Abstract
Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na+, Cs+, NH4+, and K+, with a rise in intracellular Ca2+ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH4+ and 25.0 ± 5.8 pS for Na+), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na+, 240.1 ± 3.6 pS for NH4+, 34.0 ± 1.7 pS for Ca2+, and ~ 36 pS for NMDG+. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na+, K+, Ca2+ and NH4+ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia.
Highlights
The transient receptor potential (TRP) multigene superfamily consists of 28 known sequences that are subdivided into seven subfamilies, six of which are found in mammalian tissues and encode for integral membrane proteins that function as cation-selective channels in extra- or intracellular membranes [1, 2]
We report that in symmetrical NH4Cl solution the bovine homologue of TRPV3 conducts ammonia with a single-channel conductance of 240.1 ± 3.6 pS
As reported for the human homologue [32], menthol, thymol, carvacrol, and 2-Aminoethoxydiphenyl borate (2-APB) stimulated the conductance for monovalent cations, including Na+, Cs+, K+, and NH4+
Summary
The transient receptor potential (TRP) multigene superfamily consists of 28 known sequences that are subdivided into seven subfamilies, six of which are found in mammalian tissues and encode for integral membrane proteins that function as cation-selective channels in extra- or intracellular membranes [1, 2]. Owing to the original discovery in the sensory system of drosophila flies [3], most functional research to date has focused on the role of TRP channels in Ca2+ mediated sensory signaling cascades, such as those involved in the detection of thermal signals, inflammation, or pain [4]. The fragrant constituents found in herbs have been found to interact in a specific manner with various members of the TRP. Stu258/7-1, Franziska Liebe), the Akademie fur Tiergesundheit
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