Functional and molecular biological characterization of two TRP channels of the rumen as candidate proteins mediating the transport of NH4+, Na+, K+, and Ca2+

肝脏疾病中的脑炎，牛的蛋白质效率低下，以及向环境中排放氨而对全球气候造成后果，这些都是与食物蛋白质在胃肠道降解为氨有关的紧迫问题，氨被吸收并逃脱发酵整合到微生物蛋白质中。因此，迫切需要更好地了解氨从肠道，特别是从牛的瘤胃运输的机制。基于前人对天然绵羊和牛瘤胃上皮功能的研究结果表明，选择性瞬时受体电位(Trp)通道调节剂对Na+和NH4+诱导的短路电流的影响，本研究的主要目标是在分子水平上确定潜在的转运机制。为此，在不同的模型体系中，具有与先前获得的功能数据相匹配的药理图谱的天然上皮表达的Trp通道将被过表达，以确定其对相关阳离子的通透性，包括生理上重要的氨(NH4+)离子。作为第一步，我们将使用PCR技术和Western blotts来获得牛瘤胃上皮表达的Trp通道的概述。两个有希望的候选基因TRPA1和TRPV3将被完全测序并随后在HEK 293细胞中过表达，以研究各种阳离子通过膜片钳技术的全细胞和单通道配置的通透性。研究将从测定这两个通道对单价阳离子NH4+、Na+和K+的电导开始，但将继续测量对钙离子和镁离子的渗透性。为了能够鉴定成功的转基因细胞，目的基因将与绿色荧光蛋白共表达。荧光染料将被用来测定细胞内pH、钙和镁的变化。在过度表达非洲爪哇卵母细胞时，将使用双管离子选择微电极并行监测膜电位和PHI，以获得NH4+和NH3对总氨通量的相对贡献的变化信息。最后，使用激光共聚焦显微镜和抗体将通道蛋白定位在完整的上皮细胞中，其特异性将在过度表达细胞的Western blotts中进行测试。基于药理学特征和在天然牛瘤胃上皮中的强劲表达，本研究将从牛代表TRPA1的功能研究开始，随后将对TRPV3进行研究。鉴于目前还没有关于色氨酸家族的非选择性阳离子通道对NH4+的渗透性的数据，这项研究的潜在影响可能超出了对氨的瘤胃运输的理解。

Encephalitis in hepatic disease, poor protein efficiency in cattle, and emissions of ammonia into the environment with consequences for the global climate are urgent problems associated with the gastrointestinal degradation of dietary protein into ammonia that is absorbed and escapes fermentative integration into microbial protein. There is thus an urgent need for a better understanding of the mechanisms behind the transport of ammonia from the gut in general and from the rumen of cattle in particular. Based on the results of previous functional studies of the native ovine and bovine ruminal epithelium demonstrating effects of selective transient receptor potential (TRP) channel modulators on Na+ and NH4+ induced short circuit currents, the primary goal of this study will be to identify the underlying transport mechanisms on a molecular level. For this purpose, TRP channels expressed by the native epithelium with a pharmacological profile fitting the previously obtained functional data will be overexpressed in different model systems in order to determine their permeability to relevant cations including the physiologically important ammonium (NH4+) ion.As a first step, the PCR technique and Western blots will be used to gain an overview of the TRP channels expressed by the bovine ruminal epithelium. Two promising candidates, TRPA1 and TRPV3, will be fully sequenced and subsequently overexpressed in HEK 293 cells to investigate the permeability for various cations via the whole cell and single channel configuration of the patch clamp technique. The investigations will begin with a determination of the conductance of these two channels to the monovalent cations NH4+, Na+ and K+, but will progress to measuring the permeability to Ca2+ and Mg2+. To allow identification of successfully transfected cells, the genes of interest will be co-expressed with green fluorescent protein. Fluorescent dyes will be used to determine changes in intracellular pH, Ca2+ and Mg2+. In overexpressing Xenopus oocytes, double barreled ion selective microelectrodes will be used to monitor membrane potential and pHi in parallel in order to obtain information concerning the change in the relative contributions of NH4+ and NH3 to total ammonia flux. Finally, the channel proteins will be localized in the intact epithelium using confocal laser microscopy and antibodies, the specificity of which will be tested in Western blots of overexpressing cells.Based on the pharmacological profile and the robust expression in the native bovine ruminal epithelium, the study will commence with functional investigations of the bovine representative of TRPA1, to be followed by an investigation of TRPV3. Given that there are currently no data concerning the permeability of non-selective cation channels of the TRP family to NH4+, this study has the potential to have repercussions beyond understanding the ruminal transport of ammonia.