Cecilia Canessa - US grants

Maintenance and variations in blood pressure levels are highly dependent on the total Na+ reabsorbed from the surrounding milieu. The amiloride- sensitive epithelial Na+ channel plays a fundamental role in determining the net amount of Na+ reabsorbed and thus, it is the target for many regulatory mechanisms. This proposal focuses on several aspects of the functional regulation of the amiloride-sensitive epithelial Na+ channel. The aims we are going to pursue are the following: AIM 1. Biosynthesis of Na+ channels and regulation by aldosterone. Effects of aldosterone and of salt intake in the biosynthesis of Na+ channels in the whole animal (in vivo) and in primary cultures of cortical collecting tubules (in vitro). Aldosterone effects will be examined at several time points over a time course in both the in vivo and the in vitro models. The following levels on the biosynthesis of channels will be examined:a) Transcriptional activation of the individual subunits. b)Synthesis of new channel proteins: rate of synthesis, degradation and lifetimes of the individual subunits. c)Assembly of the channel complex, distribution in different cellular compartments and cell surface expression. AIM 2. Modulation of channel activity by phosphorylation. To demonstrate biochemically protein phosphorylation and to identify the cellular pathways and kinases that mediate channel phosphorylation. a)To determine the subunit(s) that are phosphorylated and to identify the amino acids that are phosphorylated by specific kinases. b)To study the changes in channel activity induced by phosphorylation by proteinkinases (PKA, PKC, Ca2+/calmoduline, kinase, thyrosine kinases). c)Functional consequences of replacing the phosphorylated amino acids by other residues. AIM 3. Isolation of other proteins that associate with the Na+ channel. Ion channels are complex multimeric proteins with a general structure consisting of pore-forming subunit(s) and other associated proteins involved in the modulation of channel activity. Here we propose: a)To isolate and identify proteins associated to the subunits of the Na+ channel. b)To examine the functional roles of these proteins in modulating the activity of channels. c)To investigate aldosterone effects on the levels of expression of the associated proteins. d)To study the molecular determinants of the protein- protein interactions and the cellular processes that change these interaction.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a central role in the maintenance of sodium homeostasis, it constitutes the main pathway for reabsorption of sodium in tight epithelia such as the distal nephron. The importance of the sodium channel in the maintenance of extracellular volume and blood pressure is underscored by the finding of mutations in the human genes that either active channels leading to hypertension (Liddle's syndrome), or conversely, decrease activity of channels leading to salt-wasting and hypovolemic states (pseudohypoaldosteronism). Elucidation of the mechanisms that normally regulate the function and expression of ENaC is of major importance for the understanding of blood volume maintenance in physiological and pathological conditions. The long term objectives of this work are to understand the molecular mechanisms of channel function and regulation. The specific goals of this proposal are: 1) to identify functional domains using the differences in properties exhibited by channels formed by ab and ag subunits. Mapping of functional domains will be performed using chimeras generated between the b and g subunits; 2) to elucidate the mechanics by which aldosterone mediates phosphorylation of ENaC. Aldosterone increase sodium permeability by increasing the abundance of sodium channels and by activating pre-existing channels. However, the mechanism(s) that mediate the activation of channels is still unknown. We propose that aldosterone induced phosphorylation is one of the mechanisms that activates pre-existing channels; 3) to understand the regulation of expression of ENaC by ubiquitination. We will examine the hypothesis that ubiquitination participates in endocytosis at the plasma membrane and in degradation of channels in intracellular compartments. Injected Xenopus oocytes and transfected cells will provide the expression systems for wild-type and mutant channels in which we will examine the functional properties, levels of expression, cellular distribution and rates of biosynthesis and degradation of channels. Experiments are designed to examine the activity and properties of channels by electrophysiologycal techniques. Ensembles of channels will be studied using the two micro-electrode voltage clamp and single channels using the patch-clamp technique. Biochemical, immunological, and molecular biological approaches will be applied to examine the state of phosphorylation and ubiquitination of the channel. These studies will identify important functional domains in the sodium channel, and are the first to explore two novel regulatory mechanisms of the function and expression of sodium channels.

The level of expression of epithelial sodium channels (ENaC) is the main determinant of the net sodium reabsorption by the distal nephron. The first hypothesis to be evaluated is that expression of ENaC at the cell surface is actively regulated by clathrin-mediated endocytosis which, under basal conditions, maintains a low number of channels at the apical membrane. Hormones and environmental factors can increase the expression of channels by modifying the rate of their internalization. This is achieved by modifications of amino acids located in the vicinity of the endocytic signals that change the interaction between the channel proteins and the endocytic machinery. To evaluate the role of endocytosis on ENaC expression we will transfect MDCK cells with wild-type or mutant channels lacking the endocytic signals. The level of expression of channels and the rate of endocytosis will be assessed by functional assays such as short- circuit current, and biochemical ones such as biotinylation of cell surface channels. The effects on the rate of endocytosis of channels induced by aldosterone, insulin, ADH, and luminal sodium concentration will be specifically examined. The second hypothesis to be examined is that the rate limiting step in the delivery of newly synthesized channels is the assembly of subunits. To evaluate this process we will determine the rate of delivery of newly synthesized channels to the plasma membrane; what subunit combinations result in delivery of functional channels to the plasma membrane; the half-life of the subunits in cells expressing varied subunit combinations, and the domains that participate in subunit recognition and assembly. The experimental approaches to investigate subunit interactions will consist on co-immunoprecipitation experiments, sedimentation in sucrose gradients and the two-hybrid system in yeast. These studies will provide insight into mechanisms important to the regulation of ENaC expression and sodium reabsorption by the cortical collecting tubule, and the pathogenesis of salt-sensitive hypertension.

DESCRIPTION (provided by applicant): Sodium reabsorption in the distal tubule of the kidney is essential for maintenance of extracellular volume and blood pressure. Aldosterone, vasopressin and insulin stimulate sodium reabsorption by upregulating the activity of ENaC. There is growing evidence that sgkl (serum- and glucocorticoid-induced kinase) mediates the effects of these hormones; however, the signaling pathways employed by sgkl and the molecular mechanisms that modify ENaC activity have not been elucidated. Our working hypothesis stems from a model in which sgkl localizes predominantly at the basolateral membrane of epithelial cells in close proximity with incoming agonists such as insulin or AVP. Phosphorylation of sgkl by these and/or other stimuli to date unknown triggers a signal cascade that reaches the apical membrane to increase the abundance and/or activity of ENaC. The specific aims of this grant proposal are to: 1) Identify the mechanism underlying the effects of sgkl on ENaC in particular, to investigate changes in the traffic of channels, insertion and/or retrieval from the apical membrane, and changes in channel kinetics. 2) Determine the contribution of sgkl to aldosterone, vasopressin and insulin responses, and 3) To identify molecules that by associating with sgkl tether it to membranes, modulate its activity or constitute substrates of the sgkl-signaling cascade. We have developed tools and designed biochemical (turnover of ENaC subunits, phosphorylation assays, modified Ras recruitment system) and electrophysiological (patch-clamp studies and blocker-induced noise analysis) experiments to be conducted in vitro (A6 cell lines modified to express various forms of sgkl and siRNAs under the control of tetracycline) and in rat tissues that together will elucidate the proposed aims. The central role of sgkl in the integration of many signaling pathways makes it a candidate to mediate sodium retention associated with hypertensive disorders. Therefore, understanding the molecular mechanisms of this signaling pathway has potential implications for the diagnosis and development of new treatment strategies for the correction of high blood pressure.

? DESCRIPTION (provided by applicant): ASIC or Acid Sensing Ion Channel is a proton-gated sodium channel that belongs to the large family of Deg/ENaC ion channels expressed in vertebrates and invertebrates. ASIC has been implicated in various neurological disorders though their physiological roles remain poorly defined. Slow progress elucidating ASIC functions stems in part from technical difficulties of changing the interstitial pH in selected areas of the brain. To date most attempts to activate ASIC have used perfusion of acid solutions or induction of ischemia; however such maneuvers have low spatial and temporal control of the concentration of protons and also tend to suppress ASIC activity because they induce desensitization. To overcome this problem we have implemented an optogenetic-based method that acidifies selected areas of the extracellular space in the CNS with high temporospatial resolution enabling activation of ASIC in any structure of the brain. The method consists on expressing the light-driven proton pump ArchT in astrocytes that upon illumination extrude protons lowering the pH surrounding nearby neurons. The acidification activates ASIC and initiates spiking of action potentials. Another hurdle in the field is the ubiquitous expression of ASIC in the CNS - almost all neurons independent of location or functional specialization express ASIC; thus, all the neurons in a microcircuit are affected by drops in pH, making difficult to tease out the contribution of individual neurons to a behavioral response. To address this problem we have developed a genetically encoded specific inhibitor of ASIC; it consists of a `nanobody' that binds to the aminoterminus and inhibits channel activity. This proposal aims to validate the newly developed tools by examining the contribution of ASIC in a well-defined neuronal microcircuit; specifically, we will investigate how ASIC modulates processing of signals encoding fear learning and memory in the basolateral amygdala. Although ASIC is present in most areas of the brain, we will focus in the amygdala because previous reports suggest that it modulates fear responses, albeit the cell types involved and the mechanism remain largely unknown. The work will prove the value of these novel genetic approaches to studying ASIC in behaviors and sets the stage for a next phase of exploration of the physiological and pathological roles of these channels in the mammalian brain.