The mood stabilizer valproate inhibits both inositol- and diacylglycerol-signaling pathways in Caenorhabditis elegans

The antiepileptic valproate (VPA) is widely used in the treatment of bipolar disorder, although the mechanism of its action in the disorder is unclear. We show here that VPA inhibits both inositol phosphate and diacylglycerol (DAG) signaling in Caenorhabditis elegans. VPA disrupts two behaviors regulated by the inositol-1,4,5-trisphosphate (IP(3)): defecation and ovulation. VPA also inhibits two activities regulated by DAG signaling: acetylcholine release and egg laying. The effects of VPA on DAG signaling are relieved by phorbol ester, a DAG analogue, suggesting that VPA acts to inhibit DAG production. VPA reduces levels of DAG and inositol-1-phosphate, but phosphatidylinositol-4,5-bisphosphate (PIP(2)) is slightly increased, suggesting that phospholipase C-mediated hydrolysis of PIP(2) to form DAG and IP(3) is defective in the presence of VPA.     

Regulation of PIP5K activity by Arf6 and its physiological significance

The phospholipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the phosphorylation of the membrane phospholipid phosphatidylinositol 4-phosphate to generate the pleiotropic phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2) ]. To date, three mammalian PIP5K isozymes, α, β, and γ, and several splicing variants of the γ isozyme have been identified. These PIP5K isozymes and PIP5Kγ variants play critical roles in various cellular functions through their product PI(4,5)P(2) . The small GTPase Arf6 is one of the key activators of PIP5K. Increasing evidence suggests that PIP5K functions as a downstream effector of Arf6 to regulate a wide variety of cellular functions, such as exocytosis, endocytosis, endosomal recycling, membrane ruffle formation, immune response, and bacterial invasion. In this review, we place our focus on the recent advances in Arf6/PIP5K signaling and its linkage to cellular functions.     

A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression.

The generation of diacylglycerol (DAG) in response to receptor stimulation is a well-documented signalling mechanism that leads to activation of protein kinase C (PKC). Putative alternative effectors contain sequences that interact with DAGs, but the mechanisms of signal transduction are unknown. We have identified a Dictyostelium gene encoding a novel protein which contains a domain with high identity to the DAG-binding domain of PKC. It does not encode a PKC homologue as the conservation does not extend outside this region. We confirm that the proposed DAG-binding domain is sufficient to mediate interaction of a fusion protein with vesicles containing DAG. The protein also shows significant homology to mammalian phosphatidylinositol phosphate (PIP) kinases and we show that this domain has PIP kinase activity. The protein, PIPkinA, is enriched in the nucleus and abrogation of gene function by homologous recombination inhibits early developmental gene expression, blocking development at an early stage. Thus, we have identified a PIP kinase from Dictyostelium which is required for development, is a candidate effector for DAG and has the potential to synthesize nuclear PIP(2).     

Molecular mechanism underlying phosphatidylinositol 4,5-bisphosphate-induced inhibition of SpIH channels

Many ion channels have been shown to be regulated by the membrane signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here, we demonstrate that the binding of PIP(2) to SpIH, a sea urchin hyperpolarization-activated cyclic nucleotide-gated ion channel (HCN), has a dual effect: potentiation and inhibition. The potentiation is observed as a shift in the voltage dependence of activation to more depolarized voltages. The inhibition is observed as a reduction in the currents elicited by the partial agonist cGMP. These two effects were separable and arose from PIP(2) binding to two different regions. Deletion of the C-terminal region of SpIH removed PIP(2)-induced inhibition but not the PIP(2)-induced shift in voltage dependence. Mutating key positively charged amino acids in the C-terminal region adjacent to the membrane selectively disrupted PIP(2)-induced inhibition, suggesting a direct interaction between PIP(2) in the membrane and amino acids in the C-terminal region that stabilizes the closed state relative to the open state in HCN channels.     

Calmodulin-binding and autoinhibitory domains of Acanthamoeba myosin I heavy chain kinase, a p21-activated kinase (PAK)

The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MIHCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated full-length MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca(2+)-calmodulin. In contrast to Dictyostelium MIHCK, however, Ca(2+)-calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.     

PIP(2) activates KCNQ channels,and its hydrolysis underlies receptor-mediated inhibition of M currents

KCNQ channels belong to a family of potassium ion channels with crucial roles in physiology and disease. Heteromers of KCNQ2/3 subunits constitute the neuronal M channels. Inhibition of M currents, by pathways that stimulate phospholipase C activity, controls excitability throughout the nervous system. Here we show that a common feature of all KCNQ channels is their activation by the signaling membrane phospholipid phosphatidylinositol-bis-phosphate (PIP(2)). We show that wortmannin, at concentrations that prevent recovery from receptor-mediated inhibition of M currents, blocks PIP(2) replenishment to the cell surface. Moreover, we identify a C-terminal histidine residue, immediately proximal to the plasma membrane, mutation of which renders M channels less sensitive to PIP(2) and more sensitive to receptor-mediated inhibition. Finally, native or recombinant channels inhibited by muscarinic agonists can be activated by PIP(2). Our data strongly suggest that PIP(2) acts as a membrane-diffusible second messenger to regulate directly the activity of KCNQ currents.     

Identification of protein kinase B (PKB) as a phosphatidylinositol 3,4,5-trisphosphate binding protein in Dictyostelium discoideum

We have searched for phosphatidylinositol (PI)-3,4,5-trisphosphate (PIP3) binding proteins in Dictyostelium discoideum using beads bearing a PIP3 analogue, PIP3-APB. One of the binding proteins with a molecular mass of 55 kDa was purified and its amino acid sequence was partially analyzed. Database searches showed that the analyzed sequence was identical to that of protein kinase B (PKB) of D. discoideum. The specific activity of D. discoideum PKB, when expressed together with constitutively active PI-3 kinase in mammalian cells, was elevated by about three-fold, suggesting that PKB could also act downstream of PI-3 kinase in Dictyostelium cells.     

The neuroprotective agent, valproic acid, regulates the mitogen-activated protein kinase pathway through modulation of protein kinase A signalling in Dictyostelium discoideum

Activation of the mitogen-activated protein kinase (MAPK) cascade gives rise to a neuroprotective effect in a variety of cell types. The bipolar disorder treatment, valproic acid (VPA), increases the activity of this pathway by modulating extracellular signal-regulated kinase 2 (ERK2) phosphorylation through an unknown mechanism. To investigate the molecular basis of this effect, we have used the biomedical model system Dictyostelium discoideum to dissect this signalling pathway. We find that, similar to mammalian systems, VPA causes a transient increase in the activation of the MAPK signalling pathway, as shown by ERK2 phosphorylation. We show that the MAP kinase and phosphatase, protein kinase A (PKA) and glycogen synthase kinase signalling pathways all function in controlling the levels of phospho-ERK2 (pERK2). We find that VPA induces elevated pERK2 levels through attenuation of the PKA signalling pathway. Interestingly, pERK2 levels are also controlled by another bipolar disorder drug, lithium, providing a common effect of these two drugs. This work therefore suggests a conserved pathway in eukaryotes that is targeted by neuroprotective and bipolar disorder drugs and allows us to propose a model for this neuroprotective effect.     

Mechanism of PLC-mediated Kir3 current inhibition

A large number of ion channels maintain their activity through direct interactions with phosphatidylinositol bisphosphate (PIP2). For such channels, hydrolysis of PIP2 causes current inhibition. It has become controversial whether the inhibitory effects on channel activity represent direct effects of PIP2 hydrolysis or of downstream PKC action. We studied Phospholipase C (PLC)-dependent inhibition of G protein-activated inwardly rectifying K+ (Kir3) channels. By monitoring simultaneously channel activity and PIP2 hydrolysis, we determined that both direct PIP2 depletion and PKC actions contribute to Kir3 current inhibition. We show that the PKC-induced effects strongly depend on PIP2 levels in the membrane. At the same time, we show that PKC destabilizes Kir3/PIP2 interactions and enhances the effects of PIP2 depletion on channel activity. These results demonstrate that PIP2 depletion and PKC-mediated effects reinforce each other and suggest that both of these interdependent mechanisms contribute to Kir3 current inhibition. This mechanistic insight may explain how even minor changes in PIP2 levels can have profound effects on Kir3 activity. We also show that stabilization of Kir3/PIP2 interactions by Gbetagamma attenuates both PKC and Gq-mediated current inhibition, suggesting that diverse pathways regulate Kir3 activity through modulation of channel interactions with PIP2.     

Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration

Phosphatidylinositol 3-OH kinase (PI3K) has been widely studied as a principal regulator of cell polarization, migration, and chemotaxis. Surprisingly, recent studies showed that mammalian neutrophils and Dictyostelium discoideum cells can polarize and migrate in the absence of PI3K activity. Here we directly probe the roles of PI3K and its downstream effector, Rac, in HL-60 neutrophils by using a chemical biology approach whereby the endogenously present enzymes are synthetically activated in less than one minute. We show that uniform activation of endogenous PI3K is sufficient to polarize previously unpolarized neutrophils and trigger effective cell migration. After a delay following symmetrical phosphatidylinositol (3,4,5)-triphosphate (PIP(3)) production, a polarized distribution of PIP(3) was induced by positive feedback requiring actin polymerization. Pharmacological studies argue that this process does not require receptor-coupled trimeric G proteins. Contrary to the current working model, rapid activation of endogenous Rac proteins triggered effective actin polymerization but failed to feed back to PI3K to generate PIP(3) or induce cell polarization. Thus, the increase in PIP(3) concentration at the leading edge is generated by positive feedback with an AND gate logic with a PI3K-Rac-actin polymerization pathway as a first input and a PI3K initiated non-Rac pathway as a second input. This AND-gate control for cell polarization can explain how Rac can be employed for both PI3K-dependent and -independent signaling pathways coexisting in the same cell.     

Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton

The antiepileptic drug valproic acid (VPA) and teratogenic VPA analogues have been demonstrated to inhibit cell motility and affect cell morphology. We here show that disruption of microtubules or of microfilaments by exposure to nocodazole or cytochalasin D had different effects on morphology of control cells and cells treated with VPA, indicating that VPA affected the cytoskeletal determinants of cell morphology. Furthermore, VPA treatment induced an increase of F-actin, and of FAK, paxillin, vinculin, and phosphotyrosine in focal adhesion complexes. These changes were accompanied by increased adhesion of VPA-treated cells to the extracellular matrix. Treatment with an RGD-containing peptide reducing integrin binding to components of the extracellular matrix partially reverted the motility inhibition induced by VPA, indicating that altered adhesion contributed to, but was not the sole reason for the VPA mediated inhibition of motility. In addition it is shown that the actomyosin cytoskeleton of VPA-treated cells was capable of contraction upon exposure to ATP, indicating that the reduced motility of VPA-treated cells was not caused by an inhibition of actomyosin contraction. On the other hand, VPA caused a redistribution of the actin severing protein gelsolin, and left the cells unable to respond to treatment with a gelsolin-peptide known to reduce the amount of gelsolin bound to phosphatidylinositol bisphosphate (PIP2), leaving a larger amount of the protein in a potential actin binding state. These findings indicate that VPA affects cell morphology and motility through interference with the dynamics of the actin cytoskeleton.     

Apical membrane segregation of phosphatidylinositol-4,5-bisphosphate influences parathyroid hormone 1 receptor compartmental signaling and localization via direct regulation of ezrin in LLC-PK1 cells

The parathyroid hormone 1 receptor (PTH1R), a primary regulator of mineral ion homeostasis, is expressed on both the apical and basolateral membranes of kidney proximal tubules and in the LLC-PK1 kidney cell line. In LLC-PK1 cells, apical PTH1R subpopulations are far more effective at signaling via phospholipase (PLC) than basolateral counterparts, revealing the presence of compartmental signaling. Apical PTH1R localization is dependent upon direct interactions with ezrin, an actin-membrane cross-linking scaffold protein. Ezrin undergoes an activation process that is dependent upon phosphorylation and binding to phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid that is selectively concentrated to apical surfaces of polarized epithelia. Consistently, the intracellular probe for PIP2, GFP-PLCδ1-PH, localizes to the apical membranes of LLC-PK1 cells, directly overlapping ezrin and PTH1R expression. Activation of the apical PTH1R shifts the GFP-PLCδ1-PH probe from the apical membrane to the cytosol and basolateral membranes, reflecting domain-specific activation of PLC and hydrolysis of PIP2. This compartmental signaling is likely due to the polarized localization of PIP2, the substrate for PLC. PIP2 degradation using a membrane-directed phosphatase shifts ezrin localization to the cytosol and induces ezrin de-phosphorylation, processes consistent with inactivation. PIP2 degradation also shifts PTH1R expression from brush border microvilli to basolateral membranes and markedly blunts PTH-elicited activation of the MAPK pathway. Transient expression of ezrin in HEK293 cells shifts PTH1R expression from the plasma membrane to microvilli-like surface projections that also contain PIP2. As a result, ezrin enhances PTH mediated activation of the PLC pathway in this cell model with increasing total receptor surface expression. Collectively, these findings demonstrate that the apical segregation of PIP2 to the apical domains not only promotes the activation of ezrin and the subsequent formation of the PTH1R containing scaffold, but also ensures the presence of ample substrate for propagating the PLC pathway.     

Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.     

Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors

Valproic acid (VPA) has been used as an anticonvulsant agent for the treatment of epilepsy, as well as a mood stabilizer for the treatment of bipolar disorder, for several decades. The mechanism of action for these effects remains to be elucidated and is most likely multifactorial. Recently, VPA has been reported to inhibit histone deacetylase (HDAC) and HDAC has been reported to play roles in differentiation of mammalian cells. In this study, the effects of HDAC inhibitors on differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) and bone marrow stromal cells (hBMSC) were determined. VPA increased osteogenic differentiation in a dose dependent manner. The pretreatment of VPA before induction of differentiation also showed stimulatory effects on osteogenic differentiation of hMSC. Trichostatin A (TSA), another HDAC inhibitor, also increased osteogenic differentiation, whereas valpromide (VPM), a structural analog of VPA which does not possess HDAC inhibitory effects, did not show any effect on osteogenic differentiation on hADSC. RT-PCR and Real-time PCR analysis revealed that VPA treatment increased osterix, osteopontin, BMP-2, and Runx2 expression. The addition of noggin inhibited VPA-induced potentiation of osteogenic differentiation. VPA inhibited proliferation of hADSC and hBMSC. Our results suggest that VPA enhance osteogenic differentiation, probably due to inhibition of HDAC, and could be useful for in vivo bone engineering using hMSC.     

The MARCKS protein plays a critical role in phosphatidylinositol 4,5-bisphosphate metabolism and directed cell movement in vascular endothelial cells

The MARCKS protein (myristoylated alanine-rich C kinase substrate) is an actin- and calmodulin-binding protein that is expressed in many mammalian tissues. The role of MARCKS in endothelial signaling responses is incompletely understood. We found that siRNA-mediated knockdown of MARCKS in cultured endothelial cells abrogated directed cell movement in a wound healing assay. We used biochemical and cell imaging approaches to explore the role of MARCKS in endothelial signal transduction pathways activated by insulin. Insulin treatment of vascular endothelial cells promoted the dose- and time-dependent phosphorylation of MARCKS. Cell imaging and hydrodynamic approaches revealed that MARCKS is targeted to plasmalemmal caveolae and undergoes subcellular translocation in response to insulin. Insulin treatment promoted an increase in levels of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) in plasmalemmal caveolae. The insulin-stimulated increase in caveolar PIP(2) was blocked by siRNA-mediated knockdown of MARCKS, as determined using both biochemical assays and imaging studies using FRET-based PIP(2) biosensors. The critical role of PIP(2) in MARCKS responses was explored by examining the PIP(2)- and actin-binding proteins Arp2/3 and N-WASP. Insulin promoted the rapid and robust phosphorylation of both N-WASP and Arp2/3, but these phosphorylation responses were markedly attenuated by siRNA-mediated MARCKS knockdown. Moreover, MARCKS knockdown effectively abrogated N-WASP activation in response to insulin, as determined using a FRET-based N-WASP activity biosensor. Taken together, these studies show that MARCKS plays a key role in insulin-dependent endothelial signaling to PIP(2) and is a critical determinant of actin assembly and directed cell movement in the vascular endothelium.     

Cell aggregation-induced FGF8 elevation is essential for P19 cell neural differentiation

FGF8, a member of the fibroblast growth factor (FGF) family, has been shown to play important roles in different developing systems. Mouse embryonic carcinoma P19 cells could be induced by retinoic acid (RA) to differentiate into neuroectodermal cell lineages, and this process is cell aggregation dependent. In this report, we show that FGF8 expression is transiently up-regulated upon P19 cell aggregation, and the aggregation-dependent FGF8 elevation is pluripotent stem cell related. Overexpressing FGF8 promotes RA-induced monolayer P19 cell neural differentiation. Inhibition of FGF8 expression by RNA interference or blocking FGF signaling by the FGF receptor inhibitor, SU5402, attenuates neural differentiation of the P19 cell. Blocking the bone morphogenetic protein (BMP) pathway by overexpressing Smad6 in P19 cells, we also show that FGF signaling plays a BMP inhibition-independent role in P19 cell neural differentiation.     

Inhibition of neuronal nitric oxide synthase by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid.

Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent K(m) for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca(2+)-calmodulin was observed in the presence of PIP2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca(2+)-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP2 and PA. These results strongly suggest that PIP2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca(2+)-calmodulin.     

The helically extended SH3 domain of the T cell adaptor protein ADAP is a novel lipid interaction domain

Adhesion and degranulation-promoting adapter protein (ADAP) is critically involved in downstream signalling events triggered by the activation of the T cell receptor. Cytokine production, proliferation and integrin clustering of T cells are dependent on ADAP function, but the molecular basis for these processes is poorly understood. We now show the hSH3 domain of ADAP to be a lipid-interaction module that binds to acidic lipids, including phosphatidylinositides. Positively charged surface patches of the domain preferentially bind to polyvalent acidic lipids such as PIP2 or PIP3 over the monovalent PS phospholipid and this interaction is dependent on the N-terminal helix of the hSH3 domain fold. Basic amino acid side-chains from the SH3 scaffold also contribute to lipid binding. In the context of T cell signalling, our findings suggest that ADAP, upon recruitment to the cell-cell junction as part of a multiprotein complex, directly interacts with phosphoinositide-enriched regions of the plasma membrane. Furthermore, the ADAP lipid interaction defines the helically extended SH3 scaffold as a novel member of membrane interaction domains.     

A role for PtdIns(4,5)P2 and PIP5Kalpha in regulating stress-induced apoptosis

The phosphoinositide phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) is essential for many cellular processes and is linked to the etiology of numerous human diseases . PtdIns(4,5)P(2) has been indirectly implicated as a negative regulator of apoptosis ; however, it is unclear if apoptotic stimuli negatively regulate PtdIns(4,5)P(2) levels in vivo. Here, we show that two apoptotic-stress stimuli, hydrogen peroxide (H(2)O(2)) and UV irradiation, cause PtdIns(4,5)P(2) depletion during programmed cell death independently of and prior to caspase activation. Depletion of PtdIns(4,5)P(2) is essential for apoptosis because maintenance of PtdIns(4,5)P(2) levels by overexpression of PIP5Kalpha rescues cells from H(2)O(2)-induced apoptosis. PIP5Kalpha expression promotes both basal and sustained ERK1/2 activation after H(2)O(2) treatment, and importantly, pharmacological inhibition of ERK1/2 signaling blocks PIP5Kalpha-mediated cell survival. H(2)O(2) induces tyrosine phosphorylation and translocation of PIP5Kalpha away from its substrate at the plasma membrane, and both are dependent upon the activity of c-src family kinases. Furthermore, constitutively active c-src enhances tyrosine phosphorylation of PIP5Kalpha in vivo and is sufficient for the translocation of PIP5Kalpha away from the plasma membrane. These observations demonstrate that certain apoptotic stimuli initiate an essential signaling pathway during cell death, and this pathway leads to caspase-independent downregulation of PIP5Kalpha and its product PtdIns(4,5)P(2).     

Direct regulation of BK channels by phosphatidylinositol 4,5-bisphosphate as a novel signaling pathway

Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)) only through phospholipase C (PLC)-generated PIP(2) metabolites that target Ca(2+) stores and protein kinase C and, eventually, the BK channel. Here, we report that PIP(2) activates BK channels independently of PIP(2) metabolites. PIP(2) enhances Ca(2+)-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PIP(2) acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PIP(2) (diC16). The PIP(2)-channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6-S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PIP(2)-induced activation is drastically potentiated by accessory beta(1) (but not beta(4)) channel subunits. Moreover, PIP(2) robustly activates BK channels in vascular myocytes, where beta(1) subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PIP(2) effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, cotransfection of cbv1+beta(1) and PI4-kinaseIIalpha robustly activates BK channels, suggesting a role for endogenous PIP(2) in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PIP(2) antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PIP(2) levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PIP(2) directly activates vascular myocyte BK channels to control vascular tone.