Malpighian Tubule Transport Physiology: Mechanisms and Regulation

该项目的总体目标是确定和描述黄热病蚊子(埃及伊蚊)马氏管中完整主细胞的自然环境中产生电流和消散电流的运输系统。在基侧细胞膜上，我们的目的是研究钾通道，以及可能的钠和钙通道。在顶膜，重点是V型H+-ATPase的电流产生，测试电压和泵的两种底物H和ATP的影响。研究电流是如何通过这一膜散布的，可能会导致发现一类新的反转运体。除了主细胞外，这项研究建议描述跨上皮分泌过程中氯进行分流的性质。需要检验的一个假说是，星状细胞充当分流通路。另一种假说认为，昆虫的隔膜连接为氯提供了一条细胞旁途径。他们的假设模型提出，隔膜连接包含一个类似氯通道的结构，该结构受利尿肽Leucokinin的调节。该提案中描述的实验将展示质子泵、通道、符号转运体和反向转运体是如何在昆虫马氏管的主细胞中组织起来的，从而通过跨细胞途径将Na和K分泌到管腔中。对分流途径的研究将显示氯是如何分泌的，可能是通过细胞旁途径。总之，这些研究将揭示上皮运输的分子构件的组合安排：泵、通道、载体和连接，以及它们如何相互作用将氯化钠、氯化钾和水分泌到小管腔中。生物学家对完整细胞中由ATP驱动的泵的拟议研究结果将会引起广泛的兴趣。

The overall goal of this project is to identify and characterize current-generating and current-dissipating transport systems in the natural environment of an intact principal cell in the Malpighian tubule of the yellow fever mosquito (Aedes aegypti). In the basolateral cell membrane, the aim is to characterize K channels, and putative Na and Ca channels. In the apical membrane, the focus is on current generation by the V-type H+-ATPase, testing the effects of voltage and the two substrates of the pump, H and ATP. Investigating how current is dissipated across this membrane may lead to the discovery of a new family of antiporters.Outside principal cells, the grant proposes to characterize the nature of the shunt taken by Cl during transepithelial secretion. One hypothesis to be tested is that the stellate cell serves as the shunt pathway. The alternate hypothesis considers insect septate junctions to offer a paracellular pathway for Cl. They hypothetical model proposes that septate junctions house a Cl channel-like structure that is regulated by the diuretic peptide leucokinin.Experiments described in this proposal will demonstrate how proton pump, channels, symporters and antiporters are organized in principal cells in insect Malpighian tubules to secrete Na and K into the tubule lumen via a transcellular path. Studies of the shunt pathway will show how Cl is secreted, presumably via a paracellular path. Together, these studies will reveal the combinatorial arrangement of the molecular building blocks of epithelial transport: pumps, channels, carriers, and junctions and how they interact to secrete NaCl, KCl, and water into the tubule lumen. Results from the proposed studies of an ATP-driven pump in an intact cell will be of wide interest to biologists.