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

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.

The TRPA1 Agonist Cinnamaldehyde Induces the Secretion of HCO3− by the Porcine Colon

“…Accordingly, the effect of cinnamaldehyde on TRPV3 was studied using HEK-293 cells transfected with the human variant of TRPV3. As control cells, cells were transfected with the empty vector, essentially as described previously [ 40 , 41 ]. Cells were also treated with thymol, so that a subsequent comparison of the relative response of the TRPV3 expressing cells and the colonic tissues to cinnamaldehyde and thymol was possible.…”

“…In porcine colon, the responses to thymol were highly variable, with both increases in I sc and decreases observed, in marked contrast to the uniform responses observed in parallel in tissues from the same pigs after the application of cinnamaldehyde. While changes in the barrier function may have contributed, the selectivity of TRPV3 to Ca 2+ is poor, and it follows an Eisenman sequence IV with P(K + ) > P(Na + ) [ 4 , 40 ], while TRPA1 follows an Eisenman XI sequence with P(Na + ) > P(K + ) [ 1 , 2 ]. Accordingly, and depending on the gradients present, the opening of TRPV3 by thymol may lead to a secretion of K + with the hyperpolarization of the apical membrane and a drop in I sc .…”

“…A human construct of TRPV3 (hTRPV3) was used for the patch-clamp experiments, essentially as in [ 40 , 41 ]. The sequence (GeneArt, Thermo Fisher Scientific, Regensburg, Germany) was tagged with hemagglutinin (HA) and streptavidin (Strep).…”

“…Accordingly, the successfully transfected hTRPV3 cells should show green fluorescence. Furthermore, the cells were stained with a mouse TRPV3 antibody (ABIN863127, antibodies-online GmbH, Aachen, Germany) at a 1:1000 dilution, as in [ 40 ].…”

“…A low sampling rate (100 Hz) pulse protocol was used for the recording in order to assess the solution changes. This automatically switched to a classical step protocol with a high sampling rate (5 kHz) to assess the channel kinetics, as in previous studies [ 40 , 41 ]. After the measuring the osmolality, the solutions were adjusted to 300 mosmol·kg −1 with mannitol and buffered to a pH of 7.4.…”