Aylin R. Rodan

N-linked oligosaccharides are necessary and sufficient for association of glycosylated forms of bovine RNase with calnexin and calreticulin.

Calnexin and calreticulin are lectin‐like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N‐linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N‐linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N‐glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin‐like interaction, and not a protein‐protein interaction, played the central role in stabilizing RNase‐calnexin/calreticulin complexes.

Drosophila fasciclinII Is Required for the Formation of Odor Memories and for Normal Sensitivity to Alcohol

Drosophila fasciclinII (fasII) mutants perform poorly after olfactory conditioning due to a defect in encoding, stabilizing, or retrieving short-term memories. Performance was rescued by inducing the expression of a normal transgene just before training and immediate testing. Induction after training but before testing failed to rescue performance, showing that Fas II does not have an exclusive role in memory retrieval processes. The stability of odor memories in fasII mutants are indistinguishable from control animals when initial performance is normalized. Like several other mutants deficient in odor learning, fasII mutants exhibit a heightened sensitivity to ethanol vapors. A combination of behavioral and genetic strategies have therefore revealed a role for Fas II in the molecular operations of encoding short-term odor memories and conferring alcohol sensitivity. The preferential expression of Fas II in the axons of mushroom body neurons furthermore suggests that short-term odor memories are formed in these neurites.

Insulin signaling in the nervous system regulates ethanol intoxication in Drosophila melanogaster

The insulin signaling pathway regulates multiple physiological processes, including energy metabolism, organismal growth, aging and reproduction. Here we show that genetic manipulations in Drosophila melanogaster that impair the function of insulin-producing cells or of the insulin-receptor signaling pathway in the nervous system lead to increased sensitivity to the intoxicating effects of ethanol. These findings suggest a previously unknown role for this highly conserved pathway in regulating the behavioral responses to an addictive drug.

The Genetics of Behavioral Alcohol Responses in Drosophila

Drosophila melanogaster is commonly found near rotting or fermenting fruit, reflected in its name pomace, or vinegar fly. In such environments, flies often encounter significant levels of ethanol. Three observations have made Drosophila a very promising model organism to understand the genetic contributions to the behavioral responses to alcohol. First, similar to higher vertebrates, flies show hyperactivation upon exposure to a low to medium dose of alcohol, while high doses can lead to sedation. In addition, when given a choice, flies will actually prefer alcohol-containing food over regular food. Second, the genes and biochemical pathways implicated in controlling these behavioral responses in flies are also participating in determining alcohol responses, and drinking behavior in mammals. Third, the fact that flies have been studied genetically for over one hundred years means that an exceptional repertoire of genetic tools are at our disposal. Here, we will review some of these tools and experimental approaches, survey the methods for, and measures after Drosophila ethanol exposure, and discuss the different molecular components and functional pathways involved in these behavioral responses to alcohol.

Recent advances in distal tubular potassium handling

It is well known that sodium reabsorption and aldosterone play important roles in potassium secretion by the aldosterone-sensitive distal nephron. Sodium- and aldosterone-independent mechanisms also exist. This review focuses on some recent studies that provide novel insights into the sodium- and aldosterone-independent potassium secretion by the aldosterone-sensitive distal nephron. In addition, we discuss a study reporting on the regulation of the mammalian potassium kidney channel ROMK by intracellular and extracellular magnesium, which may be important in the pathogenesis of persistent hypokalemia in patients with concomitant potassium and magnesium deficiency. We also discuss outstanding questions and propose working models for future investigation.

Rsu1 regulates ethanol consumption in Drosophila and humans

Significance Genetic factors play a major role in the development of human addiction. Identifying these genes and understanding their molecular mechanisms are necessary first steps in the development of targeted therapeutic intervention. Here, we have isolated the gene encoding Ras suppressor 1 (Rsu1) in an unbiased genetic screen for altered ethanol responses in the vinegar fly, Drosophila melanogaster. Our behavioral, genetic, and biochemical experiments show that Rsu1 links signaling from the integrin cell adhesion molecule to the small GTPase Rac1 in adult neurons to regulate actin dynamics and alcohol consumption preference. We also show that variants in human RSU1 associate with altered drinking and brain activation during a reward prediction task, thereby validating the predictive power of our approach. Alcohol abuse is highly prevalent, but little is understood about the molecular causes. Here, we report that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. We show that Rsu1 is required in the adult nervous system for normal sensitivity and that it acts downstream of the integrin cell adhesion molecule and upstream of the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naïve preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naïve preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naïve and acquired ethanol preference. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla.

Distal potassium handling based on flow modulation of maxi-K channel activity

Purpose of reviewStudies on the mechanisms of distal K+ secretion have highlighted the importance of the renal outer-medullary K+ (ROMK) and maxi-K channels. This review considers several human disorders characterized by hypokalemia and hyperkalemia, as well as mouse models of these disorders, and the mechanisms by which ROMK and maxi-K may be dysregulated. Recent findingsAnalysis of knockout mice lacking ROMK, a model for type II Bartters syndrome, has shown a role for maxi-K in distal K+ secretion. Knockout mice lacking either the α or β1 subunits of maxi-K also show deficits in flow-dependent K+ secretion. Analysis of transgenic and knock-in mouse models of pseudohypoaldosteronism type II, in which mutant forms of with-no-lysine kinase 4 are expressed, suggests ways in which ROMK and maxi-K may be dysregulated to result in hyperkalemia. Modeling studies also provide insights into the role of Na+ delivery vs. flow in K+ secretion. SummaryThe importance of both ROMK and maxi-K to distal K+ secretion is now well established, but the relative role that each of these two channels plays in normal and diseased states has not been definitively established. Analysis of human and animal model data can generate hypotheses for future experiments.

Long-lasting, experience-dependent alcohol preference in Drosophila

To understand the molecular and neural mechanisms underlying alcohol addiction, many models ranging from vertebrates to invertebrates have been developed. In Drosophila melanogaster, behavioral paradigms from assaying acute responses to alcohol and to behaviors more closely modeling addiction have emerged in recent years. However, both the CAFÉ assay, similar to a two‐bottle choice consumption assay, as well as conditioned odor preference, where ethanol is used as the reinforcer, are labor intensive and have low throughput. To address this limitation, we have established a novel ethanol consumption preference assay, called FRAPPÉ, which allows for fast, high throughput measurement of consumption in individual flies, using a fluorescence plate reader. We show that naïve flies do not prefer to consume ethanol, but various pre‐exposures, such as ethanol vapor or voluntary ethanol consumption, induce ethanol preference. This ethanol‐primed preference is long lasting and is not driven by calories contained in ethanol during the consumption choice. Our novel experience‐dependent model of ethanol preference in Drosophila—a highly genetically tractable organism—therefore recapitulates salient features of human alcohol abuse and will facilitate the molecular understanding of the development of alcohol preference.

Adult neuronal Arf6 controls ethanol-induced behavior with Arfaptin downstream of Rac1 and RhoGAP18B.

Alcohol use disorders affect millions of individuals. However, the genes and signaling pathways involved in behavioral ethanol responses and addiction are poorly understood. Here we identify a conserved biochemical pathway that underlies the sedating effects of ethanol in Drosophila. Mutations in the Arf6 small GTPase signaling pathway cause hypersensitivity to ethanol-induced sedation. We show that Arf6 functions in the adult nervous system to control ethanol-induced behavior. We also find that the Drosophila Arfaptin protein directly binds to the activated forms of Arf6 and Rac1 GTPases, and mutants in Arfaptin also display ethanol sensitivity. Arf6 acts downstream of Rac1 and Arfaptin to regulate ethanol-induced behaviors, and we thus demonstrate that this conserved Rac1/Arfaptin/Arf6 pathway is a major mediator of ethanol-induced behavioral responses.

The Drosophila NKCC Ncc69 is required for normal renal tubule function

Epithelial ion transport is essential to renal homeostatic function, and it is dysregulated in several diseases, such as hypertension. An understanding of the insect renal (Malpighian) tubule yields insights into conserved epithelial ion transport processes in higher organisms and also has implications for the control of insect infectious disease vectors. Here, we examine the role of the Na(+)-K(+)-2Cl(-) (NKCC) cotransporter Ncc69 in Drosophila tubule function. Ncc69 mutant tubules have decreased rates of fluid secretion and K(+) flux, and these phenotypes were rescued by expression of wild-type Ncc69 in the principal cells of the tubule. Na(+) flux was unaltered in Ncc69 mutants, suggesting Na(+) recycling across the basolateral membrane. In unstimulated tubules, the principal role of the Na(+)-K(+)-ATPase is to generate a favorable electrochemical gradient for Ncc69 activity: while the Na(+)-K(+)-ATPase inhibitor ouabain decreased K(+) flux in wild-type tubules, it had no effect in Ncc69 mutant tubules. However, in the presence of cAMP, which stimulates diuresis, additional Na(+)-K(+)-ATPase-dependent K(+) transport pathways are recruited. In studying the effects of capa-1 on wild-type and Ncc69 mutant tubules, we found a novel antidiuretic role for this hormone that is dependent on intact Ncc69, as it was abolished in Ncc69 mutant tubules. Thus, Ncc69 plays an important role in transepithelial ion and fluid transport in the fly renal tubule and is a target for regulation in antidiuretic states.