The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal am
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ION CHANNELS, RECEPTORS AND TRANSPORTERS
The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal ammonia transport Franziska Liebe 1
&
Hendrik Liebe 1,2
&
Sabine Kaessmeyer 3
&
Gerhard Sponder 1
&
Friederike Stumpff 1
Received: 13 February 2020 / Revised: 31 March 2020 / Accepted: 6 May 2020 # The Author(s) 2020
Abstract Large quantities of ammonia (NH3 or NH4+) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH4+. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH4+ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH4+. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH4+, Na+, and K+. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na+, Ca2+, and NH4+ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed. Keywords TRPV3 . Ammonia transport . Microelectrode . Climate gas . Rumen . Xenopus oocyte
Introduction Ammonia in its two forms (NH3 and NH4+) plays a central role in the interconversion of amino acids for protein metabolism, Franziska Liebe and Hendrik Liebe contributed equally to this work. A Commentary to this article is available online at https://doi.org/10. 1007/s00424-020-02394-1 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00424-020-02393-2) contains supplementary material, which is available to authorized users. * Friederike Stumpff [email protected] 1
Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany
2
Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany
3
Institute of Veterinary Anatomy, Freie Universität Berlin, Koserstraße 20, 14195 Berlin, Germany
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