Direct Activation of ENaC by Angiotensin II: Recent Advances and New Insights

Angiotensin II (Ang II) is the principal effector of the renin–angiotensin–aldosterone system (RAAS). It initiates myriad processes in multiple organs integrated to increase circulating volume and elevate systemic blood pressure. In the kidney, Ang II stimulates renal tubular water and salt reabsorption causing antinatriuresis and antidiuresis. Activation of the RAAS is known to enhance activity of the epithelial Na⁺ channel (ENaC) in the aldosterone-sensitive distal nephron. In addition to its well described stimulatory actions on aldosterone secretion, Ang II is also capable of directly increasing ENaC activity. In this brief review, we discuss recent findings about non-classical Ang II actions on ENaC and speculate about its relevance for renal sodium handling.     

Leptin-stimulated KATP channel trafficking

Insulin secretion from pancreatic β-cells is initiated by the closure of ATP-sensitive K⁺ channels (K_ATP) in response to high concentrations of glucose, and this action of glucose is counteracted by the hormone leptin, an adipokine that signals through the Ob-R_b receptor to increase K_ATP channel activity. Despite intensive investigations, the molecular basis for K_ATP channel regulation remains uncertain, particularly from the standpoint of whether fluctuations in plasma membrane K_ATP channel content underlie alterations of K_ATP channel activity in response to glucose or leptin. Surprisingly, newly published findings reveal that leptin stimulates AMP-activated protein kinase (AMPK) in order to promote trafficking of K_ATP channels from cytosolic vesicles to the plasma membrane of β-cells. This action of leptin is mimicked by low concentrations of glucose that also activate AMPK and that inhibit insulin secretion. Thus, a new paradigm for β-cell stimulus-secretion coupling is suggested in which leptin exerts a tonic inhibitory effect on β-cell excitability by virtue of its ability to increase plasma membrane K_ATP channel density and whole-cell K_ATP channel current. One important issue that remains unresolved is whether high concentrations of glucose suppress AMPK activity in order to shift the balance of membrane cycling so that K_ATP channel endocytosis predominates over vesicular K_ATP channel insertion into the plasma membrane. If so, high concentrations of glucose might transiently reduce K_ATP channel density/current, thereby favoring β-cell depolarization and insulin secretion. Such an AMPK-dependent action of glucose would complement its established ability to generate an increase of ATP/ADP concentration ratio that directly closes K_ATP channels in the plasma membrane.     

Do gut microbial communities differ in pediatric IBS and health?

Human gastrointestinal microbial communities are recognized as important determinants of the host health and disease status. We have recently examined the distal gut microbiota of two groups of children: healthy adolescents and those diagnosed with diarrhea-predominant irritable bowel syndrome (IBS). We have revealed the common core of phylotypes shared among all children, identified genera differentially abundant between two groups and surveyed possible relationships among intestinal microbial genera and phylotypes. In this article we explored the use of supervised and unsupervised ordination and classification methods to separate and classify child fecal samples based on their quantitative microbial profile. We observed sample separation according to the participant health status, and this separation could often be attributed to the abundance levels of several specific microbial genera. We also extended our original correlation network analysis of the relative abundances of bacterial genera across samples and determined possible association networks separately for healthy and IBS groups. Interestingly, the number of significant genus abundance associations was drastically lower among the IBS samples, which can potentially be attributed to the existence of multiple routes to microbiota disbalance in IBS or to the loss of microbial interactions during IBS development.     

Quantification and demonstration of the collective constriction-by-ratchet mechanism in the dynamin molecular motor

Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.

Dynamin is a mechanochemical GTPase that plays a central role in clathrin-mediated endocytosis (CME) (1–4). The 100-kDa protein polymerizes into helical filaments that coil around the necks of vesicles budding from the membrane. In the presence of guanosine-5’-triphosphate (GTP), such filaments constrict, whereas GTP hydrolysis is necessary to cut the neck, thus allowing the vesicle to separate. This process is crucial for cellular nutrient uptake and for synaptic transmission. Mutations in dynamin are associated with severe neurodegenerative disease and muscular disorders (5). Structural studies have detailed the dynamin filament structure on membrane tubes and observed the filament’s ability to form nucleotide-dependent cross-bridges between its neighboring turns (6, 7). These studies, in tandem with biophysical experiments showing GTPase-driven torque generation by dynamin filaments (8–10), have led to proposals that dynamin functions as a molecular ratchet motor (11–13). Confirming this hypothesis requires a molecular-level understanding of the principal GTP-dependent motor function and showing that the candidate power stroke (6) can provide sufficient force to drive membrane constriction. So far, theoretical studies of dynamin have concentrated on the elastic properties of dynamin filaments (14–16) and on the effects exhibited by passive elastic filaments on the membranes (17, 18). The nonequilibrium motor activity of dynamin, based on GTP hydrolysis, was treated only in a phenomenological way (9, 19).Dynamin consists of five domains (Fig. 1A) (20, 21). Its stalks polymerize into a helical filament whose elementary units are criss-cross stalk dimers. The filament is anchored to the membrane by pleckstrin homology (PH) domains connected by flexible linkers to the stalk. GTPase (G) domains are connected to the filament via bundle signaling elements (BSEs). The disordered C-terminal proline-rich domain (PRD) is involved in recruitment to membrane necks. Hinge 1 and hinge 2 form flexible joints between BSEs and the stalk and between the G domain and BSEs, respectively. We refer to the combination of the G domain and BSE as the motor module (MM) of dynamin (Fig. 1A) (6, 22, 23).Open in a separate windowFig. 1.Kinetic characterization of the dynamin motor module. (A) The dynamin monomer has four structurally characterized domains (Protein Data Bank 3SNH) (20). The motor module (MM) contains the G domain and the BSE. A continuous MM construct was created by fusing the third helix of the BSE onto the N-terminal portion of the dynamin sequence. (B) Association rate constants are obtained from the slope of kapp versus MM concentration. See SI Appendix, Fig. S1 for mant-GTP association curves. (C) Mant-nucleotide dissociation rates were measured in stopped-flow experiments by mixing with excess unlabeled nucleotide. (D) Table of determined mant-nucleotide binding/dissociation rates of the MM construct. Kd is defined by the ratio koff/kon. *GTP on rate value is determined from kinetic modeling of GTPase activity (SI Appendix, section S1.A.2) because mant binding did not show a single exponential fluorescence increase for mant-GTP (SI Appendix, Fig. S1). (E) GDP production as a function of time in the linear regime for increasing concentrations of MM and an initial GTP concentration of 1 mM. Dashed lines are linear fits to three independent experiments. (F) Slope from E divided by the protein concentration gives the specific hydrolysis rate (khydspec). Linear dependence of the specific hydrolysis rate on protein concentration indicates that GTPase activity is controlled by dimerization (SI Appendix, section S1.A.1).When dynamin is assembled into a helical filament, G domains in adjacent rungs are optimally oriented for cross-dimerization (7), which can explain the enhancement of GTPase activity in the presence of membranes (23). Moreover, the conformation of dynamin’s MM is sensitive to its nucleotide state. In the presence of the nonhydrolyzable GTP analog β,γ-methyleneguanosine 5′-triphosphate (GMPPCP), the MM crystallized in an open BSE conformation (6), whereas a closed conformation of the BSE was observed in the nucleotide-free state or when bound to guanosine-5’-diphosphate (GDP) or to GDP⋅AlF4−, a mimic of the transition state of GTP hydrolysis (20, 21, 23, 24). While this structural shift is the obvious candidate for the power stroke driving motor function, it remains unknown how strong it is, since even small energy preferences might result in the different structural states captured in the available structures. For example, in crystallographic studies, a dynamin family member, MxA, shows open and closed states in the GMPPCP- and GDP-bound states, respectively (25), but only weak nucleotide-dependent preferences in solution (26).In this study, kinetic measurements for the GTPase cycle of the dynamin MM are first performed. After that, we explore nucleotide-dependent conformational changes in the MM using single-molecule FRET experiments. Their results, in conjunction with molecular modeling, are then used to quantify the forces generated by single dynamin motors and to refine the ratchet effect. Next, we incorporate the determined forces and the measured kinetics into a polymer-like computational model that resolves individual dynamin motors and includes a deformable membrane template with lipid flows within it. Direct simulations of the model reproduce collective tight constriction of membrane necks down to the hemifission radius by dynamin filaments in the presence of GTP.     

Shedding Light on the Blood–Brain Barrier Transport with Two-Photon Microscopy In Vivo

Pharmaceutical Research - Treatment of brain disorders relies on efficient delivery of therapeutics to the brain, which is hindered by the blood–brain barrier (BBB). The work of Prof....