Structural Basis of Human KCNQ1 Modulation and Gating.

KCNQ1, also known as Kv7.1, is a voltage-dependent K+ channel that regulates gastric acid secretion, salt and glucose homeostasis, and heart rhythm. Its functional properties are regulated in a tissue-specific manner through co-assembly with beta subunits KCNE1-5. In non-excitable cells, KCNQ1 forms a complex with KCNE3, which suppresses channel closure at negative membrane voltages that otherwise would close it. Pore opening is regulated by the signaling lipid PIP2. Using cryoelectron microscopy (cryo-EM), we show that KCNE3 tucks its single-membrane-spanning helix against KCNQ1, at a location that appears to lock the voltage sensor in its depolarized conformation. Without PIP2, the pore remains closed. Upon addition, PIP2 occupies a site on KCNQ1 within the inner membrane leaflet, which triggers a large conformational change that leads to dilation of the pore's gate. It is likely that this mechanism of PIP2 activation is conserved among Kv7 channels.

The Architecture of Talin1 Reveals an Autoinhibition Mechanism.

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.

Cryo-EM Structure of a KCNQ1/CaM Complex Reveals Insights into Congenital Long QT Syndrome.

KCNQ1 is the pore-forming subunit of cardiac slow-delayed rectifier potassium (IKs) channels. Mutations in the kcnq1 gene are the leading cause of congenital long QT syndrome (LQTS). Here, we present the cryoelectron microscopy (cryo-EM) structure of a KCNQ1/calmodulin (CaM) complex. The conformation corresponds to an "uncoupled," PIP2-free state of KCNQ1, with activated voltage sensors and a closed pore. Unique structural features within the S4-S5 linker permit uncoupling of the voltage sensor from the pore in the absence of PIP2. CaM contacts the KCNQ1 voltage sensor through a specific interface involving a residue on CaM that is mutated in a form of inherited LQTS. Using an electrophysiological assay, we find that this mutation on CaM shifts the KCNQ1 voltage-activation curve. This study describes one physiological form of KCNQ1, depolarized voltage sensors with a closed pore in the absence of PIP2, and reveals a regulatory interaction between CaM and KCNQ1 that may explain CaM-mediated LQTS.

Structure of a Pancreatic ATP-Sensitive Potassium Channel.

ATP-sensitive potassium channels (KATP) couple intracellular ATP levels with membrane excitability. These channels play crucial roles in many essential physiological processes and have been implicated extensively in a spectrum of metabolic diseases and disorders. To gain insight into the mechanism of KATP, we elucidated the structure of a hetero-octameric pancreatic KATP channel in complex with a non-competitive inhibitor glibenclamide by single-particle cryoelectron microscopy to 5.6-Å resolution. The structure shows that four SUR1 regulatory subunits locate peripherally and dock onto the central Kir6.2 channel tetramer through the SUR1 TMD0-L0 fragment. Glibenclamide-bound SUR1 uses TMD0-L0 fragment to stabilize Kir6.2 channel in a closed conformation. In another structural population, a putative co-purified phosphatidylinositol 4,5-bisphosphate (PIP2) molecule uncouples Kir6.2 from glibenclamide-bound SUR1. These structural observations suggest a molecular mechanism for KATP regulation by anti-diabetic sulfonylurea drugs, intracellular adenosine nucleotide concentrations, and PIP2 lipid.

Dual regulation of IP3 receptors by IP3 and PIP2 controls the transition from local to global Ca2+ signals.

The spatial organization of inositol 1,4,5-trisphosphate (IP3)-evoked Ca2+ signals underlies their versatility. Low stimulus intensities evoke Ca2+ puffs, localized Ca2+ signals arising from a few IP3 receptors (IP3Rs) within a cluster tethered beneath the plasma membrane. More intense stimulation evokes global Ca2+ signals. Ca2+ signals propagate regeneratively as the Ca2+ released stimulates more IP3Rs. How is this potentially explosive mechanism constrained to allow local Ca2+ signaling? We developed methods that allow IP3 produced after G-protein coupled receptor (GPCR) activation to be intercepted and replaced by flash photolysis of a caged analog of IP3. We find that phosphatidylinositol 4,5-bisphosphate (PIP2) primes IP3Rs to respond by partially occupying their IP3-binding sites. As GPCRs stimulate IP3 formation, they also deplete PIP2, relieving the priming stimulus. Loss of PIP2 resets IP3R sensitivity and delays the transition from local to global Ca2+ signals. Dual regulation of IP3Rs by PIP2 and IP3 through GPCRs controls the transition from local to global Ca2+ signals.

Phosphoinositide Regulation of TRP Channels: A Functional Overview in the Structural Era.

Transient receptor potential (TRP) ion channels have diverse activation mechanisms including physical stimuli, such as high or low temperatures, and a variety of intracellular signaling molecules. Regulation by phosphoinositides and their derivatives is their only known common regulatory feature. For most TRP channels, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] serves as a cofactor required for activity. Such dependence on PI(4,5)P2 has been demonstrated for members of the TRPM subfamily and for the epithelial TRPV5 and TRPV6 channels. Intracellular TRPML channels show specific activation by PI(3,5)P2. Structural studies uncovered the PI(4,5)P2 and PI(3,5)P2 binding sites for these channels and shed light on the mechanism of channel opening. PI(4,5)P2 regulation of TRPV1-4 as well as some TRPC channels is more complex, involving both positive and negative effects. This review discusses the functional roles of phosphoinositides in TRP channel regulation and molecular insights gained from recent cryo-electron microscopy structures.

Gßγ activates PIP2 hydrolysis by recruiting and orienting PLCß on the membrane surface.

Phospholipase C-ßs (PLCßs) catalyze the hydrolysis of phosphatidylinositol 4, 5-bisphosphate [Formula: see text] into [Formula: see text] [Formula: see text] and [Formula: see text]  [Formula: see text]. [Formula: see text] regulates the activity of many membrane proteins, while IP3 and DAG lead to increased intracellular Ca2+ levels and activate protein kinase C, respectively. PLCßs are regulated by G protein-coupled receptors through direct interaction with [Formula: see text] and [Formula: see text] and are aqueous-soluble enzymes that must bind to the cell membrane to act on their lipid substrate. This study addresses the mechanism by which [Formula: see text] activates PLCß3. We show that PLCß3 functions as a slow Michaelis-Menten enzyme ( [Formula: see text] ) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of PLCß3. Its partition coefficient is such that only a small quantity of PLCß3 exists in the membrane in the absence of [Formula: see text] . When [Formula: see text] is present, equilibrium binding on the membrane surface increases PLCß3 in the membrane, increasing [Formula: see text] in proportion. Atomic structures on membrane vesicle surfaces show that two [Formula: see text] anchor PLCß3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that [Formula: see text] activates PLCß by increasing its concentration on the membrane surface and orienting its catalytic core to engage [Formula: see text] . This principle of activation explains rapid stimulated catalysis with low background activity, which is essential to the biological processes mediated by [Formula: see text], IP3, and DAG.

The mechanism of Gαq regulation of PLCß3-catalyzed PIP2 hydrolysis.

PLCß (Phospholipase Cß) enzymes cleave phosphatidylinositol 4,5-bisphosphate (PIP2) producing IP3 and DAG (diacylglycerol). PIP2 modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca2+ levels and protein phosphorylation by protein kinase C, respectively. PLCß enzymes are under the control of G protein coupled receptor signaling through direct interactions with G proteins Gßγ and Gαq and have been shown to be coincidence detectors for dual stimulation of Gαq and Gαi-coupled receptors. PLCßs are aqueous-soluble cytoplasmic enzymes but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed that Gßγ activates PLCß3 by recruiting it to the membrane. Using these same methods, here we show that Gαq increases the catalytic rate constant, kcat, of PLCß3. Since stimulation of PLCß3 by Gαq depends on an autoinhibitory element (the X-Y linker), we propose that Gαq produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the PLCß3·Gαq and PLCß3·Gßγ(2)·Gαq complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate PLCß3 activity. The structures rationalize a finding in the enzyme assay, that costimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of PLCß3 is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.

Faulty TRPM4 channels underlie age-dependent cerebral vascular dysfunction in Gould syndrome.

Gould syndrome is a rare multisystem disorder resulting from autosomal dominant mutations in the collagen-encoding genes COL4A1 and COL4A2. Human patients and Col4a1 mutant mice display brain pathology that typifies cerebral small vessel diseases (cSVDs), including white matter hyperintensities, dilated perivascular spaces, lacunar infarcts, microbleeds, and spontaneous intracerebral hemorrhage. The underlying pathogenic mechanisms are unknown. Using the Col4a1+/G394V mouse model, we found that vasoconstriction in response to internal pressure-the vascular myogenic response-is blunted in cerebral arteries from middle-aged (12 mo old) but not young adult (3 mo old) animals, revealing age-dependent cerebral vascular dysfunction. The defect in the myogenic response was associated with a significant decrease in depolarizing cation currents conducted by TRPM4 (transient receptor potential melastatin 4) channels in native cerebral artery smooth muscle cells (SMCs) isolated from mutant mice. The minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is necessary for TRPM4 activity. Dialyzing SMCs with PIP2 and selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that converts PIP2 to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored TRPM4 currents. Acute inhibition of PI3K activity and blockade of transforming growth factor-beta (TGF-ß) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-ß signaling pathways stimulates PI3K to deplete PIP2 and impair TRPM4 channels. We conclude that age-related cerebral vascular dysfunction in Col4a1+/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP2 depletion, revealing an age-dependent mechanism of cSVD.

Acute phosphatidylinositol 4,5 bisphosphate depletion destabilizes sarcolemmal expression of cardiac L-type Ca2+ channel CaV1.2.

CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility.

PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease-protective variant PLCγ2 R522.

Variants identified in genome-wide association studies have implicated immune pathways in the development of Alzheimer's disease (AD). Here, we investigated the mechanistic basis for protection from AD associated with PLCγ2 R522, a rare coding variant of the PLCG2 gene. We studied the variant's role in macrophages and microglia of newly generated PLCG2-R522-expressing human induced pluripotent cell lines (hiPSC) and knockin mice, which exhibit normal endogenous PLCG2 expression. In all models, cells expressing the R522 mutation show a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This manifests as enhanced release of cellular calcium ion stores in response to physiologically relevant stimuli like Fc-receptor ligation or exposure to Aß oligomers. Expression of the PLCγ2-R522 variant resulted in increased stimulus-dependent PIP2 depletion and reduced basal PIP2 levels in vivo. Furthermore, it was associated with impaired phagocytosis and enhanced endocytosis. PLCγ2 acts downstream of other AD-related factors, such as TREM2 and CSF1R, and alterations in its activity directly impact cell function. The inherent druggability of enzymes such as PLCγ2 raises the prospect of PLCγ2 manipulation as a future therapeutic approach in AD.

Roles of Intramolecular Interactions in the Regulation of TRP Channels.

The transient receptor potential (TRP) channels, classified into six (-A, -V, -P, -C, -M, -ML, -N and -Y) subfamilies, are important membrane sensors and mediators of diverse stimuli including pH, light, mechano-force, temperature, pain, taste, and smell. The mammalian TRP superfamily of 28 members share similar membrane topology with six membrane-spanning helices (S1-S6) and cytosolic N-/C-terminus. Abnormal function or expression of TRP channels is associated with cancer, skeletal dysplasia, immunodeficiency, and cardiac, renal, and neuronal diseases. The majority of TRP members share common functional regulators such as phospholipid PIP2, 2-aminoethoxydiphenyl borate (2-APB), and cannabinoid, while other ligands are more specific, such as allyl isothiocyanate (TRPA1), vanilloids (TRPV1), menthol (TRPM8), ADP-ribose (TRPM2), and ML-SA1 (TRPML1). The mechanisms underlying the gating and regulation of TRP channels remain largely unclear. Recent advances in cryogenic electron microscopy provided structural insights into 19 different TRP channels which all revealed close proximity of the C-terminus with the N-terminus and intracellular S4-S5 linker. Further studies found that some highly conserved residues in these regions of TRPV, -P, -C and -M members mediate functionally critical intramolecular interactions (i.e., within one subunit) between these regions. This review provides an overview on (1) intramolecular interactions in TRP channels and their effect on channel function; (2) functional roles of interplays between PIP2 (and other ligands) and TRP intramolecular interactions; and (3) relevance of the ligand-induced modulation of intramolecular interaction to diseases.

ConFERMing the role of talin in integrin activation and mechanosignaling.

Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinity-regulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites.

Drosophila CTP synthase regulates collective cell migration by controlling the polarized endocytic cycle.

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) is involved in many biological functions. However, the mechanisms of PIP2 in collective cell migration remain elusive. This study highlights the regulatory role of cytidine triphosphate synthase (CTPsyn) in collective border cell migration through regulating the asymmetrical distribution of PIP2. We demonstrated that border cell clusters containing mutant CTPsyn cells suppressed migration. CTPsyn was co-enriched with Actin at the leading edge of the Drosophila border cell cluster where PIP2 was enriched, and this enrichment depended on the CTPsyn activity. Genetic interactions of border cell migration were found between CTPsyn mutant and genes in PI biosynthesis. The CTPsyn reduction resulted in loss of the asymmetric activity of endocytosis recycling. Also, genetic interactions were revealed between components of the exocyst complex and CTPsyn mutant, indicating that CTPsyn activity regulates the PIP2-related asymmetrical exocytosis activity. Furthermore, CTPsyn activity is essential for RTK-polarized distribution in the border cell cluster. We propose a model in which CTPsyn activity is required for the asymmetrical generation of PIP2 to enrich RTK signaling through endocytic recycling in collective cell migration.

Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy.

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.

Oligomerization of Monoamine Transporters.

Transporters of the monoamine transporter (MAT) family regulate the uptake of important neurotransmitters like dopamine, serotonin, and norepinephrine. The MAT family functions using the electrochemical gradient of ions across the membrane and comprises three transporters, dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). MAT transporters have been observed to exist in monomeric states to higher-order oligomeric states. Structural features, allosteric modulation, and lipid environment regulate the oligomerization of MAT transporters. NET and SERT oligomerization are regulated by levels of PIP2 present in the membrane. The kink present in TM12 in the MAT family is crucial for dimer interface formation. Allosteric modulation in the dimer interface hinders dimer formation. Oligomerization also influences the transporters' function, trafficking, and regulation. This chapter will focus on recent studies on monoamine transporters and discuss the factors affecting their oligomerization and its impact on their function.

Structural mechanisms for the activation of human cardiac KCNQ1 channel by electro-mechanical coupling enhancers.

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.

Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

The role of high cholesterol in SARS-CoV-2 infectivity.

Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2. The virus binds to angiotensinogen converting enzyme 2 (ACE2), which mediates viral entry into mammalian cells. COVID-19 is notably severe in the elderly and in those with underlying chronic conditions. The cause of selective severity is not well understood. Here we show cholesterol and the signaling lipid phosphatidyl-inositol 4,5 bisphosphate (PIP2) regulate viral infectivity through the localization of ACE2's into nanoscopic (<200 nm) lipid clusters. Uptake of cholesterol into cell membranes (a condition common to chronic disease) causes ACE2 to move from PIP2 lipids to endocytic ganglioside (GM1) lipids, where the virus is optimally located for viral entry. In mice, age and high-fat diet increase lung tissue cholesterol by up to 40%. And in smokers with chronic disease, cholesterol is elevated 2-fold, a magnitude of change that dramatically increases infectivity of virus in cell culture. We conclude increasing the ACE2 location near endocytic lipids increases viral infectivity and may help explain the selective severity of COVID-19 in aged and diseased populations.

Regulation of drought tolerance in Arabidopsis involves the PLATZ4-mediated transcriptional repression of plasma membrane aquaporin PIP2;8.

Plant A/T-rich protein and zinc-binding protein (PLATZ) transcription factors play important roles in plant growth, development and abiotic stress responses. However, how PLATZ influences plant drought tolerance remains poorly understood. The present study showed that PLATZ4 increased drought tolerance in Arabidopsis thaliana by causing stomatal closure. Transcriptional profiling analysis revealed that PLATZ4 affected the expression of a set of genes involved in water and ion transport, antioxidant metabolism, small peptides and abscisic acid (ABA) signaling. Among these genes, the direct binding of PLATZ4 to the A/T-rich sequences in the plasma membrane intrinsic protein 2;8 (PIP2;8) promoter was identified. PIP2;8 consistently reduced drought tolerance in Arabidopsis through inhibiting stomatal closure. PIP2;8 was localized in the plasma membrane, exhibited water channel activity in Xenopus laevis oocytes and acted epistatically to PLATZ4 in regulating the drought stress response in Arabidopsis. PLATZ4 increased ABA sensitivity through upregulating the expression of ABSCISIC ACID INSENSITIVE 3 (ABI3), ABI4 and ABI5. The transcripts of PLATZ4 were induced to high levels in vegetative seedlings under drought and ABA treatments within 6 and 3 h, respectively. Collectively, these findings reveal that PLATZ4 positively influences plant drought tolerance through regulating the expression of PIP2;8 and genes involved in ABA signaling.