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

肝脏疾病的脑炎、牛的蛋白质效率低下以及氨排放到环境中并对全球气候造成影响，这些都是与膳食蛋白质胃肠道降解成氨有关的紧迫问题，氨被吸收并逃避发酵整合成微生物蛋白质。因此，迫切需要更好地了解氨从肠道运输的机制，特别是从牛的瘤胃。基于先前对天然绵羊和牛瘤胃上皮的功能研究结果，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，本研究的主要目标将是在分子水平上确定潜在的运输机制。为此，天然上皮表达的TRP通道的药理学特征与先前获得的功能数据相符，将在不同的模型系统中过度表达，以确定它们对相关阳离子的通透性，包括生理上重要的铵（NH4+）离子。作为第一步，PCR技术和Western blots将用于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，以通过膜片钳技术的全细胞和单通道配置研究各种阳离子的通透性。研究将从确定这两个通道对单价阳离子NH4+， Na+和K+的电导开始，但将进一步测量对Ca2+和Mg2+的渗透性。为了鉴定成功转染的细胞，感兴趣的基因将与绿色荧光蛋白共表达。荧光染料将用于测定细胞内pH、Ca2+和Mg2+的变化。在过表达非洲爪蟾卵母细胞中，利用双管离子选择微电极并行监测膜电位和pHi，以获取NH4+和NH3对总氨通量相对贡献的变化信息。最后，通道蛋白将使用共聚焦激光显微镜和抗体在完整的上皮中定位，其特异性将在过表达细胞的Western blots中进行测试。基于其药理特征和在天然牛瘤胃上皮中的强劲表达，该研究将从TRPA1的牛代表的功能调查开始，随后是TRPV3的调查。鉴于目前还没有关于TRP家族的非选择性阳离子通道对NH4+的渗透性的数据，该研究有可能产生超出理解氨的瘤胃运输的影响。

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.