Die Heterodimerisierung von G- Protein gekoppelten Rezeptoren (GPCR) stellt ein aktuelles Forschungsgebiet dar, das molekulare Erklärungsmöglichkeiten für die Vielfalt der Signalwege über solche Rezeptoren aufzeigt. Die genauen Funktionen diese Konstrukte in vivo sind bisher erst in Ansätzen erforscht, ebenso wenig die molekularbiologischen Mechanismen. Für die beiden Serotoninrezeptoren 5-HT1A und 5-HT7 konnte Heterodimerisierung molekular nachgewiesen werden, in ihren physiologischen Mechanismen und Effekten sollte daher eine Charakterisierung vorgenommen werden. Mittels elektrophysiologischer Messverfahren wurden Ströme an dem heterologen Expressionsmodell der Oozyten des Krallenfrosches Xenopus laevis mittels Voltage-Clamp Technik an Kaliumionenkanälen (Kir3 und TASK-1) gemessen. Hierbei konnte gezeigt werden, dass die heterodimere Koexpression beider Rezeptoren eine signifikante Reduktion des Rezeptor-aktivierten Kanalstroms im Vergleich zur homomeren Expression zur Folge hatte. Weitere Experimente konnten dann zeigen, dass diese Effekte spezifisch für dieses Rezeptorheterodimer sind, und dass die Effekte von der Dosis bzw. dem Verhältnis der exprimierten cRNA abhängen. In Fluoreszenzmessung konnte zudem gezeigt werden, dass die Reduktion der Stromamplitude in der heterodimeren Expression nicht auf eine Reduktion von Kanalproteinen in der Zellmembran zurückzuführen ist. Zur weiteren Charakterisierung des bisher erst in Ansätzen erforschten 5-HT7 Rezeptors wurde dieser abschließend mit einem ß- adrenergen Rezeptor verglichen, der über den gleichen Signalweg bzw. Ionenkanal funktioniert. Auch hier zeigte sich eine signifikante Reduktion des Kanalstroms beim 5-HT7 Rezeptor. Die physiologische Relevanz dieser Ergebnisse liegt darin begründet, dass ein weiterführendes Verständnis von 5-HT Rezeptor vermittelten Signalwegen, insbesondere von der Bedeutung und den Mechanismen ihrer Heterodimersierung, neue pathophysiologische Zusammenhänge verdeutlicht. Speziell im Hinblick auf Erkrankungen, die mit den 5-HT Rezeptoren assoziiert sind, wie etwa Depressionen und Angststörungen, soll sich hieraus die Möglichkeit spezifischerer Therapien ergeben. / Heteromerisation of G-protein coupled receptors (GPCR)is a current object of research to find out diversity of signaling pathways. The functional details of those constructs in vivo are not yet understood, also molecular mechanisms. For the Serotonin receptors 1A and 7 heteromerisation recently could be shown, therefore intention now is to make a physiological characterization. By electrophysiological methods, i.e. voltage clamp, currents of potassium channels (Kir3 and TASK-1) could be detected using the heterologous expression system of oocytes of xenopus leavis. So we could demonstrate, that heterologous expression of both receptors leads to a reduced current amplitude in comparison to homologous expression of one receptor. Those effects where shown to be specific and dependend of cRNA dose. By using fluorescense tagged Kir-channel we could demonstrate that the effect doesn't base on less channel protein in cell surface of the oocytes. Another point of interest was the characterization of Serotonin-7 channel. Therefore we analyzed a dose-response-relationship, afterwards we compared data with a ß-adrenergic receptore in heteromeric expression with Serotonin-1A. The physiological relevance of those experiments is to understand serotonin pathways an metabolism that is very important in development of mental disorders of fear or major depression.

In the shark rectal gland (SRG), apical Cl[superscript]- secretion through CFTR channels is tightly coupled to a basolateral K[superscript]+ conductance. The identity of this K[superscript]+ conductive pathway is unknown. Studies were performed in the isolated perfused SRG with 16 K[superscript]+ channel inhibitors at their IC50 and with acidic perfusate. During maximal chloride secretion stimulated by forskolin and IBMX, secretion was inhibited >90% by barium chloride, a non-selective inhibitor of K[superscript]+ channels. Specific inhibitors of calcium sensitive, voltage sensitive, ATP sensitive, and inward rectifying K[superscript]+ channels had no effect on chloride secretion. The inhibitors quinidine, quinine, bupivicaine, anandamide, and low perfusate pH (6.0) abruptly and reversibly inhibited secretion by >90%, consistent with the presence of the Two-Pore-Domain (4TM 2P/KCNK/K2P) family of K+ channels. Degenerate primers were designed to regions of high amino acid homology in known mammal and teleost 4TM 2P K[superscript]+ channel subtypes: TWIK, THIK, TASK, TREK, and TRAAK. PCR with cDNA from several shark tissues identified a putative TASK-1 fragment (394 bp) in shark rectal gland, brain, gill, and kidney. 5and 3 RACE PCR was used to obtain the entire 3 sequence and partial 5 sequence of the shark gene. Genome walking was then used to obtain the remaining 5sequence, including 335 bp of untranslated region sequence upstream of the start codon. The full length clone (1282bp) had an open reading frame encoding a protein of 375 amino acids. This isoform was 80% identical at the amino acid level to the human TASK-1 protein (394 amino acids). Major structural features of the human protein were conserved in the shark ortholog, including the four transmembrane segments (M1-M4), the 2P domains (P1 and P2), short NH2- and long COOH-termini, and an extended extracellular loop between M1 and P1. Shark and human TASK-1 full-length clones were expressed in Xenopus oocytes and studied with two electrode voltage clamp (TEVC) techniques. Both the shark and human TASK-1 channel showed identical current voltage relationships (outward rectifying) with a reversal potential near -90 mV compared to water injected controls. The responses to the inhibitor quinine, and the TASK-1 inhibitor bupivacaine, were identical in shark and human TASK-1. However, shark TASK-1 differed from the human ortholog in two critical responses: response to pH and the metal zinc. The pKa for shark TASK-1 was 7.75 vs. 7.37 for human TASK-1, values that are exceedingly close to the arterial pH for each species, suggesting that TASK-1 channels are regulated closely by the ambient pH. An antibody specific to shark TASK-1 was generated and expression of TASK-1 protein in the rectal gland was confirmed by confocal immuno-fluorescent microscopy which revealed localization to the basolateral membrane, with some apical staining. Shark rectal gland TASK-1 appears to be the major K[superscript]+ channel coupled to secretion in the SRG, is the oldest 4TM 2P family member identified to date, and is the first TASK-1 channel identified to play an essential role in chloride secreting epithelia.

No description available.

No description available.

No description available.