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

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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Photobiomodulation on KATP Channels of Kir6.2-Transfected HEK-293 Cells

International Journal of Photoenergy ◽

10.1155/2014/898752 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7

Author(s):

Fu-qing Zhong ◽

Yang Li ◽

Xian-qiang Mi

Keyword(s):

Laser Irradiation ◽

Katp Channel ◽

Cell Metabolism ◽

Katp Channels ◽

Open Probability ◽

Hek 293 ◽

Hek 293 Cells ◽

Low Intensity ◽

293 Cells ◽

Excised Patches

Background and Objective. ATP-sensitive potassium (KATP) channel couples cell metabolism to excitability. To explore role of KATP channels in cellular photobiomodulation, we designed experiment to study effect of low intensity 808 nm laser irradiation on the activity of membrane KATP channel.Study Design/Materials and Methods. Plasmids encoding Kir6.2 was constructed and heterologously expressed in cultured mammalian HEK-293 cells. The patch-clamp and data acquisition systems were used to record KATP channel current before and after irradiation. A laser beam of Ga-As 808 nm at 5 mW/cm2was used in experiments. A one-way ANOVA test followed by apost hocStudent-Newman-Keuls test was used to assess the statistical differences between data groups.Results. Obvious openings of KATP channels of Kir6.2-transfected HEK-293 cells and excised patches were recorded during and after low intensity 808 nm laser irradiation. Compared with the channels that did not undergo irradiation, open probability, current amplitude, and dwell time of KATP channels after irradiation improved.Conclusions. Low intensity 808 nm laser irradiation may activate membrane KATP channels of Kir6.2-transfected HEK-293 cells and in excised patches.

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Structural and Functional Interactions of KCl Cotransport Proteins KCC1 and KCC3 in Sickle and Normal Erythrocyte Membranes

Blood ◽

10.1182/blood.v112.11.2474.2474 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2474-2474

Author(s):

Mary Risinger ◽

Jesse Rinehart ◽

Scott Crable ◽

Anna Ottlinger ◽

Richard Winkelmann ◽

...

Keyword(s):

Western Blot ◽

Specific Antibody ◽

Blot Analysis ◽

Western Blot Analysis ◽

Erythrocyte Membranes ◽

Western Blots ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

In Cells

Abstract The KCl cotransporter (KCC) mediates volume reduction in normal reticulocytes and exaggerated KCC activity in sickle red blood cells (SS RBC) (Joiner et al, Blood109:1728, 2007) contributes to pathological dehydration that potentiates sickling. Three separate genes (KCC1, KCC3, KCC4) are expressed in RBC (Crable et al, Exp. Hem.33:624, 2005). KCC1 and KCC3 proteins have been shown to interact in ex vivo expression systems (Simard et al, JBC282(25):18083, 2007), and co-expression of an N-terminal truncation of KCC1 reduces KCC activity mediated by full-length KCC1 or KCC3 (Casula et al. JBC276:41870, 2001), suggesting functional interaction. We show here via western blot analysis that SS RBC membranes contain more KCC1 protein (relative to KCC3) than AA RBC, independent of the reticulocytosis of sickle blood. Immunoprecipitation of solubilized SS RBC membranes with KCC3-specific antibody yielded a band at 125 kD on SDS PAGE which contained KCC1, as identified by western blotting with KCC1-specific antibody and by TOF mass spectroscopy. The effect of co-expression of KCC1 and KCC3 on KCC activity was assessed by measuring NEM-stimulated, Cl-dependent, (ouabain + bumetanide)-insensitive Rb uptake in HEK 293 cells. The Flip-In T-rex HEK 293 cell line (Invitrogen) containing a tetracycline-response promoter was transfected with a pcDNA5a plasmid containing KCC3a cDNA. Recombination of the plasmid with the integrated tet-promoter construct inserts the KCC3a gene under control of a tetracycline-responsive promoter. These cells were subsequently transduced with a retroviral vector (SF-91. Hildinger et at, Gene Ther. 5:1575, 1998) containing KCC1 cDNA linked to a GFP cassette. Control cells contained SF-91 vector lacking KCC1. Cells were selected for GFP expression and grown in the absence (un-induced, no KCC3a expression) or presence of tetracycline (induced, KCC3a expression). From this binary matrix, four types of cells were obtained: Cells with no additional KCC expression, representing endogenous KCC activity; cells with only KCC1 or KCC3a expression; cells with both KCC1 and KCC3a expression. Western blots indicated similar KCC1 expression in cells with KCC1 only and [KCC1 + KCC3] and similar KCC3 expression in cells with KCC3 only and [KCC1 + KCC3]. Thus, the expression of neither isoform was affected by the presence of the other. KCC activity in cells overexpressing KCC1 only was similar to endogenous activity in HEK 293 cells; i.e., transport activity of KCC1 alone was minimal. Cells overexpressing KCC3 only had a 5-fold increase in KCC activity over endogenous levels. When KCC1 was co-expressed with KCC3 in [KCC1 + KCC3] cells, an additional 50% increase in KCC activity was observed (p < 0.05 by paired t-test, N=4), despite similar levels of KCC3 expression by western blot analysis. This synergistic effect was dependent on the cytoplasmic N-terminus of KCC1, as it was not seen when the first 39 amino acids of KCC1 were removed. Interestingly, removal of the entire cytoplasmic N-terminal domain (117 aa) produced an inhibitory effect when co-expressed with KCC3a in HEK cells, as previously reported in Xenopus oocytes (Casula et al.). These data indicate that KCC1 and KCC3 interact structurally and functionally in RBC membranes, and provide another potential mechanism for regulation of KCC activity via multimeric associations between KCC isoforms. Thus, KCC activity could be modulated not only by transcriptional mechanisms and post-translational modification (phosphorylation), but also by altering the ratios of KCC isoforms or the kinetics of their association. We speculate that higher levels of KCC1 protein relative to KCC3 in SS RBC membranes could account for higher KCC activity in these cells relative to AA RBC.

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BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations

International Journal of Molecular Sciences ◽

10.3390/ijms221910638 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10638

Author(s):

Chayma El Khamlichi ◽

Laetitia Cobret ◽

Jean-Michel Arrang ◽

Séverine Morisset-Lopez

Keyword(s):

Cortical Neurons ◽

G Protein Coupled Receptors ◽

Constitutive Activity ◽

Western Blots ◽

Inverse Agonists ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Functional Consequences ◽

G Protein Coupled

G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states.

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Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.6.c1261 ◽

1999 ◽

Vol 276 (6) ◽

pp. C1261-C1270 ◽

Cited By ~ 115

Author(s):

Achim D. Gruber ◽

Kevin D. Schreur ◽

Hong-Long Ji ◽

Catherine M. Fuller ◽

Bendicht U. Pauli

Keyword(s):

Mammary Gland ◽

Human Lung ◽

Glycosylation Site ◽

Hek 293 ◽

Hek 293 Cells ◽

Amino Terminal ◽

Anion Conductance ◽

Whole Cell Patch Clamp ◽

293 Cells ◽

Human Trachea

The CLCA family of Ca2+-activated Cl− channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.

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Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00462.2006 ◽

2007 ◽

Vol 293 (5) ◽

pp. F1699-F1713 ◽

Cited By ~ 112

Author(s):

Ling Wu ◽

Xiaochong Gao ◽

Rachel C. Brown ◽

Stefan Heller ◽

Roger G. O'Neil

Keyword(s):

Shear Stress ◽

Calcium Influx ◽

Collecting Duct ◽

Loss Of Function ◽

Western Blots ◽

Hek 293 ◽

Hek 293 Cells ◽

Hek Cells ◽

293 Cells

Gain/loss of function studies were utilized to assess the potential role of the endogenous vanilloid receptor TRPV4 as a sensor of flow and osmolality in M-1 collecting duct cells (CCD). TRPV4 mRNA and protein were detectable in M-1 cells and stably transfected HEK-293 cells, where the protein occurred as a glycosylated doublet on Western blots. Immunofluorescence imaging demonstrated expression of TRPV4 at the cell membranes of TRPV4-transfected HEK and M-1 cells and at the luminal membrane of mouse kidney CCD. By using intracellular calcium imaging techniques, calcium influx was monitored in cells grown on coverslips. Application of known activators of TRPV4, including 4α-PDD and hypotonic medium, induced strong calcium influx in M-1 cells and TRPV4-transfected HEK-293 cells but not in nontransfected cells. Applying increased flow/shear stress in a parallel plate chamber induced calcium influx in both M-1 and TRPV4-transfected HEK cells but not in nontransfected HEK cells. Furthermore, in loss-of-function studies employing small interference (si)RNA knockdown techniques, transfection of both M-1 and TRPV4-transfected HEK cells with siRNA specific for TRPV4, but not an inappropriate siRNA, led to a time-dependent decrease in TRPV4 expression that was accompanied by a loss of stimuli-induced calcium influx to flow and hypotonicity. It is concluded that TRPV4 displays a mechanosensitive nature with activation properties consistent with a molecular sensor of both fluid flow (or shear stress) and osmolality, or a component of a sensor complex, in flow-sensitive renal CCD.

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Heterodimerization, trafficking and membrane topology of the two proteins, Ostα and Ostβ, that constitute the organic solute and steroid transporter

Biochemical Journal ◽

10.1042/bj20070716 ◽

2007 ◽

Vol 407 (3) ◽

pp. 363-372 ◽

Cited By ~ 45

Author(s):

Na Li ◽

Zhifeng Cui ◽

Fang Fang ◽

Jin Young Lee ◽

Nazzareno Ballatori

Keyword(s):

Plasma Membrane ◽

Membrane Topology ◽

Bimolecular Fluorescence Complementation ◽

Mrna Levels ◽

Western Blots ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Pngase F ◽

Organic Solute

Co-immunoprecipitation studies using mouse ileal proteins and transfected HEK-293 (human embryonic kidney-293) cells revealed that the two proteins, Ostα and Ostβ, which generate the organic-solute transporter are able to immunoprecipitate each other, indicating a heteromeric complex. Mouse ileal Ostα protein appeared on Western blots largely as bands of 40 and 80 kDa, the latter band consistent with an Ostα homodimer, and both of these bands were sensitive to digestion by the glycosidase PNGase F (peptide:N-glycosidase F). Ostβ appeared as bands of 17 and 19 kDa, and these bands were not sensitive to PNGase F. Both the 40 and 80 kDa forms of Ostα, and only the 19 kDa form of Ostβ, were detected among the immunoprecipitated proteins, indicating that the interaction between Ostα and Ostβ is associated with specific post-translational processing. Additional evidence for homodimerization of Ostα and for a direct interaction between Ostα and Ostβ was provided by BiFC (bimolecular fluorescence complementation) analysis of HEK-293 cells transfected with Ostα and Ostβ tagged with yellow-fluorescent-protein fragments. BiFC analysis and surface immunolabelling of transfected HEK-293 cells also indicated that the C-termini of both Ostα and Ostβ are facing the intracellular space. The interaction between Ostα and Ostβ was required not only for delivery of the proteins to the plasma membrane, but it increased their stability, as noted in transfected HEK-293 cells and in tissues from Ostα-deficient (Ostα−/−) mice. In Ostα−/− mice, Ostβ mRNA levels were maintained, yet Ostβ protein was not detectable, indicating that Ostβ protein is not stable in the absence of Ostα. Overall, these findings identify the membrane topology of Ostα and Ostβ, demonstrate that these proteins are present as heterodimers and/or heteromultimers, and indicate that the interaction between Ostα and Ostβ increases the stability of the proteins and is required for delivery of the heteromeric complex to the plasma membrane.

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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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Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

AJP Cell Physiology ◽

10.1152/ajpcell.00115.2010 ◽

2011 ◽

Vol 300 (3) ◽

pp. C576-C587 ◽

Cited By ~ 21

Author(s):

Deblina Dey ◽

Andrew Shepherd ◽

Judith Pachuau ◽

Miguel Martin-Caraballo

Keyword(s):

Membrane Trafficking ◽

Leukemia Inhibitory Factor ◽

Fluorescent Protein ◽

Expression System ◽

Functional Expression ◽

Dominant Negative ◽

Inhibitory Factor ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca2+ channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca2+ channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca2+ channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca2+ channel α1H-subunit resulted in the generation of transient Ca2+ currents. Overnight treatment of α1H-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca2+ channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca2+ channels. Trafficking of α1H-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α1H-GFP subunits. Trafficking of T-type Ca2+ channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca2+ channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca2+ channels.

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

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-021-02616-0 ◽

2021 ◽

Author(s):

Hendrik Liebe ◽

Franziska Liebe ◽

Gerhard Sponder ◽

Sarah Hedtrich ◽

Friederike Stumpff

Keyword(s):

Cell Membrane ◽

Human Skin ◽

Single Channel ◽

Ruthenium Red ◽

Channel Measurements ◽

Hek 293 ◽

Hek 293 Cells ◽

Intestinal Epithelia ◽

Skin Equivalents ◽

293 Cells

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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