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Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lₒ) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lₒ phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lₒ domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lₒ and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lₒ and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P₂ and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lₒ than in Ld. Further, PtdIns(4,5)P₂ was restored more rapidly in Lₒ than in Ld. Thus destruction and restoration of PtdIns(4,5)P₂ are faster in Lₒ than in Ld. This suggests that Lₒ is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.

Brain capillaries play a critical role in sensing neural activity and translating it into dynamic changes in cerebral blood flow to serve the metabolic needs of the brain. The molecular cornerstone of this mechanism is the capillary endothelial cell inward rectifier K⁺ (Kir2.1) channel, which is activated by neuronal activity–dependent increases in external K⁺ concentration, producing a propagating hyperpolarizing electrical signal that dilates upstream arterioles. Here, we identify a key regulator of this process, demonstrating that phosphatidylinositol 4,5-bisphosphate (PIP₂) is an intrinsic modulator of capillary Kir2.1-mediated signaling. We further show that PIP₂ depletion through activation of Gq protein-coupled receptors (GqPCRs) cripples capillary-to-arteriole signal transduction in vitro and in vivo, highlighting the potential regulatory linkage between GqPCR-dependent and electrical neurovascular-coupling mechanisms. These results collectively show that PIP₂ sets the gain of capillary-initiated electrical signaling by modulating Kir2.1 channels. Endothelial PIP₂ levels would therefore shape the extent of retrograde signaling and modulate cerebral blood flow.

Phosphoinositides play a crucial role in regulating many cellular functions, such as actin dynamics, signaling, intracellular trafficking, membrane dynamics, and cell–matrix adhesion. Central to this process is phosphatidylinositol bisphosphate (PIP2). The levels of PIP2 in the membrane are rapidly altered by the activity of phosphoinositide-directed kinases and phosphatases, and it binds to dozens of different intracellular proteins. Despite the vast literature dedicated to understanding the regulation of PIP2 in cells over past 30 years, much remains to be learned about its cellular functions. In this review, we focus on past and recent exciting results on different molecular mechanisms that regulate cellular functions by binding of specific proteins to PIP2 or by stabilizing phosphoinositide pools in different cellular compartments. Moreover, this review summarizes recent findings that implicate dysregulation of PIP2 in many diseases

G-protein-gated inward rectifier potassium (GIRK) channels are regulated by G proteins and PIP 2 . Here, using cryo-EM single particle analysis we describe the equilibrium ensemble of structures of neuronal GIRK2 as a function of the C8-PIP 2 concentration. We find that PIP 2 shifts the equilibrium between two distinguishable structures of neuronal GIRK (GIRK2), extended and docked, towards the docked form. In the docked form the cytoplasmic domain, to which G βγ binds, becomes accessible to the cytoplasmic membrane surface where G βγ resides. Furthermore, PIP 2 binding reshapes the G βγ binding surface on the cytoplasmic domain, preparing it to receive G βγ . We find that cardiac GIRK (GIRK1/4) can also exist in both extended and docked conformations. These findings lead us to conclude that PIP 2 influences GIRK channels in a structurally similar manner to Kir2.2 channels. In Kir2.2 channels, the PIP 2 -induced conformational changes open the pore. In GIRK channels, they prepare the channel for activation by G βγ .

The cardiac KCNQ1 potassium channel carries the important IKs current and controls the heart rhythm. Hundreds of mutations in KCNQ1 can cause life-threatening cardiac arrhythmia. Although KCNQ1 structures have been recently resolved, the structural basis for the dynamic electro-mechanical coupling, also known as the voltage sensor domain–pore domain (VSD-PD) coupling, remains largely unknown. In this study, utilizing two VSD-PD coupling enhancers, namely, the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and a small-molecule ML277, we determined 2.5–3.5 Å resolution cryo-electron microscopy structures of full-length human KCNQ1-calmodulin (CaM) complex in the apo closed, ML277-bound open, and ML277-PIP₂-bound open states.ML277 binds at the “elbow” pocket above the S4-S5 linker and directly induces an upward movement of the S4-S5 linker and the opening of the activation gate without affecting the C-terminal domain (CTD) of KCNQ1. PIP₂ binds at the cleft between the VSD and the PD and brings a large structural rearrangement of the CTD together with the CaM to activate the PD. These findings not only elucidate the structural basis for the dynamic VSD-PD coupling process during KCNQ1 gating but also pave the way to develop new therapeutics for anti-arrhythmia.

Gradients of PtdIns4P between organelle membranes and the endoplasmic reticulum (ER) are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel pathway requires the SAC1 phosphatase to degrade PtdIns4P in a ‘cis’ configuration at the ER to maintain the gradient. However, SAC1 has also been proposed to act in ‘trans’ at membrane contact sites, which could oppose lipid traffic. It is therefore crucial to determine which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 causes accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane contact sites, and that SAC1 has little activity in ‘trans’, unless a linker is added between its ER-anchored and catalytic domains. The data reveal an obligate ‘cis’ activity of SAC1, supporting its role in non-vesicular lipid traffic and implicating lipid traffic more broadly in inositol lipid homeostasis and function.

Protein–lipid interactions are a key element of the function of many integral membrane proteins. These potential interactions should be considered alongside the complexity and diversity of membrane lipid composition. Inward rectifier potassium channel (Kir) Kir2.2 has multiple interactions with plasma membrane lipids: Phosphatidylinositol (4, 5)-bisphosphate (PIP₂) activates the channel; a secondary anionic lipid site has been identified, which augments the activation by PIP₂; and cholesterol inhibits the channel. Molecular dynamics simulations are used to characterize in molecular detail the protein–lipid interactions of Kir2.2 in a model of the complex plasma membrane. Kir2.2 has been simulated with multiple, functionally important lipid species. From our simulations we show that PIP₂ interacts most tightly at the crystallographic interaction sites, outcompeting other lipid species at this site. Phosphatidylserine (PS) interacts at the previously identified secondary anionic lipid interaction site, in a PIP2 concentration-dependent manner. There is interplay between these anionic lipids: PS interactions are diminished when PIP₂ is not present in the membrane, underlining the need to consider multiple lipid species when investigating protein–lipid interactions.

We recently reported that the inward-rectifier Kir2.1 channel in brain capillary endothelial cells (cECs) plays a major role in neurovascular coupling (NVC) by mediating a neuronal activity-dependent, propagating vasodilatory (hyperpolarizing) signal. We further demonstrated that Kir2.1 activity is suppressed by depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2). Whether cECs express depolarizing channels that intersect with Kir2.1-mediated signaling remains unknown. Here, we report that Ca2+/Na+-permeable TRPV4 (transient receptor potential vanilloid 4) channels are expressed in cECs and are tonically inhibited by PIP2. We further demonstrate that depletion of PIP2 by agonists, including putative NVC mediators, that promote PIP2 hydrolysis by signaling through Gq-protein-coupled receptors (GqPCRs) caused simultaneous disinhibition of TRPV4 channels and suppression of Kir2.1 channels. These findings collectively support the concept that GqPCR activation functions as a molecular switch to favor capillary TRPV4 activity over Kir2.1 signaling, an observation with potentially profound significance for the control of cerebral blood flow. Capillaries form branching networks that surround all cells of the body. They allow oxygen and nutrient exchange between blood and tissue, but this is not their only role. Capillaries in the brain form a tight barrier that prevents components carried in the blood from easily reaching the brain compartment. They also detect the activity of neurons and trigger on-demand increases in blood flow to active regions of the brain. This role, revealed only recently, depends upon ion channels on the surface of the capillary cells. Active neurons release potassium ions, which open a type of ion channel called Kir2.1 that allows potassium inside the cell to flow out. This process is repeated in neighboring capillary cells until it reaches an upstream vessel, where it causes the vessel to relax and increase the blood flow. Kir2.1 channels sit astride the membranes of capillary cells, where they can interact with other membrane molecules. One such molecule, called PIP2, plays several roles in relaying signals from the outside to the inside of cells. It also physically interacts with channels in the membrane, including Kir2.1 channels. If PIP2 levels are low, Kir2.1 channel activity decreases. Here, Harraz et al. discovered that capillary cells contain another type of ion channel, called TRPV4, which is also regulated by PIP2. But unlike Kir2.1, its activity increases when PIP2 levels drop. Moreover, TRPV4 channels allow sodium and calcium ions to flow into the cell, which has an effect opposite to that of potassium flowing out of the cell. Capillary cells also have receptor proteins called GqPCRs that are activated by chemical signals released by active neurons in the brain. GqPCRs break down PIP2, so their activity turns Kir2.1 channels off and TRPV4 channels on. This resets the system so that it is ready to respond to new signals from active neurons. GqPCRs work as molecular switches to control the balance between Kir2.1 and TRPV4 channels and turn brain blood flow up and down. GqPCRs and ion channels that depend on PIP2 can also be found in other types of cells. These findings could reveal clues about how signals are switched on and off in different cells. Understanding the role of PIP2 in signaling could also unveil what happens when signaling go wrong.

To assess the effect of triacontanol (TRIA) on rice plants grown under normal or drought conditions, rice seeds were presoaked in TRIA (35 ppm) for two hours. After 20 days of sowing, rice seedlings developed from TRIA-treated or untreated seeds were subjected to drought stress. After 10 days of plant exposure to drought stress, data of major growth attributes and the content of photosynthetic pigments were recorded. Moreover, the effect of drought stress on stomatal conductance and the photochemical efficiency of PSII (Fv/Fm) were followed. The data obtained indicated that the species of rice (Oryza sativa L.) cultivar Giza 177 under investigation was sensitive to drought stress where there were significant decreases in the fresh and dry weights of shoots and roots and in stomatal conductance, as well as in the content of chlorophyll a, chlorophyll b, and carotenoids. Seed priming with TRIA enhanced both growth and acquired plant tolerance to drought stress. Thus, TRIA via the enhancement of stomatal conductance through the regulation of stomatal closure, the rate of water loss, ABA metabolism, the accumulation of osmolytes, and the regulation of aquaporins genes improved the water status of plants grown under water scarcity. Moreover, TRIA via increasing the content of free amino acids and sugars under drought stress may increase the chance of plant tissues to retain more water under scarcity conditions.

Na + /Ca 2+ exchangers (NCXs) transport Ca 2+ across the plasma membrane in exchange for Na + and play a vital role in maintaining cellular Ca 2+ homeostasis. Our previous structural study of human cardiac NCX1 (HsNCX1) reveals the overall architecture of the eukaryotic exchanger and the formation of the inactivation assembly by the intracellular regulatory domain that underlies the cytosolic Na + -dependent inactivation and Ca 2+ activation of NCX1. Here, we present the cryo-EM structures of HsNCX1 in complex with a physiological activator phosphatidylinositol 4,5-bisphosphate (PIP 2 ), or pharmacological inhibitor SEA0400, that enhances the inactivation of the exchanger. We demonstrate that PIP 2 binding stimulates NCX1 activity by inducing a conformational change at the interface between the transmembrane (TM) and cytosolic domains that destabilizes the inactivation assembly. In contrast, SEA0400 binding in the TM domain of NCX1 stabilizes the exchanger in an inward-facing conformation that facilitates the formation of the inactivation assembly, thereby promoting the Na + -dependent inactivation of NCX1. Thus, this study reveals the structural basis of PIP 2 activation and SEA0400 inhibition of NCX1 and provides some mechanistic understandings of cellular regulation and pharmacology of NCX family proteins.