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

瘤胃两个 TRP 通道作为介导 NH4 、 Na 、 K 和 Ca2 转运的候选蛋白的功能和分子生物学特征

• 批准号: 318480266
• 负责人: Professorin Dr. Friederike Stumpff
• 金额: --
• 依托单位: Fakultät Medizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/318480266?language=en
• 关键词: Functional; molecular; biological; characterization; two

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.

肝病中的脑炎、牛中蛋白质效率差以及氨排放到环境中对全球气候造成的后果是与膳食蛋白质胃肠道降解成氨相关的紧迫问题，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，特别是从牛的瘤胃运输的机制。基于先前对天然绵羊和牛瘤胃上皮的功能研究结果，证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转运机制。为此目的，将在不同的模型系统中过表达由天然上皮表达的具有与先前获得的功能数据拟合的药理学特征的TRP通道，以确定它们对相关阳离子（包括生理上重要的铵（NH4+）离子）的渗透性。PCR技术和蛋白质印迹将用于获得由牛瘤胃上皮表达的TRP通道的概况。两个有前途的候选人，TRPA1和TRPV3，将被完全测序，随后在HEK 293细胞中过表达，以研究通过膜片钳技术的全细胞和单通道配置对各种阳离子的渗透性。研究将开始，测定这两个通道对一价阳离子NH 4+、Na+和K+的电导率，但将进展到测量对Ca 2+和Mg 2+的渗透性。为了鉴定成功转染的细胞，将目的基因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。在过度表达非洲爪蟾卵母细胞，双管离子选择性微电极将被用来监测膜电位和pH值平行，以获得有关的变化的信息NH4+和NH3总氨通量的相对贡献。最后，利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，并通过Western印迹法检测其特异性。基于药理学特征和天然牛瘤胃上皮细胞中的稳健表达，本研究将从TRPA 1的牛代表性功能研究开始，随后研究TRPV 3。鉴于目前没有关于TRP家族的非选择性阳离子通道对NH4+的渗透性的数据，本研究有可能产生超出理解氨的瘤胃运输的影响。

项目成果

A comparative study of ammonia transport across ruminal epithelia from Bos indicus crossbreds versus Bos taurus.

印度牛与金牛杂交瘤胃上皮氨转运的比较研究

• DOI: 10.1111/asj.13107
• 发表时间: 2018
• 期刊: Animal science journal = Nihon chikusan Gakkaiho
• 作者: Rabbani;Akhtar;Rosendahl;Grunau;Tietjen;Masood;Kaessmeyer;Gunzel;Rehman;Stumpff
• 通讯作者: Stumpff

TRPV3 and TRPV4 as candidate proteins for intestinal ammonium absorption

• DOI: 10.1111/apha.13694
• 发表时间: 2021-06-07
• 期刊: ACTA PHYSIOLOGICA
• 影响因子: 6.3
• 作者: Manneck, David;Braun, Hannah-Sophie;Stumpff, Friederike
• 通讯作者: Stumpff, Friederike

The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal ammonia transport

牛瘤胃的 TRPV3 通道：与瘤胃氨转运相关的蛋白质的定位和功能表征

• DOI: 10.1007/s00424-020-02393-2
• 发表时间: 2020
• 期刊: Pflugers Archiv
• 作者: Kaessmeyer;Sponder;Stumpff
• 通讯作者: Stumpff

Evidence for the functional involvement of members of the TRP channel family in the uptake of Na+ and NH4+ by the ruminal epithelium

• DOI: 10.1007/s00424-016-1835-4
• 发表时间: 2016-05
• 期刊: Pflügers Archiv - European Journal of Physiology
• 作者: J. Rosendahl;H. Braun;K. T. Schrapers;Holger Martens;F. Stumpff