Biomedical subjects

Thomas Voets

Publications and source records attributed to Thomas Voets.

At least 19 recordsLinked to original sources

PubMed2006

TRPM8-independent menthol-induced Ca2+ release from endoplasmic reticulum and Golgi.

Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food and pharmaceutical industries as a cooling/soothing compound and odorant. It induces Ca2+ influx in a subset of sensory neurons from dorsal root and trigeminal ganglia, due to activation of TRPM8, a Ca2+-permeable, cold-activated member of the TRP superfamily of cation channels. Menthol also induces Ca2+ release from intracellular stores in several TRPM8-expressing cell types, which has led to the suggestion that TRPM8 can function as an intracellular Ca2+-release channel. Here we show that menthol induces Ca2+ release from intracellular stores in four widely used cell lines (HEK293, lymph node carcinoma of the prostate (LNCaP), Chinese hamster ovary (CHO), and COS), and provide several lines of evidence indicating that this release pathway is TRPM8-independent: 1) menthol-induced Ca2+ release was potentiated at higher temperatures, which contrasts to the cold activation of TRPM8; 2) overexpression of TRPM8 did not enhance the menthol-induced Ca2+) release; 3) menthol-induced Ca2+ release was mimicked by geraniol and linalool, which are structurally related to menthol, but not by the more potent TRPM8 agonists icilin or eucalyptol; and 4) TRPM8 expression in HEK293 cells was undetectable at the protein and mRNA levels. Moreover, using a novel TRPM8-specific antibody we demonstrate that both heterologously expressed TRPM8 (in HEK293 cells) and endogenous TRPM8 (in LNCaP cells) are mainly localized in the plasma membrane, which contrast to previous localization studies using commercial anti-TRPM8 antibodies. Finally, aequorin-based measurements demonstrate that the TRPM8-independent menthol-induced Ca2+ release originates from both endoplasmic reticulum and Golgi compartments.

Animals↗

PubMed2006

Calbindin-D28K dynamically controls TRPV5-mediated Ca2+ transport.

In Ca(2+)-transporting epithelia, calbindin-D(28K) (CaBP(28K)) facilitates Ca(2+) diffusion from the luminal Ca(2+) entry side of the cell to the basolateral side, where Ca(2+) is extruded into the extracellular compartment. Simultaneously, CaBP(28K) provides protection against toxic high Ca(2+) levels by buffering the cytosolic Ca(2+) concentration ([Ca(2+)](i)) during high Ca(2+) influx. CaBP(28K) consistently colocalizes with the epithelial Ca(2+) channel TRPV5, which constitutes the apical entry step in renal Ca(2+)-transporting epithelial cells. Here, we demonstrate using protein-binding analysis, subcellular fractionation and evanescent-field microscopy that CaBP(28K) translocates towards the plasma membrane and directly associates with TRPV5 at a low [Ca(2+)](i). (45)Ca(2+) uptake measurements, electrophysiological recordings and transcellular Ca(2+) transport assays of lentivirus-infected primary rabbit connecting tubule/distal convolute tubule cells revealed that associated CaBP(28K) tightly buffers the flux of Ca(2+) entering the cell via TRPV5, facilitating high Ca(2+) transport rates by preventing channel inactivation. In summary, CaBP(28K) acts in Ca(2+)-transporting epithelia as a dynamic Ca(2+) buffer, regulating [Ca(2+)] in close vicinity to the TRPV5 pore by direct association with the channel.

Animals↗

PubMed2006

The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate.

Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.

Amino Acid Sequence↗

PubMed2006

Permeation and selectivity of TRP channels.

Ion channels are pore-forming transmembrane proteins that allow ions to permeate biological membranes. Pore structure plays a crucial role in determining the ion permeation and selectivity properties of particular channels. In the past few decades, efforts have been undertaken to identify key elements of the pore regions of different classes of ion channels. In this review, we summarize current knowledge about permeation and selectivity of channel proteins from the transient receptor potential (TRP) superfamily. Whereas all TRP channels are permeable for cations, only two TRP channels are impermeable for Ca2+ (TRPM4, TRPM5), and two others are highly Ca2+ permeable (TRPV5, TRPV6). Despite the great advances in the TRP channel field during the past decade, only a limited number of reports have dealt with functional characterization of pore properties, biophysical aspects of cation permeation, or description of pore structures of TRP channels. This review gives an overview of available experimental and theoretical data and discusses the functional impact of pore-structure modifications on TRP channel properties.

Algorithms↗

PubMed2005

Heat activation of TRPM5 underlies thermal sensitivity of sweet taste.

TRPM5, a cation channel of the TRP superfamily, is highly expressed in taste buds of the tongue, where it has a key role in the perception of sweet, umami and bitter tastes. Activation of TRPM5 occurs downstream of the activation of G-protein-coupled taste receptors and is proposed to generate a depolarizing potential in the taste receptor cells. Factors that modulate TRPM5 activity are therefore expected to influence taste. Here we show that TRPM5 is a highly temperature-sensitive, heat-activated channel: inward TRPM5 currents increase steeply at temperatures between 15 and 35 degrees C. TRPM4, a close homologue of TRPM5, shows similar temperature sensitivity. Heat activation is due to a temperature-dependent shift of the activation curve, in analogy to other thermosensitive TRP channels. Moreover, we show that increasing temperature between 15 and 35 degrees C markedly enhances the gustatory nerve response to sweet compounds in wild-type but not in Trpm5 knockout mice. The strong temperature sensitivity of TRPM5 may underlie known effects of temperature on perceived taste in humans, including enhanced sweetness perception at high temperatures and 'thermal taste', the phenomenon whereby heating or cooling of the tongue evoke sensations of taste in the absence of tastants.

Animals↗

PubMed2005

TRP channels in disease.

The mammalian TRP (transient receptor potential) family consists of six main subfamilies termed the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) groups. These subfamilies encompass 28 ion channels that function as diverse cellular sensors. All of the channels are permeable to monovalent cations, and most are also permeable to Ca(2+). There are strong indications that TRP channels are involved in many diseases. At this point, four channelopathies have been identified in which a defect in a TRP channel-encoding gene is the direct cause of disease. TRPs are also involved in some systemic diseases because of their role as receptors for irritants, inflammation products, and xenobiotic toxins. Other indications of the involvement of TRPs in several diseases come from correlations between the levels of channel expression and disease symptoms or from the mapping of TRP-encoding genes to susceptible chromosome regions. Finally, the phenotypes of TRP knockout mice and other transgenic models allow a degree of extrapolation to human diseases. We present an overview of current knowledge about the role of TRP channels in human disease and highlight some TRP "suspects" for which a role in disease can be anticipated. An understanding of the genetics of disease may lead to the development of targeted new therapies.

Animals↗

PubMed2005

TRP channels: a TR(I)P through a world of multifunctional cation channels.

The "transient receptor potential" (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed from yeast to man. On the basis of structural homology, the TRP family can be subdivided in to seven main subfamilies: the TRPC ('Canonical') group, the TRPV ('Vanilloid') group, the TRPM ('Melastatin') group, the TRPP ('Polycystin'), the TRPML ('Mucolipin'), the TRPA ('Ankyrin') and the TRPN ('NOMP') family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of cell types, tissues and species. This paper briefly reviews the TRP superfamily and the basic properties of its many members as a reader's guide in this Special Issue. Hopefully, a better understanding of TRP channel physiology will provide important insight into the relationship between TRP channel dysfunction and human diseases.

Animals↗

PubMed2005

Gating of TRP channels: a voltage connection?

TRP channels represent the main pathways for cation influx in non-excitable cells. Although TRP channels were for a long time considered to be voltage independent, several TRP channels now appear to be weakly voltage dependent with an activation curve extending mainly into the non-physiological positive voltage range. In connection with this voltage dependence, there is now abundant evidence that physical stimuli, such as temperature (TRPV1, TRPM8, TRPV3), or the binding of various ligands (TRPV1, TRPV3, TRPM8, TRPM4), shift this voltage dependence towards physiologically relevant potentials, a mechanism that may represent the main functional hallmark of these TRP channels. This review discusses some features of voltage-dependent gating of TRPV1, TRPM4 and TRPM8. A thermodynamic principle is elaborated, which predicts that the small gating charge of TRP channels is a crucial factor for the large voltage shifts induced by various stimuli. Some structural considerations will be given indicating that, although the voltage sensor is not yet known, the C-terminus may substantially change the voltage dependence of these channels.

Animals↗

PubMed2005

The selectivity filter of the cation channel TRPM4.

Transient receptor potential channel melastatin subfamily (TRPM) 4 and its close homologue, TRPM5, are the only two members of the large transient receptor potential superfamily of cation channels that are impermeable to Ca(2+). In this study, we located the TRPM4 selectivity filter and investigated possible structural elements that render it Ca(2+)-impermeable. Based on homology with known cation channel pores, we identified an acidic stretch of six amino acids in the loop between transmembrane helices TM5 and TM6 ((981)EDMDVA(986)) as a potential selectivity filter. Substitution of this six-amino acid stretch with the selectivity filter of TRPV6 (TIIDGP) resulted in a functional channel that combined the gating hallmarks of TRPM4 (activation by Ca(2+), voltage dependence) with TRPV6-like sensitivity to block by extracellular Ca(2+) and Mg(2+) as well as Ca(2+) permeation. Neutralization of Glu(981) resulted in a channel with normal permeability properties but a strongly reduced sensitivity to block by intracellular spermine. Neutralization of Asp(982) yielded a functional channel that exhibited extremely fast desensitization (tau < 5 s), possibly indicating destabilization of the pore. Neutralization of Asp(984) resulted in a non-functional channel with a dominant negative phenotype when coexpressed with wild type TRPM4. Combined neutralization of all three acidic residues resulted in a functional channel whose voltage dependence was shifted toward very positive potentials. Substitution of Gln(977) by a glutamate, the corresponding residue in divalent cation-permeable TRPM channels, altered the monovalent cation permeability sequence and resulted in a pore with moderate Ca(2+) permeability. Our findings delineate the selectivity filter of TRPM channels and provide the first insight into the molecular basis of monovalent cation selectivity.

Alanine↗

PubMed2005

CAPS1 regulates catecholamine loading of large dense-core vesicles.

CAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs.

Animals↗

PubMed2005

Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice.

Non-selective cation (NSC) channels activated by intracellular Ca2+ ([Ca2+]i) play an important role in Ca2+ signaling and membrane excitability in many cell types. TRPM4 and TRPM5, two Ca2+-activated cation channels of the TRP superfamily, are potential molecular correlates of NSC channels. We compared the functional properties of mouse TRPM4 and TRPM5 heterologously expressed in HEK 293 cells. Dialyzing cells with different Ca2+ concentrations revealed a difference in Ca2+ sensitivity between TRPM4 and TRPM5, with EC50 values of 20.2+/-4.0 microM and 0.70+/-0.1 microM, respectively. Similarly, TRPM5 activated at lower Ca2+ concentration than TRPM4 when [Ca2+]i was raised by UV uncaging of the Ca2+-cage DMNP-EDTA. Current amplitudes of TRPM4 and TRPM5 were not correlated to the rate of changes in [Ca2+]i. The Ca2+ sensitivity of both channels was strongly reduced in inside-out patches, resulting in approximately 10-30 times higher EC50 values than under whole-cell conditions. Currents through TRPM4 and TRPM5 deactivated at negative and activated at positive potentials with similar kinetics. Both channels were equally sensitive to block by intracellular spermine. TRPM4 displayed a 10-fold higher affinity for block by flufenamic acid. Importantly, ATP4- blocked TRPM4 with high affinity (IC50 of 0.8+/-0.1 microM), whereas TRPM5 is insensitive to ATP4- at concentrations up to 1 mM.

3T3 Cells↗

PubMed2005

Sensing with TRP channels.

Drosophila melanogaster flies carrying the trp (transient receptor potential) mutation are rapidly blinded by bright light, because of the absence of a Ca2+-permeable ion channel in their photoreceptors. The identification of the trp gene and the search for homologs in yeast, flies, worms, zebrafish and mammals has led to the discovery of a large superfamily of related cation channels, named TRP channels. Activation of TRP channels is highly sensitive to a variety of chemical and physical stimuli, allowing them to function as dedicated biological sensors that are essential in processes such as vision, taste, tactile sensation and hearing.

Animals↗

PubMed2004

Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4.

TRPM4, a Ca(2+)-activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca(2+). The mechanisms underlying the alterations in Ca(2+) sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca(2+) sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca(2+) sensitivity of wild-type TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca(2+) dramatically reduced TRPM4 activation. We identified five Ca(2+)-calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca(2+) sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca(2+) sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus.

Adenosine Triphosphate↗

PubMed2004

Decavanadate modulates gating of TRPM4 cation channels.

We have tested the effects of decavanadate (DV), a compound known to interfere with ATP binding in ATP-dependent transport proteins, on TRPM4, a Ca(2+)-activated, voltage-dependent monovalent cation channel, whose activity is potently blocked by intracellular ATP(4-). Application of micromolar Ca(2+) concentrations to the cytoplasmic side of inside-out patches led to immediate current activation followed by rapid current decay, which can be explained by an at least 30-fold decreased apparent affinity for Ca(2+). Subsequent application of DV (10 microm) strongly affected the voltage-dependent gating of the channel, resulting in large sustained currents over the voltage range between -180 and +140 mV. The effect of DV was half-maximal at a concentration of 1.9 microm. The Ca(2+)- and voltage-dependent gating of the channel was well described by a sequential kinetic scheme in which Ca(2+) binding precedes voltage-dependent gating. The effects of DV could be explained by an action on the voltage-dependent closing step. Surprisingly, DV did not antagonize the effect of ATP(4-) on TRPM4, but caused a nearly 10-fold increase in the sensitivity of the ATP(4-) block. TRPM5, which is the most homologous channel to TRPM4, was not modulated by DV. The effect of DV was lost in a TRPM4 chimera in which the C-terminus was substituted with that of TRPM5. Deletion of a cluster in the C-terminus of TRPM4 containing positively charged amino acid residues with a high homology to part of the decavanadate binding site in SERCA pumps, completely abolished the DV effect but also accelerated desensitization. Deletion of a similar site in the N-terminus had no effects on DV responses. These results indicate that the C-terminus of TRPM4 is critically involved in mediating the DV effects. In conclusion, decavanadate modulates TRPM4, but not TRPM5, by inhibiting voltage-dependent closure of the channel.

Adenosine Triphosphate↗

PubMed2004

The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels.

The mammalian sensory system is capable of discriminating thermal stimuli ranging from noxious cold to noxious heat. Principal temperature sensors belong to the TRP cation channel family, but the mechanisms underlying the marked temperature sensitivity of opening and closing ('gating') of these channels are unknown. Here we show that temperature sensing is tightly linked to voltage-dependent gating in the cold-sensitive channel TRPM8 and the heat-sensitive channel TRPV1. Both channels are activated upon depolarization, and changes in temperature result in graded shifts of their voltage-dependent activation curves. The chemical agonists menthol (TRPM8) and capsaicin (TRPV1) function as gating modifiers, shifting activation curves towards physiological membrane potentials. Kinetic analysis of gating at different temperatures indicates that temperature sensitivity in TRPM8 and TRPV1 arises from a tenfold difference in the activation energies associated with voltage-dependent opening and closing. Our results suggest a simple unifying principle that explains both cold and heat sensitivity in TRP channels.

Capsaicin↗

PubMed2004

Intracellular nucleotides and polyamines inhibit the Ca2+-activated cation channel TRPM4b.

TRPM4b (in contrast to the short splice variant TRPM4a) is a Ca(2+)-activated but Ca(2+)-impermeable cation channel. We have studied TRPM4 currents in inside-out patches. Supramicromolar Ca(2+) concentrations applied at the inner side, [Ca(2+)](i), activated TRPM4 with an EC(50) value of 0.37 mM, a value that is much higher than that of whole-cell currents. Current amplitudes decreased above 1 mM [Ca(2+)](i), (IC(50) 9.3 mM). Sr(2+) but not Ba(2+)could partially substitute for Ca(2+). ATP, ADP, AMP and AMP-PNP all quickly and reversibly inhibited TRPM4 with IC(50) values between 2 and 19 microM (at +100 mV). Adenosine also blocked TRPM4 at 630 microM. The block at high ATP concentrations was incomplete and was not affected by the presence of free Mg(2+). ADP induced the most sensitive block with an IC(50) of 2.2 microM. For inhibition of TRPM4 by free ATP(4-), an IC(50) value of 1.7+/-0.3 microM was calculated. GTP, UTP and CTP at concentrations up to 1 mM did not induce a similar block. Spermine blocked TRPM4 currents with an IC(50) of 61 microM. In conclusion, TRPM4 is a channel that can be effectively modulated by intracellular nucleotides and polyamines.

Adenine Nucleotides↗

PubMed2004

Outer pore architecture of a Ca2+-selective TRP channel.

The TRP superfamily forms a functionally important class of cation channels related to the product of the Drosophila trp gene. TRP channels display an unusual diversity in activation mechanisms and permeation properties, but the basis of this diversity is unknown, as the structure of these channels has not been studied in detail. To obtain insight in the pore architecture of TRPV6, a Ca(2+)-selective member of the TRPV subfamily, we probed the dimensions of its pore and determined pore-lining segments using cysteine-scanning mutagenesis. Based on the permeability of the channel to organic cations, we estimated a pore diameter of 5.4 A. Mutating Asp(541), a residue involved in high affinity Ca(2+) binding, altered the apparent pore diameter, indicating that this residue lines the narrowest part of the pore. Cysteines introduced in a region preceding Asp(541) displayed a cyclic pattern of reactivity to Ag(+) and cationic methylthio-sulfanate reagents, indicative of a pore helix. The anionic methanethiosulfonate ethylsulfonate showed only limited reactivity in this region, consistent with the presence of a cation-selective filter at the outer part of the pore helix. Based on these data and on homology with the bacterial KcsA channel, we present the first structural model of a TRP channel pore. We conclude that main structural features of the outer pore, namely a selectivity filter preceded by a pore helix, are conserved between K(+) channels and TRPV6. However, the selectivity filter of TRPV6 is wider than that of K(+) channels and lined by amino acid side chains rather than main chain carbonyls.

Amino Acid Sequence↗

PubMed2004

Invertebrate TRP proteins as functional models for mammalian channels.

Transient receptor potential (TRP) channels constitute a large and diverse family of channel proteins that are expressed in many tissues and cell types in both vertebrates and invertebrates. While the biophysical features of many of the mammalian TRP channels have been described, relatively little is known about their biological roles. Invertebrate TRPs offer valuable genetic handles for characterizing the functions of these cation channels in vivo. Importantly, studies in model organisms can help to identify fundamental mechanisms involved in normal cellular functions and human disease. In this review, we give an overview of the different TRP channels known in the two most utilized invertebrate models, the nematode Caenorhabditis elegans and the fruit-fly Drosophila melanogaster, and discuss briefly the heuristic impact of these invertebrate channels with respect to TRP function in mammals.

Animals↗