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

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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.