PIP2 Resynthesis Research Articles

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the β2 adrenergic receptor (β2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on β2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of β2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, β2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of β2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease β2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

IMPA1 dependent regulation of phosphatidylinositol 4,5-bisphosphate and calcium signalling by lithium.

Lithium (Li) is widely used as a mood stabilizer to treat bipolar affective disorder. However, the molecular targets of Li that underpin its therapeutic effect remain unresolved. Inositol monophosphatase (IMPA1) is an enzyme involved in phosphatidylinositol 4,5-bisphosphate (PIP2) resynthesis after PLC signaling. In vitro, Li inhibits IMPA1, but the relevance of this inhibition within neural cells remains unknown. Here, we report that treatment with therapeutic concentrations of Li reduces receptor-activated calcium release from intracellular stores and delays PIP2 resynthesis. These effects of Li are abrogated in IMPA1 deleted cells. We also observed that in human forebrain cortical neurons, treatment with Li reduced neuronal excitability and calcium signals. After Li treatment of human cortical neurons, transcriptome analyses revealed down-regulation of signaling by glutamate, a key excitatory neurotransmitter in the human brain. Collectively, our findings suggest that inhibition of IMPA1 by Li reduces receptor-activated PLC signaling and neuronal excitability.

Regulation of PIP2 homeostasis at ER‐plasma membrane contacts by Nir proteins

Phosphatidylinositol 4,5‐bisphosphate (PIP2) in the plasma membrane is a critical lipid molecule that regulates nearly all functional processes occurring at the cell surface. Additionally, PIP2 is hydrolyzed following receptor‐induced activation of phospholipase C to generate second messengers for signal transduction. It is crucial to quickly replenish PIP2 in the plasma membrane following receptor‐induced hydrolysis to sustain signaling outputs and to maintain PIP2‐dependent cellular functions. Replenishment of plasma membrane PIP2 in receptor‐stimulated cells involves transport of phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the plasma membrane for PIP2 re‐synthesis. ER‐plasma membrane contacts are subcellular loci characterized by the close apposition of the ER to the plasma membrane. Recent findings revealed that the PI transfer proteins, Nir2 and Nir3, dynamically localize at ER‐plasma membrane contacts to mediate replenishment of PIP2 hydrolyzed following receptor stimulation. Deficiency in Nir2 and Nir3 suppressed replenishment of PIP2 in the plasma membrane and Ca2+ signaling in receptor‐stimulated cells. A third mammalian Nir protein, Nir1, has been linked to several human diseases including retinal dystrophy. Unlike Nir2 and Nir3, Nir1 lacks a PI transfer protein domain and readily localizes at ER‐plasma membrane contacts in resting cells. The presence of Nir1 promotes Nir2 localization at ER‐plasma membrane contacts to mediate PIP2 replenishment following receptor stimulation. Together, Nir proteins maintain plasma membrane PIP2 homeostasis at ER‐plasma membrane contacts to support cell signaling and plasma membrane functions in receptor‐stimulated cells.

Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment

Fertility relies upon pulsatile release of gonadotropin-releasing hormone (GnRH) that drives pulsatile luteinizing hormone secretion. Kisspeptin (KP) neurons in the arcuate nucleus are at the center of the GnRH pulse generation and the steroid feedback control of GnRH secretion. However, KP evokes a long-lasting response in GnRH neurons that is hard to reconcile with periodic GnRH activity required to drive GnRH pulses. Using calcium imaging, we show that 1) the tetrodotoxin-insensitive calcium response evoked by KP relies upon the ongoing activity of canonical transient receptor potential channels maintaining voltage-gated calcium channels in an activated state, 2) the duration of the calcium response is determined by the rate of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), and 3) nitric oxide terminates the calcium response by facilitating the resynthesis of PIP2 via the canonical pathway guanylyl cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G. In addition, our data indicate that exposure to nitric oxide after KP facilitates the calcium response to a subsequent KP application. This effect was replicated using electrophysiology on GnRH neurons in acute brain slices. The interplay between KP and nitric oxide signaling provides a mechanism for modulation of the refractory period of GnRH neurons after KP exposure and places nitric oxide as an important component for tonic GnRH neuronal pulses.

Extended synaptotagmin regulates membrane contact site structure and lipid transfer function invivo.

Inter-organelle communication between closely apposed membranes is proposed at membrane contact sites (MCS). However, the regulation of MCS structure and their functional relevance invivo remain debated. The extended synaptotagmins (Esyt) are evolutionarily conserved proteins proposed to function at MCS. However, loss of all three Esyts in yeast or mammals shows minimal phenotypes questioning the functional importance of Esyt. We report that in Drosophila photoreceptors, MCS number is regulated by PLCβ activity. Photoreceptors of a null allele of Drosophila extended synaptotagmin (dEsyt) show loss of ER-PM MCS. Loss of dEsyt results in mislocalization of RDGB, an MCS localized lipid transfer protein, required for photoreceptor structure and function, ultimately leading to retinal degeneration. dEsyt depletion enhanced the retinal degeneration, reduced light responses and slower rates of plasma membrane PIP2 resynthesis seen in rdgB mutants. Thus, dEsyt function and PLCβ signaling regulate ER-PM MCS structure and lipid transfer in Drosophila photoreceptors.

Genetic dissection of the phosphoinositide cycle in Drosophila photoreceptors.

Phototransduction in Drosophila is mediated by phospholipase C-dependent hydrolysis of PIP2-, and is an important model for phosphoinositide signalling. Although generally assumed to operate by generic machinery conserved from yeast to mammals, some key elements of the phosphoinositide cycle have yet to be identified in Drosophila photoreceptors. Here, we used transgenic flies expressing fluorescently tagged probes (P4M and TbR332H), which allow in vivo quantitative measurements of PI4P and PIP2 dynamics in photoreceptors of intact living flies. Using mutants and RNA interference for candidate genes potentially involved in phosphoinositide turnover, we identified Drosophila PI4KIIIα (CG10260) as the PI4-kinase responsible for PI4P synthesis in the photoreceptor membrane. Our results also indicate that PI4KIIIα activity requires rbo (the Drosophila orthologue of Efr3) and CG8325 (orthologue of YPP1), both of which are implicated as scaffolding proteins necessary for PI4KIIIα activity in yeast and mammals. However, our evidence indicates that the recently reported central role of dPIP5K59B (CG3682) in PIP2 synthesis in the rhabdomeres should be re-evaluated; although PIP2 resynthesis was suppressed by RNAi directed against dPIP5K59B, little or no defect was detected in a reportedly null mutant (dPIP5K18 ).

Phospholipase C-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate underlies agmatine-induced suppression of N-type Ca2+ channel in rat celiac ganglion neurons

Agmatine suppresses peripheral sympathetic tone by modulating Cav2.2 channels in peripheral sympathetic neurons. However, the detailed cellular signaling mechanism underlying the agmatine-induced Cav2.2 inhibition remains unclear. Therefore, in the present study, we investigated the electrophysiological mechanism for the agmatine-induced inhibition of Cav2.2 current (ICav2.2) in rat celiac ganglion (CG) neurons. Consistent with previous reports, agmatine inhibited ICav2.2 in a VI manner. The agmatine-induced inhibition of the ICav2.2 current was also almost completely hindered by the blockade of the imidazoline I2 receptor (IR2), and an IR2 agonist mimicked the inhibitory effect of agmatine on ICav2.2, implying involvement of IR2. The agmatine-induced ICav2.2 inhibition was significantly hampered by the blockade of G protein or phospholipase C (PLC), but not by the pretreatment with pertussis toxin. In addition, diC8-phosphatidylinositol 4,5-bisphosphate (PIP2) dialysis nearly completely hampered agmatine-induced inhibition, which became irreversible when PIP2 resynthesis was blocked. These results suggest that in rat peripheral sympathetic neurons, agmatine-induced IR2 activation suppresses Cav2.2 channel voltage-independently, and that the PLC-dependent PIP2 hydrolysis is responsible for the agmatine-induced suppression of the Cav2.2 channel.

Kv 7.4 Channel Activity is Dependent upon PIP2 and Gβγ Subunits

KCNQ4-encoded voltage-gated potassium channels, Kv 7.4, are important regulators of vascular tone at rest and in response to endogenous vasodilators (Hypertension, 59:877-884, 2012). The activity of these channels is dependent upon phosphatidylinositol 4,5-bisphosphate, PIP2 (J.Gen.Physiol. 140:41-53, 2012) and by G-protein βγ subunits (PNAS, 112:6497-6502). HEK293 cells heterologously expressing Kv 7.4 were used in standard whole-cell, cell-attached and inside-out configurations of patch-clamp technique to investigate mechanisms of action of these molecules on the channel. We found that after application of wortmannin, an inhibitor of PIP2 resynthesis, whole-cell currents through Kv 7.4 evoked by step depolarization from −60 mV to 0 mV gradually decreased from 34.4±4.3 pA/pF (n=7, mean±SEM) by 85% during 20 minutes and established at steady level. Further inhibition was achieved by short activation of HEK293 native G-protein-coupled proteinase-activated receptors or purinoceptors. Gallein, an inhibitor of Gβγ subunits, produced the same level of inhibition. Structurally different activators of Kv 7 channels (ML213, NS15370, Retigabine, S-1) enhanced currents in both PIP2- and Gβγ subunits- depleted cells, but the level of increase for all of them was less than 19% in comparison to the controls. In cell-attached experiments wortmannin/gallein reduced single channel with NPo decreasing by more than 95%. Application of Gβγ subunits to PIP2- depleted inside-out patches or PIP2 applied to Gβγ subunits- depleted patches produced insignificant effects on low channel activity. This study shows that PIP2 and Gβγ subunits are crucial for Kv 7.4 function and modulation by pharmacological activators.Supported by the British Heart Foundation (PG/12/63/29824) and by the Medical Research Council (MR/K019074/1).

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca(2+) concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca(2+) oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca(2+)-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca(2+) oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li(+), an inhibitor of inositol monophosphatases that prevents PIP2 resynthesis. In Li(+)-treated cells, cytoplasmic Ca(2+) signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca(2+) oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca(2+) oscillations, and these could not be rescued by inositol or PI4P. In Li(+)-treated cells, recovery of the cytoplasmic Ca(2+) oscillations in the presence of inositol or PI4P was suppressed when Ca(2+) influx through store-operated Ca(2+) channels was inhibited. After rundown of the Ca(2+) signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca(2+) release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP2 pools. Our findings show that store-operated Ca(2+) entry is needed to sustain cytoplasmic Ca(2+) signaling following leukotriene receptor activation both by refilling the Ca(2+) stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.

Synergistic modulation of KCNQ1/KCNE1 K+ channels (IKs) by phosphatidylinositol 4,5-bisphosphate (PIP2) and [ATP

Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed. Recovery of IKs from inhibition induced by muscarinic stimulation was incomplete despite full PIP2 resynthesis. Recovery of IKs was completely suppressed under ATP-free conditions, but partially restored by the ATP analog AMP-PCP, providing evidence that depletion of intracellular ATP inhibits IKs independent of PIP2-depletion.Simultaneous patch-clamp and FRET measurements in cells co-expressing Ci-VSP and the PIP2-FRET sensor revealed a component of IKs inhibition directly related to dynamic PIP2-depletion. A second component of inhibition was independent of acute changes in PIP2 and could be mimicked by ATP-free pipette solution, suggesting that it results from intracellular ATP-depletion. The reduction of intracellular ATP upon Ci-VSP activation appears to be independent of its activity as a phosphoinositide phosphatase. Our data demonstrate that ATP-depletion slowed IKs activation but had no short-term effect on PIP2 regeneration, suggesting that impaired PIP2-resynthesis cannot account for the rapid IKs inhibition by ATP-depletion. Furthermore, the second component of IKs inhibition by Ci-VSP was reduced by AMP-PCP in the pipette filling solution, indicating that direct binding of ATP to the KCNQ1/KCNE1 complex is required for voltage activation of IKs. We suggest that fluctuations of the cellular metabolic state regulate IKs in parallel with Gq-coupled PLC activation and PIP2-depletion.

Phosphatidylinositol 4,5-bisphosphate degradation inhibits the Na+/bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus oocytes.

We previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 ) directly stimulates heterologously expressed electrogenic Na(+)/bicarbonate cotransporter NBCe1-A in an excised macropatch from the Xenopus oocyte, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate/Ca(2+). In the current study, we expand on a previous observation that PIP2 may also directly stimulate NBCe1 in the intact oocyte. In this study on oocytes, we co-expressed either NBCe1-B or -C and a voltage-sensitive phosphatase (VSP), which depletes PIP2 without changing inositol 1,4,5-trisphosphate, and monitored NBCe1-mediated currents with the two-electrode voltage-clamp technique or pHi changes using Vm/pH-sensitive microelectrodes. Activating VSP inhibited NBCe1-B and -C outward currents and NBCe1-mediated pHi increases, and changes in NBCe1 activity paralleled changes in surface PIP2. This study is a quantitative assessment of PIP2 itself as a regulator of NBCe1-B and -C in the intact cell, and represents the first use of VSP to characterize the PIP2 sensitivity of a transporter. These data combined with our previous work demonstrate that NBCe1-B and -C are regulated by two PIP2-mediated signalling pathways. Specifically, a decrease in PIP2 per se can inhibit NBCe1, whereas hydrolysis of PIP2 to inositol 1,4,5-trisphosphate/Ca(2+) can stimulate the transporter. The electrogenic Na(+)/bicarbonate cotransporter (NBCe1) of the Slc4 gene family is a powerful regulator of intracellular pH (pHi) and extracellular pH (pHo), and contributes to solute reabsorption and secretion in many epithelia. Using Xenopus laevis oocytes expressing NBCe1 variants, we have previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) directly stimulates NBCe1-A in an excised macropatch, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate (InsP3)/Ca(2+). In the current study, we used the two-electrode voltage-clamp technique alone or in combination with pH/voltage-sensitive microelectrodes or confocal fluorescence imaging of plasma membrane PIP2 to characterize the PIP2 sensitivity of NBCe1-B and -C in whole oocytes by co-expressing a voltage-sensitive phosphatase (VSP) that decreases PIP2 and bypasses the InsP3/Ca(2+) pathway. An oocyte depolarization that activated VSP only transiently stimulated the NBCe1-B/C current, consistent with an initial rapid depolarization-induced NBCe1 activation, and then a subsequent slower VSP-mediated NBCe1 inhibition. Upon repolarization, the NBCe1 current decreased, and then slowly recovered with an exponential time course that paralleled PIP2 resynthesis as measured with a PIP2-sensitive fluorophore and confocal imaging. A subthreshold depolarization that minimally activated VSP caused a more sustained increase in NBCe1 current, and did not lead to an exponential current recovery following repolarization. Similar results were obtained with oocytes expressing a catalytically dead VSP mutant at all depolarized potentials. Depleting endoplasmic reticulum Ca(2+) did not inhibit the NBCe1 current recovery following repolarization from VSP activation, demonstrating that changes in InsP3/Ca(2+) were not responsible. This study demonstrates for the first time that depleting PIP2 per se inhibits NBCe1 activity. The data in conjunction with previous findings implicate a dual PIP2 regulatory pathway for NBCe1 involving both PIP2 itself and generated InsP3/Ca(2+).

A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors.

Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

PIP2 hydrolysis is responsible for voltage independent inhibition of CaV2.2 channels in sympathetic neurons

GPCRs regulate CaV2.2 channels through both voltage dependent and independent inhibition pathways. The aim of the present work was to assess the phosphatidylinositol-4,5-bisphosphate (PIP2) as the molecule underlying the voltage independent inhibition of CaV2.2 channels in SCG neurons. We used a double pulse protocol to study the voltage independent inhibition and changed the PIP2 concentration by means of blocking the enzyme PLC, filling the cell with a PIP2 analogue and preventing the PIP2 resynthesis with wortmannin. We found that voltage independent inhibition requires the activation of PLC and can be hampered by internal dialysis of exogenous PIP2. In addition, the recovery from voltage independent inhibition is blocked by inhibition of the enzymes involved in the resynthesis of PIP2. These results support that the hydrolysis of PIP2 is responsible for the voltage independent inhibition of CaV2.2 channels.

Hydrolysis of PIP2 is Responsible for the Voltage-Independent Inhibition of Cav2.2 Channels

GPCRs regulate CaV2.2 channels through both voltage-dependent and -independent inhibition pathways. Gβγ subunits are the effectors of the voltage-dependent inhibition while the molecular mechanism for the voltage-independent inhibition remains to be elucidated. Here, we addressed whether the hydrolysis of PIP2 is responsible for the inhibition remaining after a conditioning pulse, named as voltage-independent inhibition. We recorded CaV2.2 currents from rat superior cervical ganglion neurons. We isolated the voltage-independent inhibition with a double-pulse protocol and assumed that the inhibition remaining after a conditioning pulse corresponds to the voltage-independent pathway. To test our hypothesis, we changed the PIP2 concentration by means of blocking phospholipase C, filling the cell with a PIP2 analogue, and preventing PIP2 re-synthesis. The internal dialysis of short-chain PIP2 was firstly assessed during the muscarinic inhibition of KCNQ2/3 channels. First, we determined the required magnitude, duration, and interval between the conditioning and second pulses to isolate the voltage-independent inhibition. Our results show that the inhibition of phospholipase C reduces the inhibition remaining after a conditioning pulse upon noradrenaline or muscarinic application. Dialyzing into the cell micromolar concentration of short-chain PIP2, through the recording pipette, hampers the muscarinic inhibition of KCNQ2/3 current in tsA-201 cells as well as the voltage independent inhibition of CaV2.2 channels in neurons. Also, blocking the phosphatidylinositol 4-kinase with wortmannin avoids the recovery from the voltage independent inhibition. These results support the hypothesis that the hydrolysis of PIP2 is responsible of the voltage-independent inhibition of CaV2.2. Regulation of levels of PIP2 in nerve terminals can result in the regulation of presynaptic calcium channels and in the subsequent modulation of exocytosis.This work was supported by UNAM- DGAPA-PAPIIT (IN200710).

Facilitation of sympathetic neurotransmission by phosphatidylinositol-4,5-bisphosphate-dependent regulation of KCNQ channels in rat mesenteric arteries

Sympathetic nerves regulate vascular tone by releasing neurotransmitters into the vasculature. We previously demonstrated that bradykinin facilitates sympathetic neurotransmission in rat mesenteric arteries. Although little is known about the intracellular mechanism modulating this neurotransmission, recent cell line experiments have shown that the KCNQ channel, which is inhibited by the depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP₂), participates in the control of neurotransmission by bradykinin. In the present study, we examined the mechanism regulating neurotransmitter release from rat perivascular sympathetic nerves. Excitatory junction potentials (EJPs) elicited by repetitive nerve stimulation (1 Hz, 11 pulses, 20 μs, 20-50 V), a measure of sympathetic purinergic neurotransmission, were recorded with a conventional microelectrode technique in rat mesenteric arteries. Bradykinin (10⁻⁷ mol l⁻¹) significantly enhanced the amplitude of EJPs (n=22, P<0.05). This enhancing effect was abolished by N-type calcium-channel inhibition with ω-conotoxin GVIA (2 × 10⁻⁹ mol ⁻¹l, n=8). The blockade of phospholipase C with U-73122 (10(-6) mol l⁻¹, n=17) also eliminated the facilitatory effect of bradykinin. In addition, the effects of bradykinin were diminished by the prevention of PIP₂ resynthesis with wortmannin (10⁻⁵ mol l⁻¹ n=7) or KCNQ channel inhibition with XE-991 (10⁻⁵ mol l⁻¹, n=7). On the other hand, depletion of intracellular calcium stores with cyclopiazonic acid (3 × 10⁻⁶ mol l⁻¹, n=6) or the inhibition of protein kinase C with bisindolylmaleimide-I (10⁻⁶ mol l⁻¹, n=9) did not alter the action of bradykinin. These data demonstrate that the hydrolysis of PIP₂ by phospholipase C, which is activated by G(q/11)-coupled receptors, and subsequent KCNQ channel inhibition enhance sympathetic purinergic neurotransmission presumably via the activation of N-type calcium channels in rat mesenteric arteries.

Direct Activation of TRPV6 Channels by Phosphatidylinositol 4,5-Bisphosphate

Transient Receptor Potential Vanilloid 6 (TRPV6) is an inwardly rectifying Ca2+ selective ion channel that plays a role in Ca2+ absorption in the intestines. The activity of these channels have been shown to depend on the presence of phosphatidylinositol 4,5-bisphosphate (PIP2). Increased in cytoplasmic Ca2+ concentrations inhibit these channels, protecting cells from toxic Ca2+ overload. Both Ca2+ Calmodulin (CaCaM) and depletion of PIP2 have been implicated in Ca2+ induced inactivation. In addition, it has been proposed that cytoplasmic ATP binds directly to the channels, and this binding is important for maintaining TRPV6 channel activity in whole-cell patch clamp experiments. To evaluate whether PIP2, CaCaM and ATP affects channel activity directly or indirectly, we have performed excised inside-out patch clamp measurements with these compounds. Channel activity upon excision showed marked current run-down, characteristic of PIP2 dependent channels. ATP reactivated the channels, but only in the presence of Mg2+. This could mean that MgATP provides substrate for lipid kinases present in the patch membrane, allowing the resynthesis of PIP2. To test this hypothesis, we have used wortmannin and LY294002 at concentrations where they inhibit phosphatidylinositol 4-kinases, and found that they both inhibit the effect of MgATP in excised patches. At lower concentrations, where they specifically inhibit PI3Kinases, they did not inhibit the effects of MgATP. We also found that PIP2 reactivates TRPV6 in excised patches, and this effect is inhibited by CaCaM. To further establish PIP2 as a direct activator of TRPV6, we purified the channel protein and reconstituted it in planar lipid bilayers. We found that the reconstituted protein showed inward rectification, and its activity depended on the presence of PIP2. Our data establish PIP2 as a direct activator of TRPV6 and raise doubt that intracellular ATP regulates the channel through direct binding.

Voltage-Sensitive PIP2 Phosphatase Shows PIP2 Requirements for KCNQ and Ca 2+ Channels

The four talks in this symposium highlight the dependence of ion channel function on membrane phospholipids and fatty acids. Although of low abundance (1-2%), the phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) is a signature lipid of plasma membranes. The PIP2 lipid aids ion channel function there, and depleting PIP2 can decrease the activity of >80 types of channels (Logothetis et al, Pflugers 2010). A voltage sensitive 5-phosphatase (VSP) converts PIP2 to PI(4)P rapidly during strong depolarizing voltage steps (Okamura et al, J Physiol 2009). Exogenously expressed, this VSP serves to test the PIP2 dependence of plasma membrane ion channels and the kinetics of resynthesis of PIP2. Using the PLCdelta1 PH (pleckstrin homology) domain as a reporter, we measure the membrane PIP2 concentration as a change in FRET (Forster resonance energy transfer) between YFP-PH and CFP-PH. The PH-domain assay reports that strongly activated VSP depletes PIP2 with a time constant <150 ms. Activating VSP for 500 ms with a +100 mV depolarization reduces current in KCNQ channels by 90% (Falkenburger et al, JGP 2010) and reduces current in four subtypes of voltage-gated Ca2+ channels by 30-60% (Suh et al, Neuron 2010). The channel currents and the PH domain signals recover with time constants of 7-15 s. Such observations imply that: (1) KCNQ channels and several Ca2+ channels need PIP2 for best operation, (2) the PIP2 likely binds directly to the channel protein, (3) PH domains and channels form low-affinity complexes with PIP2 that last < 10 ms, and (4) resynthesis of PIP2 from plasma membrane PI(4)P takes only ∼10 s. This resynthesis from PI(4)P is much faster than the 150-s time seen for two-step resynthesis from PI after activation of receptors coupled to PLC. Support: NIH grants NS08174, GM83913.

Depolarization Increases Phosphatidylinositol (PI) 4,5-Bisphosphate Level and KCNQ Currents through PI 4-Kinase Mechanisms

A growing body of evidence shows that membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P(2), PIP(2)) play an important role in cell signaling. The presence of PIP(2) is fundamentally important for maintaining the functions of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility, and endo- and exocytosis. PIP(2) levels in the membrane are dynamically modulated, which is an important signaling mechanism for modulation of PIP(2)-dependent cellular processes. In this study, we describe a novel mechanism of membrane PIP(2) modulation. Membrane depolarization induces an elevation in membrane PIP(2), and subsequently increases functions of PIP(2)-sensitive KCNQ potassium channels expressed in Xenopus oocytes. Further evidence suggests that the depolarization-induced elevation of membrane PIP(2) occurs through increased activity of PI4 kinase. With increased recognition of the importance of PIP(2) in cell function, the effect of membrane depolarization in PIP(2) metabolism is destined to have important physiological implications.

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.

A lipid kinase controls the maintenance of dendritic spines.

A lipid kinase controls the maintenance of dendritic spines.