Publikationsdatenbank

Establishing an antibody to verify expression of the bovine TRPV3 channel by the rumen of cattle (2018)

Art

Poster

Autoren

Liebe, F (WE 2)

Liebe, Hendrik (WE 2)

Vitzthum, C (WE 2)

Kaessmeyer, Sabine (WE 1)

Sponder, G. (WE 2)

Stumpff, F (WE 2)

Kongress

Europhysiology 2018

London, 13. – 16.09.2018

Quelle

Proceedings of the Physiological Society

Seiten: 357P

ISSN: 1749-6187

Sprache

Englisch

Verweise

URL (Volltext): https://static.physoc.org/app/uploads/2019/03/22193716/Europhysiology-2018_ABSTRACTS_ONLINE.pdf

Kontakt

Institut für Veterinär-Anatomie

Koserstr. 20
14195 Berlin
+49 30 838 75784
anatomie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Detection of TRPV3 channels in the ruminal epithelium on the protein level is of considerable interest, since the bovine analogue of this non-selective cation channel, bTRPV3, has emerged from functional studies and mRNA data as a candidate protein for the uptake of various cations from the rumen of cattle. Among these is ammonium (NH4+), the degradation product of dietary protein (1, 2). After absorption, excess ammonium needs detoxification via the liver and excretion via the kidney, with the resulting emissions of nitrogen by dairy cattle playing a considerable role in climate change. Additionally, bTRPV3 is the first known candidate suitable for mediating the uptake of calcium by the rumen (1,2), which is essential for meeting demand in dairy cattle. However, antibodies suitable for use in the bovine species are lacking.In a first step, the epitopes of commercial antibodies against the human TRPV3 channel were aligned with the sequence of the bovine homologue. To confirm the efficiency of the antibody, standard expression systems were used. Xenopus oocytes were either injected with RNAse-free water (control) or linearized strep-tagged bTRPV3-cRNA or hTRPV3-cRNA. Likewise, HEK-293 cells were either transiently transfected with a pIRES2-AcGFP1 vector (control) or with the same vector construct including a strep-tagged bTRPV3 or hTRPV3 sequence. Successful expression was confirmed by immunodetection (n = 8) of the strep tag. In a second step, the antibody was tested in western blots (n = 7) for both expression systems and native ruminal protein. To test the specificity of the antibody, immunohistological stainings of bTRPV3, hTRPV3, and controls in Xenopus oocytes (n = 3) and HEK-293 cells (n = 2) were performed. We could confirm the expression of the channel protein in the cell membrane. Finally, the commercial TRPV3 antibody revealed staining of the transporting layers of the ruminal epithelium.In conclusion, we could establish an antibody useful for staining the bovine TRPV3 channel. Furthermore, we show that the bTRPV3 channel protein is highly expressed by the transporting syncytium of the ruminal epithelium. Additionally, successful expression of bovine and human TRPV3 was confirmed in the membrane of cellular models, which enables further functional experiments to characterize bTRPV3 and hTRPV3. Detection of TRPV3 on the protein level in the rumen is in line with functional data indicating a diverse role of the bTRPV3 channel in ruminal transport, exceeding its commonly known sensory function in other tissues.