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Master of Science / Department of Anatomy and Physiology / Daniel C. Marcus / Sensory transduction in the cochlea depends on regulated ion secretion and absorption. Results of whole-organ experiments suggested that Reissner’s membrane may play a role in the control of luminal Cl-. We tested for the presence of Cl- transport pathways in isolated mouse Reissner’s membrane using whole-cell patch clamp recordings and gene transcript analyses using RT-PCR. The current-voltage (I-V) relationship in the presence of symmetrical NMDG-Cl was strongly inward-rectifying at negative voltages, with a small outward current at positive voltages. The inward-rectifying component of the I-V curve had several properties similar to those of the ClC-2 Cl- channel. It was stimulated by extracellular acidity and inhibited by extracellular Cd2+, Zn2+, and intracellular ClC-2 antibody. Channel transcripts expressed in Reissner’s membrane include ClC-2, Slc26a7 and ClC-Ka, but not Cftr, ClC-1, ClCa1, ClCa2, ClCa3, ClCa4, Slc26a9, ClC-Kb, Best1, Best2, Best3 or the beta-subunit of ClC-K, barttin. ClC-2 is the only molecularly-identified channel present that is a strong inward rectifier. This thesis incorporates the publication by K.X. Kim and D.C. Marcus, Inward-rectifier chloride currents in Reissner’s membrane epithelial cells. Biochem. Biophys. Res. Commun. 394 (2010) 434-438, with permission of the publisher Elsevier, and is the first report of conductive Cl- transport in epithelial cells of Reissner’s membrane and is consistent with an important role in endolymph anion homeostasis.

<p>Mosquitoes pose significant threats to human health because they act as vectors for disease causing viruses and protozoans. Indeed, <em>Aedes aegypti</em> is known as the Yellow Fever Mosquito because of its role as a vector for viral infections that kill thousands of people each year. A more thorough understanding of mosquito physiology will aid development of novel control strategies. Previous work on ion transport across the midgut has been focused primarily on larval <em>A. aegypti</em>, while research on the midgut of the adult stage is less complete. The posterior midgut of the adult female is of particular interest because it is used for the storage and digestion of the blood meal which is required for the production of eggs. This study used an array of electrophysiological methodologies such as the Scanning Ion Electrode Technique (SIET) in order to elucidate the patterns and mechanisms of Na⁺, H⁺ and K⁺ transport across the posterior midgut at intervals during postprandial diuresis and digestion of the blood meal. Measurements of transepithelial potential indicated that the lumen was at its most negative (-13.2 mV) three hours after the blood meal and then gradually became less negative during the time course of digestion. Na⁺ was absorbed (from lumen to bath) at all intervals after the blood meal (6 min, 30 min, 2h, 24 h); calculations of the electrochemical potential indicated that absorption required active transport. H⁺ absorption at all times (6 min – 48 h) after the blood meal was also active (<em>i.e.</em> against the electrochemical gradient for H⁺) and was greatly reduced by inhibition of carbonic anhydrase. K⁺ transport across the midgut exhibited two distinct phases. During diuresis, luminal concentrations of K⁺ were in the range 24 – 28 mM and secretion into the midgut was opposed by the electrochemical gradient, indicating active transport. After diuresis, during blood meal digestion, concentrations of K⁺ in the midgut contents were high (95 – 134 mM) and absorption of K⁺ was favoured by the electrochemical gradient. K⁺ absorption was sensitive to the channel blocker Ba²⁺ during this period.</p> / Master of Science (MSc)

The proton dependent oligopeptide transporters (POT family) are members of the Major Facilitator Superfamily of secondary active transporter proteins. They use the transmembrane proton gradient to drive the uptake of di- and tripeptides into the cytoplasm. Members of the family are highly conserved in pro- and eukaryotic genomes, and in humans they are responsible for the oral absorption of many drug families, including -lactam antibiotics. Recently, the crystal structures of PepTSo and PepTSt, two prokaryotic homologues of the human proteins PepT1 and PepT2, captured the proteins in two distinct conformations, providing insight into the structural aspects of the transport mechanism. A protocol was designed for functional liposome reconstitution of POT proteins, and transport assays were conducted to characterise their substrate specificity, pH dependence and kinetic properties. Using site-directed mutagenesis, we identified binding site residues involved in peptide recognition and proton translocation, and distinguished between the two roles by comparing protein activity in proton- and peptide-driven conditions. We also investigated the roles of key residues in the conformational transitions that accompany the transport cycle, using data from biochemical assays, molecular dynamics simulations and modeling, as well as electron paramagnetic resonance measurements. In addition, several bacterial POT members were screened for crystallisation, in order to assess their stability and crystal diffraction quality in different detergents. Further work was performed with bacterial POT homologues YdgR and GkPOT, including binding studies using NMR spectroscopy and assaying drug transport in vivo and in vitro. Together, the data establish bacterial POTs as model systems for studying the mammalian oligopeptide transporters, and a mechanistic model for peptide transport is proposed.

Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldrüse der Schabe <i>Periplaneta americana</i> durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldrüse sezernieren einen Primärspeichel, der in den Ausführgängen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Flüssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt.<br> Mikrofluorometrische Ca²⁺-, Na⁺- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivitäten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgeführt. Sie sollten Informationen über die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. <br><br> Wesentliche Ergebnisse dieser Arbeit waren:<br><br> <ul> <li>Messungen des intrazellulären pH (pH_i) in Gangzellen zeigten, dass isolierte Ausführgänge mit Acini bei Stimulierung mit DA und 5-HT stark ansäuerten. In isolierten Ausführgängen ohne Acini verursachte nur DA eine schwache Ansäuerung. Da nur die Ausführgänge dopaminerg innerviert sind, die Acini jedoch dopaminerg und serotonerg, zeigt dieses Ergebnis, dass die DA- und/oder 5-HT-induzierte Primärspeichelbildung die Ursache für die pHi-Änderungen in den Gangzellen ist. pH_i-Messungen in den Gangzellen geben also auch Hinweise auf Transportvorgänge in den Acini.</li> <li> Der Na⁺-K⁺-2Cl^--Symporter und der Cl^--HCO₃^--Antiporter, gekoppelt mit dem Na⁺ H⁺-Antiporter (NHE) waren an der NaCl-Aufnahme in die peripheren Zellen der Acini zur Bildung des NaCl-reichen Primärspeichels beteiligt. Die Aktivität dieser Transporter hing von der CO₂/HCO₃^--Verfügbarkeit ab und war Ca²⁺-abhängig.</li> <li>Die starke Ansäuerung in den Gangzellen hing nicht von der Aktivität der apikalen vakuolären Protonen-ATPase (V-H⁺-ATPase), aber von der Aktivität der basolateralen Na⁺-K⁺-ATPase ab, die anscheinend in den Ausführgängen die Speichelmodifikation energetisiert.</li> <li>In isolierten Ausführgängen mit Acini waren die V-H⁺-ATPase und Na⁺-abhängige Transporter (u. a. NHE) an der Erholung von einer DA-induzierten oder einer NH₄Cl-Vorpuls-induzierten Ansäuerung in den Gangzellen beteiligt. Bei der Regulation des pH_i in unstimulierten Gangzellen spielten diese Transporter keine Rolle.</li> <li>In isolierten Ausführgängen mit Acini induzierte DA in den Gangzellen einen Anstieg der [Na⁺]_i und, zeitlich verzögert, auch der [Ca²⁺]_i. Der [Na⁺]_i-Anstieg war von der Aktivität der Acini abhängig und erfolgte möglicherweise über apikale Na+-Kanäle. Der [Ca²⁺]_i-Anstieg war graduiert und tonisch. Der DA-induzierte [Na⁺]_i-Anstieg in den Gangzellen und deren Depolarisation führten dazu, dass der basolaterale Na⁺-Ca²⁺-Antiporter in den Ca²⁺-Influx-Modus umkehrte. Die daraus resultierende tonische [Ca²⁺]_i-Erhöhung könnte an der Regulation der Na⁺-Rückresorption beteiligt sein.</li> <li>Zum Nachweis transepithelialer Flüssigkeitsbewegungen in isolierten Ausführgängen wurde eine videomikroskopische Methode entwickelt. Isolierte Ausführgänge ohne Acini resorbierten im unstimulierten Zustand Flüssigkeit aus dem Ausführganglumen. Möglicherweise sezernieren die Acini auch im unstimulierten Zustand mit geringerer Rate einen Primärspeichel, der in den Ausführgängen resorbiert wird. Die Resorption war ATP-abhängig. Der ATP-verbrauchende Transportmechanismus konnte nicht identifiziert werden. Weder die Na⁺-K⁺-ATPase noch die V-H⁺-ATPase waren an der Resorption beteiligt.</li> </ul> <br> Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldrüsen in Insekten bei und erweiterte das lückenhafte Wissen über die zellulären Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldrüsen in Vertebraten deutlich. / The acinar salivary glands in the cockroach <i>Periplaneta americana</i> are innervated by dopaminergic and serotonergic fibers and secrete a NaCl-rich primary saliva upon stimulation with the biogenic amines dopamine (DA) or serotonin (5-HT). The ducts downstream of the acini are thought to modify the primary saliva by Na⁺reabsorption and K⁺ secretion. The electrolyte and fluid transport processes activated by DA and 5-HT as well as the second messenger pathways mediating between the biogenic amine receptors and the effector transport mechanisms are poorly understood.In this sudy, microfluorometrical Ca²⁺, Na⁺ and pH measurements were performed in combination with pharmacological experiments. Furthermore, ATPase activity assays and microscopical analyses of transepithelial fluid transport were done. The aim of this work has been the characterisation of the DA-induced transport mechanisms in the cockroach salivary glands in order to improve our understanding of the cellular actions of biogenic amines in insects. <br><br> Intracellular pH measurements in duct cells of isolated small lobes of salivary glands consiting of several acini and ducts showed a strong intracellular acidification upon DA or 5-HT stimulation. On the other hand, only a small intracellular acidification could be recognised in isolated ducts without acini. The acini are innervated by dopaminergic and serotonergic fibers, whereas the ducts are innervated only by dopaminergic fibers. Thus, this result demonstrates, that the DA- or 5-HT-induced production of primary saliva in the acini causes the intracellular pH changes in the ducts. Consequently, intracellular pH measurements in ducts are also useful to characterise transport processes in the acini.<br><br> The Na⁺-K⁺-2Cl^- cotransport and/or the Cl^--HCO₃^- exchange combined with the Na⁺ H⁺ exchange (NHE) were responsible for the NaCl uptake at the basolateral membrane in the peripheral cells of the acini during production of primary saliva. The activity of these transporters was regulated by the CO₂/HCO₃^--availability and was Ca²⁺-dependent. The activity of the basolateral Na⁺-K⁺-ATPase, but not of the apical vacuolar-type proton pump (V-H⁺-ATPase) in the duct cells was necessary for the strong intracellular acidification in the ducts with acini. Thus, the Na⁺-K⁺-ATPase seems to energise the saliva modification in the ducts. In ducts with acini, the V-H⁺-ATPase and Na⁺-dependent transporters (e.g. NHE) were responsible for the pH-recovery after a DA- or NH₄Cl-induced intracellular acidification in the duct cells. In the regulation of the intracellular resting pH these transporters played a minor role. In addition, DA induced an increase in the intracellular Na⁺ concentration, followed by an increase in the intracellular Ca²⁺ concentration in duct cells with acini, but never in duct cells without acini. The Na⁺ elevation was probably the result of the activity of apical Na⁺ channels. The DA-induced Na⁺ elevation and a depolarisation of the basolateral membrane of the duct cells reversed a Na⁺-Ca²⁺ exchange activity into the reverse mode causing a graded Ca²⁺ elevation in duct cells. The Ca²⁺ elevation is probably involved in the regulation of the Na⁺ reabsorption during saliva modification. Transepithelial fluid transport in isolated ducts was detected with a fluorescent microscopical method. Already unstimulated isolated ducts reabsorbed fluid from the duct lumen to the bath side. Perhaps unstimulated acini possess a basic secretion rate and this primary saliva is than reabsorbed in the ducts. The fluid reabsorption was ATP-dependent, but the ATP-consuming transport mechanism could not be identified. Neither the basolateral Na⁺-K⁺-ATPase, nor the apical V-H⁺-ATPase were involved in fluid reabsorption. This work extends our knowledge about the complex function of insect salivary glands and about the cellular action of biogenic amines in insects. Additionally, it indicates lots of similarities between the functions of salivary glands in vertebrates and invertebrates.