Beyond Ca2+ signalling: the role of TRPV3 in the transport of NH4+

AbstractMutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH4+. It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH4+, to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH4+ was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH4+ in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca2+ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH4+ via TRPV3 might stimulate skin cornification or intestinal NH4+ transport are discussed.

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

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-020-02393-2 ◽

2020 ◽

Vol 472 (6) ◽

pp. 693-710 ◽

Cited By ~ 2

Author(s):

Franziska Liebe ◽

Hendrik Liebe ◽

Sabine Kaessmeyer ◽

Gerhard Sponder ◽

Friederike Stumpff

Keyword(s):

Single Channel ◽

Functional Characterization ◽

Nitrogen Efficiency ◽

Channel Measurements ◽

Hek 293 ◽

Bovine Rumen ◽

Ruminal Ph ◽

293 Cells ◽

Ammonia Transport ◽

Ruminal Epithelium

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.

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Selective Open-Channel Block of Shaker (Kv1) Potassium Channels by S-Nitrosodithiothreitol (Sndtt)

The Journal of General Physiology ◽

10.1085/jgp.118.1.113 ◽

2001 ◽

Vol 118 (1) ◽

pp. 113-134 ◽

Cited By ~ 20

Author(s):

Mathew W. Brock ◽

Chris Mathes ◽

William F. Gilly

Keyword(s):

Open Channel ◽

Single Channel ◽

Time Dependent ◽

Channel Block ◽

Giant Fiber ◽

Open Channel Block ◽

Hek 293 ◽

Hek 293 Cells ◽

Kv Channels ◽

293 Cells

Large quaternary ammonium (QA) ions block voltage-gated K+ (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH2CH(OH)CH(OH)CH2SNO) produces qualitatively similar “open-channel block” in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na+ or Ca2+ channels. Inactivation-removed ShakerB (ShBΔ) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose–response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (Kd2 = 0.14 mM) is lower than that of the first molecule (Kd1 = 0.67 mM), indicating cooperativity. The half-blocking concentration (K1/2) is ∼0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about −0.3 e0) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (τ ≈ 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1–1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.

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Proximal C-terminal domain of sulphonylurea receptor 2A interacts with pore-forming Kir6 subunits in KATP channels

Biochemical Journal ◽

10.1042/bj20031087 ◽

2004 ◽

Vol 379 (1) ◽

pp. 173-181 ◽

Cited By ~ 14

Author(s):

Richard D. RAINBOW ◽

Marian JAMES ◽

Diane HUDMAN ◽

Mohammed AL JOHI ◽

Harprit SINGH ◽

...

Keyword(s):

Cell Membrane ◽

Katp Channel ◽

Direct Interaction ◽

Katp Channels ◽

Full Length ◽

Hek 293 ◽

Sulphonylurea Receptor ◽

Hek 293 Cells ◽

Terminal Domain ◽

293 Cells

Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein–SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254–1545), rSUR2A-CTB (residues 1254–1403) and rSUR2A-CTC (residues 1294–1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358–1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294–1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294–1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.

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Unique modulation of L-type Ca2+ channels by short auxiliary β1d subunit present in cardiac muscle

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00348.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. H2363-H2374 ◽

Cited By ~ 24

Author(s):

Risa M. Cohen ◽

Jason D. Foell ◽

Ravi C. Balijepalli ◽

Vaibhavi Shah ◽

Johannes W. Hell ◽

...

Keyword(s):

Membrane Trafficking ◽

Single Channel ◽

Surface Membrane ◽

Full Length ◽

Open Probability ◽

Western Blots ◽

Hek 293 ◽

Hek 293 Cells ◽

Amino Terminal ◽

293 Cells

Recent studies have identified a growing diversity of splice variants of auxiliary Ca2+ channel Cavβ subunits. The Cavβ1d isoform encodes a putative protein composed of the amino-terminal half of the full-length Cavβ1 isoform and thus lacks the known high-affinity binding site that recognizes the Ca2+ channel α1-subunit, the α-binding pocket. The present study investigated whether the Cavβ1d subunit is expressed at the protein level in heart, and whether it exhibits any of the functional properties typical of full-length Cavβ subunits. On Western blots, an antibody directed against the unique carboxyl terminus of Cavβ1d identified a protein of the predicted molecular mass of 23 kDa from canine and human hearts. Immunocytochemistry and surface-membrane biotinylation experiments in transfected HEK-293 cells revealed that the full-length Cavβ1b subunit promoted membrane trafficking of the pore-forming α1C (Cav1.2)-subunit to the surface membrane, whereas the Cavβ1d subunit did not. Whole cell patch-clamp analysis of transfected HEK-293 cells demonstrated no effect of coexpression of the Cavβ1d with the α1C-subunit compared with the 15-fold larger currents and leftward shift in voltage-dependent activation induced by full-length Cavβ1b coexpression. In contrast, cell-attached patch single-channel studies demonstrated that coexpression of either Cavβ1b or Cavβ1d significantly increased mean open probability four- to fivefold relative to the α1C-channels alone, but only Cavβ1b coexpression increased the number of channels observed per patch. In conclusion, the Cavβ1d isoform is expressed in heart and can modulate the gating of L-type Ca2+ channels, but it does not promote membrane trafficking of the channel complex.

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Activation of Vanilloid Receptor 1 (VR1) by Eugenol

Journal of Dental Research ◽

10.1177/154405910308201004 ◽

2003 ◽

Vol 82 (10) ◽

pp. 781-785 ◽

Cited By ~ 116

Author(s):

B.H. Yang ◽

Z.G. Piao ◽

Y.-B. Kim ◽

C.-H. Lee ◽

J.K. Lee ◽

...

Keyword(s):

Molecular Mechanisms ◽

Expression System ◽

Sensory Nerve ◽

Ruthenium Red ◽

Vanilloid Receptor ◽

Hek 293 ◽

Hek 293 Cells ◽

Vanilloid Receptor 1 ◽

293 Cells ◽

Inward Currents

The structural similarity of eugenol with capsaicin suggests that these two agents may share molecular mechanisms to produce their effects. We investigated the effects of eugenol in comparison with those of capsaicin using whole-cell patch clamp and Fura-2-based calcium-imaging techniques in a heterologous expression system and with sensory neurons. In vanilloid receptor 1 (VR1)-expressing human embryonic kidney (HEK) 293 cells and trigeminal ganglion (TG) neurons, eugenol activated inward currents, whereas capsazepine, a competitive VR antagonist, and ruthenium red (RR), a functional VR antagonist, completely blocked eugenol-induced inward currents. Moreover, eugenol caused elevation of [Ca2+]i, and this was completely abolished by both capsazepine and ruthenium red in VR1-expressing HEK 293 cells and TG neurons. Our results provide strong evidence that eugenol produces its effects, at least in part, via VR1 expressed by the sensory nerve endings in the teeth.

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Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽

10.4161/auto.25455 ◽

2013 ◽

Vol 9 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 41

Author(s):

Patience Musiwaro ◽

Matthew Smith ◽

Maria Manifava ◽

Simon A. Walker ◽

Nicholas T. Ktistakis

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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