscle

Voltage-sensing modules (VSMs) of similar molecular structure underlie voltage-sensitivity in ion channels, the voltage-sensor of EC coupling (the DHPR), and voltage-sensitive phosphatases (VSPs). Upon sustained depolarization (or “antipolarization” of VSPs) all VSMs enter a functionally disabled inactivated or relaxed state. This is accompanied in every case by a change in voltage dependence of the sensor charge movement, including a large negative shift of its central voltage VT. In frog muscle, voltage-driven transitions of the EC coupling VSM elicit charge movements that result in two charge distributions with different VT: Q1 in the normally polarized fiber; Q2 when the membrane is held depolarized. Here we show in mammalian muscle —single fibers of mouse FDB— a similar interconversion of voltage-sensor charge. In 2 mouse strains VT at rest was ∼-20 mV, but shifted to ∼-80 mV upon sustained depolarization. The change was a conversion between two distributions (Q1 and Q2) with distinct but fixed VT‘s. The observations require a minimum model with 4 states: C (cis), T (trans), CR (cis relaxed), TR (trans relaxed). The transitions C->T and CR->TR are fast and mobilize respectively Q1 and Q2. C->T opens the RyR, but CR->TR does not. The transitions to and from relaxed states are slow and voltage-independent. Low extracellular [Ca2+] promoted the C->CR and T->TR transitions, while high [Ca2+] opposed it. Observations of similar antagonism between inactivation of Ca2+ and K+ channels and the pore occupancy by ions led to the notion that inactivation involves pore collapse. DHPR pore opening, however, is not required for proper operation of the EC coupling VSM. The effects of extracellular Ca2+ on voltage-dependent inactivation must be reinterpreted in view of these findings. (Support: ANII, CSIC and NIGMS).

Intracellular Zinc Potentiates KCNQ Channels through Modulation of their Sensitivity to PIP2

KCNQ potassium channels which underlie neuronal M current play dominant roles in controlling neuronal excitability. Inhibition or genetic loss of M channels are implicated in many excitability disorders (e.g. epilepsy, pain, deafness, arrhythmia), while M channel openers retigabine and flupirtine are used clinically as anticonvulsant drugs and analgesics. Zinc pyrithione (ZnPy) is one of the recently discovered M channel openers which activity was previously attributed to the pyrithione moiety; ZnPy is also used as zinc ionophore. Here we show that the likely mechanism of ZnPy action on KCNQ channels is elevation of intracellular zinc which, in turn, activates the channels. Using patch-clamp recordings we show that 10μM ZnPy increased the recombinant KCNQ4 and KCNQ2/3 currents as well as native M current in DRG neurons by 2.4±0.4, 1.6±0.1 and 1.6±0.2 fold respectively; these effects were completely reversed by zinc chelator TPEN (20μM) added in the presence of ZnPy. Fluorescent zinc imaging with FluoZin3TM in HEK293 cells and DRG neurons demonstrated that ZnPy induced robust intracellular zinc rises which were completely reversed by TPEN. Four other chemically unrelated zinc ionophores PDTC, DEDTC, DIQ and Zinc ionophore I had similar to ZnPy effects: all four compounds augmented KCNQ4 and KCNQ2/3 currents and induced intracellular zinc rises; TPEN completely abolished these effects. Using voltage sensitive phosphatase CiVSP we found that the Ci-VSP-dependent PIP2 hydrolysis strongly inhibited recombinant KCNQ4 and KCNQ2/3, however, ZnPy and PDTC completely abolished these effect rendering channels insensitive to Ci-VSP activation. TIRF imaging using optical PIP2 reporter PLCδPH-GFP confirmed that neither ZnPy nor PDTC affected the ability of CiVSP to hydrolyze PIP2, as determined by the depolarization-induced PLCδPH-GFP translocation. In sum, our data suggest that intracellular zinc potently augment KCNQ channel activity by reducing channel requirement for PIP2.