Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions

Direct interactions of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) with inwardly rectifying potassium channels are stronger with channels rendered constitutively active by binding to PtdIns(4,5)P2, such as IRK1, than with G-protein-gated channels (GIRKs). As a result, PtdIns(4,5)P2 alone can activate IRK1 but not GIRKs, which require extra gating molecules such as the beta gamma subunits of G proteins or sodium ions. Here we identify two conserved residues near the inner-membrane interface of these channels that are critical in interactions with PtdIns(4,5)P2. Between these two arginines, a conservative change of isoleucine residue 229 in GIRK4 to the corresponding leucine found in IRK1 strengthens GIRK4-PtdIns(4,5)P2 interactions, eliminating the need for extra gating molecules. A negatively charged GIRK4 residue, two positions away from the most strongly interacting arginine, mediates stimulation of channel activity by sodium by strengthening channel-PtdIns(4,5)P2 interactions. Our results provide a mechanistic framework for understanding how distinct gating mechanisms of inwardly rectifying potassium channels allow these channels to subserve their physiological roles.     

Distinct specificities of inwardly rectifying K(+) channels for phosphoinositides

Activation of several inwardly rectifying K(+) channels (Kir) requires the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The constitutively active Kir2.1 (IRK1) channels interact with PtdIns(4,5)P(2) strongly, whereas the G-protein activated Kir3.1/3.4 channels (GIRK1/GIRK4), show only weak interactions with PtdIns(4,5)P(2). We investigated whether these inwardly rectifying K(+) channels displayed distinct specificities for different phosphoinositides. IRK1, but not GIRK1/GIRK4 channels, showed a marked specificity toward phosphates in the 4,5 head group positions. GIRK1/GIRK4 channels were activated with a similar efficacy by PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). In contrast, IRK1 channels were not activated by PtdIns(3,4)P(2) and only marginally by high concentrations of PtdIns(3,5)P(2). Similarly, high concentrations of PtdIns(3,4,5)P(3) were required to activate IRK1 channels. For either channel, PtdIns(4)P was much less effective than PtdIns(4,5)P(2), whereas PtdIns was inactive. In contrast to the dependence on the position of phosphates of the phospholipid head group, GIRK1/GIRK4, but not IRK1 channel activation, showed a remarkable dependence on the phospholipid acyl chains. GIRK1/GIRK4 channels were activated most effectively by the natural arachidonyl stearyl PtdIns(4,5)P(2) and much less by the synthetic dipalmitoyl analog, whereas IRK1 channels were activated equally by dipalmitoyl and arachidonyl stearyl PtdIns(4,5)P(2). Incorporation of PtdInsP(2) into the membrane is necessary for activation, as the short chain water soluble diC(4) PtdIns(4,5)P(2) did not activate either channel, whereas activation by diC(8) PtdIns(4, 5)P(2) required high concentrations.     

Movin' on up: the role of PtdIns(4,5)P(2) in cell migration

Cell migration requires the coordination of many biochemical events, including cell-matrix contact turnover and cytoskeletal restructuring. Recent advances further implicate phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)] in the control of these events. Many proteins that are crucial to the assembly of the migration machinery are regulated by PtdIns(4,5)P(2). Coordinated synthesis of PtdIns(4,5)P(2) at these sites is dependent on the precise targeting of the type I phosphatidylinositol phosphate kinases (PIPKs). Two PIPKI isoforms target to, and generate, PtdIns(4,5)P(2) at membrane ruffles and focal adhesions during cell migration. Here, we discuss our current understanding of PtdIns(4,5)P(2) in the regulation of cell responses to migratory stimuli and how the migrating cell controls PtdIns(4,5)P(2) availability.     

Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools

Regulation of PIPK (phosphatidylinositol phosphate kinase) and PtdIns(4,5)P2 signalling by small G-proteins and their effectors is key to many biological functions. Through selective recruitment and activation of different PIPK isoforms, small G-proteins such as Rho, Rac and Cdc42 modulate actin dynamics and cytoskeleton-dependent cellular events in response to extracellular signalling. These activities affect a number of processes, including endocytosis, bacterial penetration into host cells and cytolytic granule-mediated targeted cell killing. Small G-proteins and their modulators are also regulated by phosphoinositides through translocation and conformational changes. Arf family small G-proteins act at multiple sites as regulators of membrane trafficking and actin cytoskeletal remodelling, and regulate a feedback loop comprising phospholipase D, phosphatidic acid, PIPKs and PtdIns(4,5)P2, contributing to enhancement of PtdIns(4,5)P2-mediated cellular events and receptor signalling. Na+, Kir (inwardly rectifying K+), Ca2+ and TRP (transient receptor potential) ion channels are regulated by small G-proteins and membrane pools of PtdIns(4,5)P2. Yeast phosphatidylinositol 4-phosphate 5-kinases Mss4 and Its3 are involved in resistance against disturbance of sphingolipid biosynthesis and maintenance of cell integrity through the synthesis of PtdIns(4,5)P2 and downstream signalling through the Rom2/Rho2 and Rgf1/Rho pathways. Here, we review models for regulated intracellular targeting of PIPKs by small G-proteins and other modulators in response to extracellular signalling. We also describe the spatial and temporal cross-regulation of PIPKs and small G-proteins that is critical for a number of cellular functions.     

Phosphatidylinositol 4,5-bisphosphate (PIP2) modulation of ATP and pH sensitivity in Kir channels. A tale of an active and a silent PIP2 site in the N terminus

Phosphatidylinositol polyphosphates (PIPs) are potent modulators of Kir channels. Previous studies have implicated basic residues in the C terminus of Kir6.2 channels as interaction sites for the PIPs. Here we examined the role of the N terminus and identified an arginine (Arg-54) as a major determinant for PIP(2) modulation of ATP sensitivity in K(ATP) channels. Mutation of Arg-54 to the neutral glutamine (R54Q) and, in particular, to the negatively charged glutamate (R54E) impaired PIP(2) modulation of ATP inhibition, while mutation to lysine (R54K) had no effect. These data suggest that electrostatic interactions between PIP(2) and Arg-54 are an essential step for the modulation of ATP sensitivity. This N-terminal PIP(2) site is highly conserved in Kir channels with the exception of the pH-gated channels Kir1.1, Kir4.1, and Kir5.1 that contain a neutral residue at the corresponding positions. Introduction of an arginine at this position in Kir1.1 channels rendered the N-terminal PIP(2) site functional largely increasing the PIP(2) affinity. Moreover, Kir1.1 channels lose the ability to respond to physiological changes of the intracellular pH. These results explain the need of a silent N-terminal PIP(2) site in pH-gated channels and highlight the N terminus as an important region for PIP(2) modulation of Kir channel gating.     

CRAC channel is inhibited by neomycin in a Ptdlns(4,5)P2‐independent manner

Depletion of intracellular Ca²⁺ stores evokes store‐operated Ca²⁺ entry through the Ca²⁺ release‐activated Ca²⁺ (CRAC) channels. In this study, we found that the store‐operated Ca²⁺ entry was inhibited by neomycin, an aminoglycoside that strongly binds phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Patch clamp recordings revealed that neomycin blocked the CRAC currents reconstituted by co‐expression of Orai1 and Stim1 in HEK293 cells. Using a rapamycin‐inducible PtdIns(4,5)P2‐specific phosphatase (Inp54p) system to manipulate the PtdIns(4,5)P2 in the plasma membrane, we found that the CRAC current was not altered by PtdIns(4,5)P2 depletion. This result suggests that PtdIns(4,5)P2 is not required for CRAC channel activity, and thereby, neomycin inhibits CRAC channels in a manner that is independent of neomycin–PtdIns(4,5)P2 binding. Copyright © 2015 John Wiley & Sons, Ltd.     

PtdIns(4,5)P2 turnover is required for multiple stages during clathrin- and actin-dependent endocytic internalization

The lipid phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P(2)) appears to play an important role in endocytosis. However, the timing of its formation and turnover, and its specific functions at different stages during endocytic internalization, have not been established. In this study, Sla2 ANTH-GFP and Sjl2-3GFP were expressed as functional fusion proteins at endogenous levels to quantitatively explore PtdIns(4,5)P(2) dynamics during endocytosis in yeast. Our results indicate that PtdIns(4,5)P(2) levels increase and decline in conjunction with coat and actin assembly and disassembly, respectively. Live-cell image analysis of endocytic protein dynamics in an sjl1Delta sjl2Delta mutant, which has elevated PtdIns(4,5)P(2) levels, revealed that the endocytic machinery is still able to assemble and disassemble dynamically, albeit nonproductively. The defects in the dynamic behavior of the various endocytic proteins in this double mutant suggest that PtdIns(4,5)P(2) turnover is required for multiple stages during endocytic vesicle formation. Furthermore, our results indicate that PtdIns(4,5)P(2) turnover may act in coordination with the Ark1/Prk1 protein kinases in stimulating disassembly of the endocytic machinery.     

A single amino acid mutation attenuates rundown of voltage-gated calcium channels

The activity of voltage-gated calcium channels (VGCCs) decreases with time in whole-cell and inside-out patch-clamp recordings. In this study we found that substituting a single amino acid (I1520) at the intracellular end of IIIS6 in the alpha(1) subunit of P/Q-type Ca(2+) channels with histidine or aspartate greatly attenuated channel rundown in inside-out patch-clamp recordings. The homologous mutations also slowed rundown of N- and L-type Ca(2+) channels, albeit to a lesser degree. In P/Q-type channels, the attenuation of rundown is accompanied by an increased apparent affinity for phosphatidylinositol-4,5-bisphosphate, which has been shown to be critical for maintaining Ca(2+) channel activity [L. Wu, C.S. Bauer, X.-G. Zhen, C. Xie, J. Yang, Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419 (2002) 947-952]. Furthermore, the histidine mutation significantly stabilized the open state, making the channels easier to open, slower to close, harder to inactivate and faster to recover from inactivation. Our finding that mutation of a single amino acid can greatly attenuate rundown provides an easy and efficient way to slow the rundown of VGCCs, facilitating functional studies that require direct access to the cytoplasmic side of the channel.     

Structure and dynamics of the pore of inwardly rectifying K(ATP) channels

Inwardly rectifying K(+) currents are generated by a complex of four Kir (Kir1-6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteine-interacting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying K(ATP) channels. The ability of Cd(2+) ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd(2+) accessibility is consistent with an alpha-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. In several mutants, single channel measurements indicated that inhibition occurred by a single transition from the open state to a zero-conductance state. Analysis of currents expressed from mixtures of control and L164C mutant subunits indicated that at least three cysteines are required for coordination of the Cd(2+) ion. Application of phosphatidylinositol 4,5-diphosphate to inside-out membrane patches stabilized the open state of all mutants and also reduced cadmium sensitivity. Moreover, the Cd(2+) sensitivity of several mutants was greatly reduced in the presence of inhibitory ATP concentrations. Taken together, these results are consistent with state-dependent accessibility of single Cd(2+) ions to coordination sites within a relatively narrow inner vestibule.     

Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella

Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.     

PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.     

Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate

Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein-coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P(2)) from the plasma membrane. Knockdown of phospholipase Cbeta3 (PLCbeta3) inhibits PtdIns(4,5)P(2) hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5)P(2) depletion is sufficient to close Cx43 channels. When PtdIns(4,5)P(2) is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We find that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLCbeta3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5)P(2)-hydrolyzing receptors to inhibit cell-cell communication, but not for receptor-PLC coupling. Our results show that PtdIns(4,5)P(2) is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLCbeta3 and Cx43 into a signaling complex to allow regulation of cell-cell communication by localized changes in PtdIns(4,5)P(2).     

Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro.

We studied the possibility that hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] may be the initiating event for the increase in [32P]Pi incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) during carbachol and pancreozymin (cholecystokinin-octapeptide) action in the rat pancreas. After prelabelling acini for 2h, [32P]Pi incorporation into PtdA, PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P) had reached equilibrium. Subsequent addition of carbachol or pancreozymin caused 32P in PtdIns(4,5)P2 to decrease by 30-50% within 10-15 s, and this was followed by sequential increases in [32P]Pi incorporation into PtdA and PtdIns. Similar changes in 32P-labelling of PtdIns4P were not consistently observed. Confirmation that the decrease in 32P in chromatographically-purified PtdIns(4,5)P2 reflected an actual decrease in this substance was provided by the fact that similar results were obtained (a) when PtdIns(4,5)P2 was prelabelled with [2-3H]inositol, and (b) when PtdIns(4,5)P2 was measured as its specific product (glycerophosphoinositol bisphosphate) after methanolic alkaline hydrolysis and ion-exchange chromatography. The secretogogue-induced breakdown of PtdIns(4,5)P2 was not inhibited by Ca2+ deficiency (severe enough to inhibit amylase secretion and Ca2+-dependent hydrolysis of PtdIns), and ionophore A23187 treatment did not provoke PtdIns(4,5)P2 hydrolysis. The increase in the hydrolysis of PtdIns(4,5)P2 and the increase in [32P]Pi incorporation into PtdA commenced at the same concentration of carbachol in dose-response studies. Our findings suggest that the hydrolysis of PtdIns(4,5)P2 is an early event in the action of pancreatic secretogogues that mobilize Ca2+, and it is possible that this hydrolysis may initiate the Ca2+-independent labelling of PtdA and PtdIns. Ca2+ mobilization may follow these responses, and subsequently cause Ca2+-dependent hydrolysis of PtdIns and exocytosis.     

Differential roles of phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in plasma membrane targeting of C2 domains. Molecular dynamics simulation, membrane binding, and cell translocation studies of the PKCalpha C2 domain

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. PtdIns(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P(2) and/or PtdIns(3,4,5)P(3) from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.     

Distant Cytosolic Residues Mediate a Two-way Molecular Switch That Controls the Modulation of Inwardly Rectifying Potassium (Kir) Channels by Cholesterol and Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. An emerging feature of several Kir channels is that they are regulated by cholesterol. However, the mechanism by which cholesterol affects channel function is unclear. Here we show that mutations of two distant Kir2.1 cytosolic residues, Leu-222 and Asn-251, form a two-way molecular switch that controls channel modulation by cholesterol and affects critical hydrogen bonding. Notably, these two residues are linked by a residue chain that continues from Asn-251 to connect adjacent subunits. Furthermore, our data indicate that the same switch also regulates the sensitivity of the channels to phosphatidylinositol 4,5-bisphosphate, a phosphoinositide that is required for activation of Kir channels. Thus, although cholesterol and phosphatidylinositol 4,5-bisphosphate do not interact with the same region of Kir2.1, these different modulators induce a common gating pathway of the channel.     

Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin

PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.     

Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2 synthesis.     

Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains

Epsin and AP180/CALM are endocytotic accessory proteins that have been implicated in the formation of clathrin-coated pits. Both proteins have phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding domains in their N termini, but these domains are structurally and functionally different. To understand the basis of their distinct properties, we measured the PtdIns(4,5)P2-dependent membrane binding of the epsin N-terminal homology (ENTH) domain and the AP180 N-terminal homology (ANTH) domain by means of surface plasmon resonance and monolayer penetration techniques and also calculated the effect of PtdIns(4,5)P2 on the electrostatic potential of these domains. PtdIns(4,5)P2 enhances the electrostatic membrane association of both domains; however, PtdIns(4,5)P2 binding exerts distinct effects on their membrane dissociation. Specifically, PtdIns(4,5)P2 induces the membrane penetration of the N-terminal alpha-helix of the ENTH domain, which slows the membrane dissociation of the domain and triggers the membrane deformation. These results provide the biophysical explanation for the membrane bending activity of epsin and its ENTH domain.     

Interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 is important for actin-dependent endocytic internalization

A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.     

Integrin adhesion and force coupling are independently regulated by localized PtdIns(4,5)2 synthesis

The 90-kDa isoform of the lipid kinase PIP kinase Type I γ (PIPKIγ) localizes to focal adhesions (FAs), where it provides a local source of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Although PtdIns(4,5)P(2) regulates the function of several FA-associated molecules, the role of the FA-specific pool of PtdIns(4,5)P(2) is not known. We report that the genetic ablation of PIPKIγ specifically from FAs results in defective integrin-mediated adhesion and force coupling. Adhesion defects in cells deficient in FAPtdIns(4,5)P(2) synthesis are corrected within minutes while integrin-actin force coupling remains defective over a longer period. Talin and vinculin, but not kindlin, are less efficiently recruited to new adhesions in these cells. These data demonstrate that the specific depletion of PtdIns(4,5)P(2) from FAs temporally separates integrin-ligand binding from integrin-actin force coupling by regulating talin and vinculin recruitment. Furthermore, it suggests that force coupling relies heavily on locally generated PtdIns(4,5)P(2) rather than bulk membrane PtdIns(4,5)P(2).