Active Ion Transport Research Articles

Mislocalization of CFTR expression in acute pancreatitis and the beneficial effect of VX-661+VX-770 treatment on disease severity.

Cystic fibrosis transmembrane conductance regulator (CFTR) has an essential role in maintaining pancreatic ductal function. Impaired CFTR function can trigger acute pancreatitis (AP) and exacerbate disease severity. We aimed to investigate the localization and expression of CFTR during AP, and determined the effects of a CFTR corrector (VX-661) and potentiator (VX-770) on disease severity. AP was induced in FVB/n mice by 6-10 hourly intraperitoneal injections of 50μg/kg cerulein. Some mice were pre-treated with five to six daily injections of 2mg/kg VX-661+VX-770. Control animals were administered physiological saline instead of cerulein and dimethyl sulfoxide instead of VX compounds. AP severity was determined by measuring laboratory and histological parameters; CFTR and CK19 expression was measured. Activity of ion transporters was followed by intracellular pH or fluid secretion measurement of isolated pancreatic intra-/interlobular ducts. Cerulein-induced AP severity was greatest between 12 and 24h. CFTR mRNA expression was significantly increased 24h after AP induction. Immunohistochemistry demonstrated disturbed staining morphology of CFTR and CK19 proteins in AP. Mislocalization of CFTR protein was observed from 6h, while expression increased at 24h compared to control. Ductal HCO3- transport activity was significantly increased 6h after AP induction. AP mice pre-treatment with VX-661+VX-770 significantly reduced the extent of tissue damage by about 20-30%, but other parameters were unchanged. Interestingly, VX-661+VX-770 in vitro administration significantly increased the fluid secretion of ducts derived from AP animals. This study described the course of the CFTR expression and mislocalization in cerulein-induced AP. Our results suggest that the beneficial effects of CFTR correctors and potentiators should be further investigated in AP. KEY POINTS: Cystic fibrosis transmembrane conductance regulator (CFTR) is an important ion channel in epithelial cells. Its malfunction has several serious consequences, like developing or aggravating acute pancreatitis (AP). Here, the localization and expression of CFTR during cerulein-induced AP in mice were investigated and the effects of CFTR corrector (VX-661) and a potentiator (VX-770) on disease severity were determined. CFTR mRNA expression was significantly increased and mislocalization of CFTR protein was observed in AP compared to the control group. Interestingly, pre-treatment of AP mice with VX-661+VX-770 significantly reduced the extent of pancreatic tissue damage by 20-30%. In vitro administration of VX-661+VX-770 significantly increased the fluid secretion of ducts derived from AP animals. Based on these results, the utilization of CFTR correctors and potentiators should be further investigated in AP.

Exploration of Pyrazolo[1,5-a]pyrimidines as Membrane-Bound Pyrophosphatase Inhibitors.

Inhibition of membrane‐bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure‐activity relationships around a new scaffold having a pyrazolo[1,5‐a]pyrimidine core. The most promising pyrazolo[1,5‐a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay.

Enhancement of hydrogen evolution reaction activity using metal–rich molybdenum sulfotelluride with graphene support: A combined experimental and computational study

‘Green hydrogen’ can be one of the promising energy technologies in near future. Therefore, the importance of discovering the efficient electrocatalysts for hydrogen generation can never be refuted. The present work demonstrates a facile, ultrafast (60 s) microwave-assisted synthesis approach to develop an electrocatalyst of molybdenum sulfotelluride on graphene support, which is denoted as MoSxTey/Gr. The abundant interfaces in hybrid nanostructure of MoSxTey/Gr enable more exposed active sites for electrochemical reaction, facilitating the ion and charge transport activities. Among the resultant nanocomposites with different elemental ratios of Mo, S and Te, the MoS0.46Te0.58/Gr exhibits the best hydrogen evolution property with a low overpotential of 62.2 mV at 10 mA cm−2, a small Tafel slope of 61.1 mV dec−1, and long–term stability in 0.5 M H2SO4 electrolyte. Moreover, the tool of periodic plane-wave density functional theory (DFT) has been used to elucidate hydrogen binding energetics on various molybdenum sulfotelluride (stoichiometric and non-stoichiometric molybdenum-rich) on graphene nanocomposite systems. According to the computational results, high-performing catalytically active sites are found to be comprised of primarily exposed Mo atoms, thus showing Mo enrichment as a potential method for electrocatalyst engineering. Furthermore, in a volcano plot constructed with both computational and experimental values, the position of the MoS0.46Te0.58/Gr nanocomposite is found to be close to the apex with near thermoneutral catalytic activity.

Advances in plant heavy metal transporter P1B-ATPases

P1B-ATPases are a group of proteins that can transport heavy metal ions across membranes by hydrolyzing ATP and they are a subclass of the P-type ATPase family. It was found that P1B-ATPases are mainly responsible for the active transport of heavy metal ions in plants and play an important role in the regulation of heavy metal homeostasis in plants. In this paper, we dissusses the mechanism of P1B-ATPases from the structure and classification of P1B-ATPases, and review the current research progress in the function of P1B-ATPases, in order to provide reference for future research and application of P1B-ATPases in improving crop quality and ecological environment management.

Light-Driven Molecular Motors Boost the Selective Transport of Alkali Metal Ions through Phospholipid Bilayers.

A hydrophobic light-driven rotary motor is functionalized with two 18-crown-6 macrocycles and incorporated into phospholipid bilayers. In the presence of this molecular construct, fluorescence assays and patch clamp experiments show the formation of selective alkali ion channels through the membrane. Further, they reveal a strongly accelerated ion transport mechanism under light irradiation. This increase of the fractional ion transport activity (up to 400%) is attributed to the out-of-equilibrium actuation dynamics of the light-driven rotary motors, which help to overcome the activation energy necessary to achieve translocation of alkali ions between macrocycles along the artificial channels.

Stimuli-Responsive Anion Transport through Acylhydrazone-Based Synthetic Anionophores.

Photoswitchable acylhydrazone-based synthetic anionophores are reported. Single-crystal X-ray structure and 1H NMR titration studies confirmed the chloride binding in solid and solution states. The ion transport activity of 1a was greatly attenuated through a phototriggered E to Z photoisomerization process, and the photoisomerized deactivated state showed high kinetic stability due to an intramolecular hydrogen bond. Switchable "OFF-ON" transport activity was achieved by the application of light and acid-catalyzed reactivation process.

Ion transport activity and optogenetics capability of light-driven Na+-pump KR2.

KR2 from marine bacteria Krokinobacter eikastus is a light-driven Na+ pumping rhodopsin family (NaRs) member that actively transports Na+ and/or H+ depending on the ionic state. We here report electrophysiological studies on KR2 to address ion-transport properties under various electrochemical potentials of Δ[Na+], ΔpH, membrane voltage and light quality, because the contributions of these on the pumping activity were less understood so far. After transient expression of KR2 in mammalian cultured cells (ND7/23 cells), photocurrents were measured by whole-cell patch clamp under various intracellular Na+ and pH conditions. When KR2 was continuously illuminated with LED light, two distinct time constants were obtained depending on the Na+ concentration. KR2 exhibited slow ion transport (τoff of 28 ms) below 1.1 mM NaCl and rapid transport (τoff of 11 ms) above 11 mM NaCl. This indicates distinct transporting kinetics of H+ and Na+. Photocurrent amplitude (current density) depends on the intracellular Na+ concentration, as is expected for a Na+ pump. The M-intermediate in the photocycle of KR2 could be transferred into the dark state without net ion transport by blue light illumination on top of green light. The M intermediate was stabilized by higher membrane voltage. Furthermore, we assessed the optogenetic silencing effect of rat cortical neurons after expressing KR2. Light power dependency revealed that action potential was profoundly inhibited by 1.5 mW/mm2 green light illumination, confirming the ability to apply KR2 as an optogenetics silencer.

A candidate gene of Alzheimer diseases was mutated in senescence-accelerated mouse prone (SAMP) 8 mice

The senescence-accelerated mouse prone (SAMP) 8 strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. We have found strong association of chromosome 12 locus with learning memory deficit (LMD) phenotype in SAMP8 strain. In the course of searching candidate gene, here we identified solute carrier family 24 sodium/potassium/calcium exchanger member 4 (Slc24a4) in SAMP8 chromosome 12 LMD possessing one single nucleotide polymorphism causing amino acid replacement of Threonine at 413 position with Methionine. Since SLC24A4 has been postulated as a candidate of late onset Alzheimer's diseases (LOAD), we further analyze the functional importance of this polymorphism. By expressing Slc24a4 protein in HEK293 cells, here we showed polymorphic SAMP8 type Slc24a4-T413 M causing significant loss of calcium ion (Ca2+) transporter activity in cells compared with that of wild type mouse (Slc24a4-WT). However, no study yet shows any functional association of human SLC24A4 polymorphism with the onset of LOAD pathogenesis. Thus, our present finding may further help to clarify the importance of this ion exchanger with age related cognitive dysfunction.

Electrochemically Switchable Double-Gate Nanofluidic Logic Device as Biomimetic Ion Pumps

Biological ion pumps with two separate gates can actively transport ions against the concentration gradient. Developing an artificial nanofluidic device with multiple responsive sites is of great importance to improve its controllability over ion transport to further explore its logic function and mimic the biological process. Here, we propose an electrochemical polymerization method to fabricate electrochemically switchable double-gate nanofluidic devices. The ion transport of the double-gate nanofluidic device can be in situ and reversibly switched among four different states. The logic function of this nanofluidic device is systematically investigated by assuming the gate state as the input and the transmembrane ionic conductance as the output. A biomimetic electrochemical ion pump is then established by alternately applying two different specific logic combinations, realizing an active ion transport under a concentration gradient. This work would inspire further studies to construct complex logical networks and explore bioinspired ion pump systems.

Cystic ovary disease impairs transport speed, smooth muscle contraction, and epithelial ion transport in the bovine oviduct.

Cystic ovary disease (COD) is a common cause of bovine infertility but the impact of this disease on the oviduct is unknown. The aim of this study was to analyze the effects of COD on particle transport speed (PTS), ciliary beat frequency, myosalpinx contraction, and epithelial ion transport. Oviducts were obtained from cows affected by COD and compared with those of healthy, mid-diestrus cows. PTS and CBF were examined using live-cell imaging. Smooth muscle contraction and epithelial ion transport were investigated using organ baths and Ussing chambers. Our results showed that muscarinic receptors are involved in cholinergic signaling in the oviduct and that forskolin-induced cyclic AMP production is involved in active ion transport in the oviductal epithelium. Oviducts from cows with luteal cysts revealed significantly decreased PTS (p = 0.02). Further to that, in the oviducts of COD cows, the cholinergic regulation of smooth muscle contractions and active epithelial ion transport were significantly diminished (p < 0.0001). These results imply that in COD cows, oviductal transport is compromised by decreased fluid flow speed and reduced cholinergic regulation of smooth muscle contraction and ion transport. This knowledge contributes to a more comprehensive understanding of COD supporting the development of novel therapeutic concepts for infertility treatment.

Recent Advances in Bioactive Artificial Ionophores.

Several life‐threatening diseases, also known as ‘Channelopathies’ are linked to irregularities in ion transport proteins. Significant research efforts have fostered the development of artificial transport systems that facilitates to restore the functions of impaired natural transport proteins. Indeed, a few of these artificial ionophores demonstrate the rare combination of transmembrane ion transport and important biological activity, offering early promises of suitability in ‘channel replacement therapy’. In this review, structural facets and functions of both cationophores and anionophores are discussed. Ionophores that are toxic to various bacteria and yeast, could be exploited as antimicrobial agent. Nevertheless, few non‐toxic ionophores offer the likelihood of treating a wide range of genetic diseases caused by the gene mutations. In addition, their ability to disrupt cellular homeostasis and to alter lysosomal pH endow ionophores as promising candidates for cancer treatment. Overall, critically outlining the advances in artificial ionophores in terms of in vitro ion transport, possible modes of action and biological activities enables us to propose possible future roadmaps in this research area.

Fe-Doped CoP holey nanosheets as bifunctional electrocatalysts for efficient hydrogen and oxygen evolution reactions

Two-dimensional porous nanosheet material has attracted great attention as a promising electrocatalyst for water splitting. Herein, mesh-like Fe-doped CoP holey nanosheets (Fe–CoP HNSs) is synthesized by a facile phosphorization treatment. The Fe–CoP HNSs catalyst features abundant active sites, short charge and ion transport pathways, and favorable mass transfer kinetics, which are conductive to promote the water splitting reaction. Remarkably, the Fe–CoP HNSs catalyst exhibits superior catalytic activity towards hydrogen and oxygen evolution reaction with low overpotentials of 79 and 220 mV to reach 10 mA cm−2, respectively. Furthermore, the assembled Fe–CoP HNSs||Fe–CoP HNSs electrolyzer needs a low cell voltage of 1.60 V to deliver 20 mA cm−2, which is higher than many reported transition metal phosphides electrocatalysts. This work sheds light on the fabrication of transition-metal compound electrocatalyst with holey nanosheet structure by element doping for renewable energy production.

Crown ether-thiourea conjugates as ion transporters

Na+, Cl− and K+ are the most abundant electrolytes present in biological fluids that are essential to the regulation of pH homeostasis, membrane potential and cell volume in living organisms. Herein, we report synthetic crown ether-thiourea conjugates as a cation/anion symporter, which can transport both Na+ and Cl− across lipid bilayers with relatively high transport activity. Surprisingly, the ion transport activities were diminished when high concentrations of K+ ions were present outside the vesicles. This unusual behavior resulted from the strong affinity of the transporters for K+ ions, which led to predominant partitioning of the transporters as the K+ complexes in the aqueous phase preventing the transporter incorporation into the membrane. Synthetic membrane transporters with Na+, Cl− and K+ transport capabilities may have potential biological and medicinal applications.

Transcriptomics analysis of differentially expressed genes in subcutaneous and perirenal adipose tissue of sheep as affected by their pre- and early postnatal malnutrition histories

BackgroundEarly life malnutrition is known to target adipose tissue with varying impact depending on timing of the insult. This study aimed to identify differentially expressed genes in subcutaneous (SUB) and perirenal (PER) adipose tissue of 2.5-years old sheep to elucidate the biology underlying differential impacts of late gestation versus early postnatal malnutrition on functional development of adipose tissues. Adipose tissues were obtained from 37 adult sheep born as twins to dams fed either NORM (fulfilling energy and protein requirements), LOW (50% of NORM) or HIGH (110% of protein and 150% of energy requirements) diets in the last 6-weeks of gestation. From day 3 to 6 months of age, lambs were fed high-carbohydrate-high-fat (HCHF) or moderate low-fat (CONV) diets, and thereafter the same moderate low-fat diet.ResultsThe gene expression profile of SUB in the adult sheep was not affected by the pre- or early postnatal nutrition history. In PER, 993 and 186 differentially expressed genes (DEGs) were identified in LOW versus HIGH and NORM, respectively, but no DEG was found between HIGH and NORM. DEGs identified in the mismatched pre- and postnatal nutrition groups LOW-HCHF (101) and HIGH-HCHF (192) were largely downregulated compared to NORM-CONV. Out of 831 DEGs, 595 and 236 were up- and downregulated in HCHF versus CONV, respectively. The functional enrichment analyses revealed that transmembrane (ion) transport activities, motor activities related to cytoskeletal and spermatozoa function (microtubules and the cytoskeletal motor protein, dynein), and responsiveness to the (micro) environmental extracellular conditions, including endocrine and nervous stimuli were enriched in the DEGs of LOW versus HIGH and NORM. We confirmed that mismatched pre- and postnatal feeding was associated with long-term programming of adipose tissue remodeling and immunity-related pathways. In agreement with phenotypic measurements, early postnatal HCHF feeding targeted pathways involved in kidney cell differentiation, and mismatched LOW-HCHF sheep had specific impairments in cholesterol metabolism pathways.ConclusionsBoth pre- and postnatal malnutrition differentially programmed (patho-) physiological pathways with implications for adipose functional development associated with metabolic dysfunctions, and PER was a major target.

Self-Assembled H-Bonding Superstructures for Alkali Cation and Proton Transport.

Transmembrane protein channels are of significant importance for the design of biomimetic artificial ion channels. Regarding the transport principles, they may be constructed from amphiphilic compounds undergoing self-assembly that synergistically generate directional superstructures across bilayer membranes. Particularly interesting, these alignments may impose an artificial pore structure that may control the ionic conduction and translocate water and ions sharing one pathway across the cell membrane. Herein, we report that the imidazole and 3-amino-triazole amphiphiles self-assemble via multiple H-bonding to form stable artificial networks within lipid bilayers. The alignment of supramolecular assemblies influences the conduction of ions, envisioned to diffuse along the hydrophilic pathways. Compounds 1-8 present subtle variations on the ion transport activities, depending the structure of hydrophilic head and hydrophobic components. Fluorinated compounds 3, 4 and 7, 8 outperform the corresponding non-fluorinated counterparts 1, 2 and 5, 6. Under the same conditions, the R enantiomers present a higher activity vs. the S enantiomers. The present systems associating supramolecular self-assembly with ion-transport behaviors may represent very promising unexplored alternatives for ion-transport along with their transient superstructures within bilayer membranes, paralleling to that of biology.

The role of the V‐type H + ‐ATPase in CAPA‐treated Malpighian tubules in the yellow fever mosquito, Aedes aegypti

Haematophagus insects, such as the female Aedes aegypti mosquito, face the challenge of excess ion and water intake after engorgement on a blood meal. To cope with this, adult female A. aegypti utilize their highly adapted excretory system that includes the Malpighian ‘renal’ tubules (MTs), which are under rigorous control by several hormones to regulate transepithelial movement of ions/water. Originating in the nervous system, the mosquito anti-diuretic hormone is a member of the CAPA peptide family and is a homolog of the vertebrate neuromedin U peptide. CAPA peptides inhibit fluid secretion of MTs stimulated by select diuretic factors, 5HT and DH31 through the NOS/cGMP/PKG pathway. However, the anti-diuretic signalling mechanism and downstream cellular targets, such as ion channels and transporters, remain unclear. Active ion transport in A. aegypti MTs is driven by the principal cells, whose brush-border membrane is densely packed by the V-type H+-ATPase (VA), serving as the primary energizer for transepithelial secretion of electrolytes and water. Due to its predominant role in fluid secretion, the VA is a likely target for diuretic and anti-diuretic factors. The effect of endocrine factors and the VA inhibitor, bafilomycin, on fluid secretion rate by MTs was measured using an in vitro bioassay. Bafilomycin was found to inhibit fluid secretion stimulated by serotonin (5HT) and the calcitonin-related diuretic hormone (DH31), while having no inhibitory action on MTs stimulated with the corticotropin-releasing factor (CRF)-related diuretic hormone. CAPA and bafilomycin treatment led to alkalization of the secreted fluid suggesting inhibition of the apical VA, which may lead to constrained entry of cations (Na+ and K+) across the apical membrane of MTs. VA activity was increased in adult female MTs treated with DH31, whereas CAPA treatment reduced VA activity, comparable to saline levels. Furthermore, a VA antibody was used to determine protein localization and expression in DH31 and CAPA-treated MTs. In DH31-incubated MTs, the VA was exclusively localized to the apical memrane of the MTs, whereas VA immunoreactivity was observed in both the apical membrane and cytosol of CAPA-treated tubules. These results suggest a novel mechanism for CAPA inhibition, blocking the VA to hinder fluid secretion. A. aegypti mosquitoes are vectors of a variety of pathogens leading to illnesses, such as Dengue fever, Yellow fever, and the Zika virus. The current results advance our knowledge of the anti-diuretic control of the A. aegypti MTs, suggesting that CAPA targets the VA to inhibit diuresis. Investigating the pathway of CAPA inhibition and its role in countering diuresis will help provide a deeper understanding of this critical physiological process. Studying the excretory system and discerning the underlying mechanisms behind ion and fluid transport may aid in developing novel pest management strategies to better control Aedes population and lessen its prominence as a disease vector.

Electrophysiological Effects of Ionophore-induced Increases in Intracellular Na+ in Cardiomyocytes

Na ionophores increase intracellular Na+ ([Na+]i). Membrane potentials and currents were measured using microelectrode and whole-cell patch-clamp techniques. Monensin (10-6-3×10-5 M) reduced the slope of the pacemaker potentials and shortened the action potential duration (APD) in sino-atrial nodal and Purkinje cells. Monensin (10-5 M) shortened the APD and reduced the amplitude of the plateau phase in ventricular myocytes. Monensin decreased the hyperpolarization-activated inward current (If), and it increased the transient outward potassium current (Ito) in Purkinje cells. In addition, monensin decreased the sodium current (INa), shifting the inactivation curve to the hyperpolarized direction. Moreover, monensin decreased the L-type calcium current (ICa) in ventricular myocytes. The Na+-Ca2+ exchange current (INa-Ca) was augmented particularly in the reverse mode, and the Na+-K+ pump current (INa-K) was also activated by monensin in cardiomyocytes. The ATP-activated potassium current (IK,ATP) could be induced by monensin. Notably, the inward rectifying K+ current (IK1), and the slow delayed outward K+ current (IKs) were not affected evidently by monensin. Collectively, alteration of [Na+]i can influence the activities of various ion channels and transporters. Thus, the significance of altered [Na+]i should be taken into consideration in the action of drugs affecting [Na+]i such as digitalis, Na+ channel blockers, and Na+ channel activating agents.

Tonic Inhibition of Sodium Reabsorption by Na + /K + ‐ATPase in the Renal Proximal Tubule

In the renal proximal tubule (PT), Na+/K+-ATPase (NKA) is exclusively located in the basolateral domain. Through its classic ATP-dependent ion-pumping function, NKA generates the Na+ gradient that drives apical Na+ reabsorption, mostly through Na+/H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical Na+ reabsorption (NHE3). In contrast, pharmacological evidence suggests that activation of the more recently discovered NKA signaling function triggers a cellular redistribution of NKA and NHE3 that decreases transcellular Na+ flux in cultured PT cells. To obtain genetic evidence of this NKA/Src mechanism in the PT and asses its physiological importance, we used a knockdown and rescue approach in pig renal epithelial cells (LLC-PK1). Additionally, we genetically targeted NKA α1 in the mouse PT by crossing mice expressing a sodium glucose co-transporter 2 promoter driven Cre transgene with Floxed NKA α1 mice (PTα1-/-). Knockdown of 90% of NKA α1 in PT LLC-PK1 cells increased transepithelial 22Na flux by 2-fold, activated NHE3 (50% decrease in inhibitory phosphorylation), and increased basolateral Na+/HCO3- cotransporter (NBCe1A) protein content. In the PTα1-/- mouse (4-month males and females in a 1:1 ratio), 70% decrease in PT NKA α1 expression decreased urine output (0.51±0.14 vs 1.57±0.21 mL/24h in PTα1+/+, p<0.001, n=16) and absolute Na+ excretion (0.14±0.05 vs 0.36±0.05 mmol/24h in PTα1+/+, p<0.05, n=8) by 65%, without histological or functional evidence of renal injury. Those changes were driven by increased PT Na+ reabsorption, as indicated by a 65% decrease in lithium clearance (4-month males, 1344±220 vs 3932±697 mL/24h in PTα1+/+, p<0.001, n=12) with unchanged GFR. This hyper-reabsorptive phenotype of PTα1-/- mice was coupled to increased membrane abundance of NHE3 and NBCe1A, and rescued upon crossing with floxed NHE3 mice, consistent a NKA/NHE3-dependent mechanism. A dismantlement of caveolar NKA/Src receptor complex and intracellular redistribution of pY418Src occurred in knockdown NKA α1 PT cells, and was also observed in the PTα1-/- hypomorphic mouse. Rescue of PT cells with wild-type but not Src signaling-null NKA α1 restored NHE3 and NBCe1A to basal levels, indicative of a role for NKA/Src receptor function in the tonic inhibition of Na+ transporters in the PT. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, which is lifted upon NKA genetic suppression in cells and in vivo, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA/Src signaling is not only physiologically relevant, it is functionally dominant over NKA ion-pumping in the control of PT reabsorption. NKA signaling therefore provides a long sought-after mechanism for the natriuretic action of endogenous NKA ligands such as cardiotonic steroids.

The Effect of Diet on Aquaporin Abundance and Localization in the Female and Male Disease Vector Mosquito, Aedes aegypti

The disease vector mosquito, Aedes aegypti, is found in tropical and sub-tropical regions around the globe and is responsible for the transmission of many deadly diseases such as dengue fever and chikungunya. Adult A. aegypti are sexually dimorphic terrestrial animals since both the males and females feed on nectar for basic nutrients, females mosquitoes require a blood meal from a vertebrate host to produce viable eggs. The blood meal poses an osmoregulatory challenge to the female mosquito because the meal is about 3x its body weight in water, plasma, and proteins. The Malpighian tubules (MTs) are the primary osmoregulatory organ, responsible for producing an ion-rich primary urine through hormone activated ion transport driven by an H+-ATPase. Through diuretic hormone control, the MTs are able to begin secretion of primary urine almost immediately following the onset of blood feeding and this process requires intermembrane transporters called aquaporins (AQPs). The concentration of ions in the lumen of the MTs drives water from the hemolymph into the lumen of the MTs by osmosis to produce primary urine. Osmosis is facilitated by the expression of AQPs in the cells of the MTs. Six AQPs have been identified in A. aegypti, most of which are all selective for water and some solutes. It is hypothesized that a blood meal in female A. aegypti, with knowledge that mRNA levels of AQP1 are increased in the MTs ~3-24hrs post blood meal, will have an effect on the localization and protein abundance of AQPs in the MTs. It was also hypothesized that the starvation of male A. aegypti would have an effect on the localization and abundance of AQPs in the MTs. Thus far, AQP1 & 4 have been localized throughout the body of both the male and female A. aegypti using immunohistochemistry. Immunolocalization of AQPs shows evidence that a blood meal has a significant effect on the localization in the female A. aegypti. It has also been found that, following Western blot analysis, a blood meal may significantly increase the protein abundance AQP1 and 4 in the MTs of the females. It has been shown that in the male A. aegypti, AQP4 abundance is significantly increased in the fat body.

Regulation of NOS3 by Novel Acetylation Sites

Nitric oxide (NO) is important for cardiovascular and renal health. NO is primarily produced via the enzymatically catalyzed reaction of L-arginine and oxygen to produce NO and L-citrulline by nitric oxide synthase (NOS) isoforms, NOS1, NOS2, and NOS3. NOS3 was first identified in the endothelium but is also present in the kidney epithelium. Endothelial NOS3 maintains cardiovascular health by preventing platelet aggregation, smooth muscle proliferation, and leukocyte adhesion. Epithelial NOS3 controls water and electrolyte homeostasis by regulating ion transporter activity along the nephron. NOS3 activity is tightly controlled by posttranslational modifications. While phosphorylation sites have been well characterized, less is known about regulation by acetylation. Lysine acetylation is a dynamic posttranslational modification observed in both the nucleus and cytosol to regulate intracellular signaling. Our lab and others have shown NOS3 to be regulated by multiple deacetylases. We therefore sought to uncover novel lysine acetylation sites on NOS3 which modulate its activity. Based on posttranslational modification prediction software we chose seven lysines to investigate: K486, K497, K504, K607, K610, K727, and K731. We hypothesized that deacetylation of any or all of these lysine sites increases NOS3-mediated NO production. In order to test this hypothesis, we created NOS3 constructs containing lysine to arginine mutations at these sites mimicking deacetylation. These plasmids were transfected into COS7 cells and expression was confirmed with Western blot. NO production was assessed by measuring nitrite accumulation in the media over 48 hours. Constructs containing either K486R, K497R, or K504R did not show a significant increase in NO production compared to WT (WT: 1703±499.3 pmol NO2/mg protein; K486R: 1984 ± 1003 pmol NO2/mg protein; K497: 1528 ± 1032 pmol NO2/mg protein; K504: 4337±576.7 pmol NO2/mg protein; p < 0.05 vs WT). However, mutations of four lysines, K607R, K610R, K727R, and K731R (K607/610/727/731R), dramatically increased NO production (21971 ± 1941 pmol NO2/mg protein; WT: 1703±499.3 pmol NO2/mg protein; p < 0.0001 vs WT). To further identify which of the four lysines are responsible for the observed phenotype, we made two new constructs containing either K607/610R or K727/731R. K607/610R was similar to WT (WT: 2271 ± 729.3 pmol NO2/mg protein; K607/610R: 3523 ± 805.8 pmol NO2/mg protein: p > 0.05), yet mutations of K727/731R demonstrated a large increase in NO production (9431 ± 1772 pmol NO2/mg protein; WT: 2271 ± 729.3 pmol NO2/mg protein; p<0.05). These studies suggest K727 and/or K731 is basally acetylated and deacetylation increases NO production uncovering novel posttranslational modifications which regulate NOS3 activity. Future studies will identify whether K727, K731, or both sites are responsible for the increase in NO production.