Biomedical subjects

Ulrich Wissenbach

Publications and source records attributed to Ulrich Wissenbach.

14 recordsLinked to original sources

PubMed2006

Trafficking and assembly of the cold-sensitive TRPM8 channel.

TRPM (transient receptor potential melastatin-like) channels are distinct from many other members of the transient receptor potential family in regard to their overall size (>1000 amino acids), the lack of N-terminal ankyrin-like repeats, and hydrophobicity predictions that may allow for more than six transmembrane regions. Common to each TRPM member is a prominent C-terminal coiled coil region. Here we have shown that TRPM8 channels assemble as multimers using the putative coiled coil region within the intracellular C terminus and that this assembly can be disturbed by a single point mutation within the coiled coil region. This mutant neither gives rise to functional channels nor do its subunits interact or form protein complexes that correspond to a multimer. However, they are still transported to the plasma membrane. Furthermore, wild-type currents can be suppressed by expressing the membrane-attached C-terminal region of TRPM8. To separate assembly from trafficking, we investigated the maturation of TRPM8 protein by identifying and mutating the relevant N-linked glycosylation site and showing that glycosylation is neither essential for multimerization nor for transport to the plasma membrane per se but appears to facilitate efficient multimerization and transport.

Animals↗

PubMed2005

TRPV6 potentiates calcium-dependent cell proliferation.

The Ca(2+) homeostasis within cells controls a diversity of cellular processes including gene transcription, proliferation and apoptosis. Perturbance of Ca(2+) signaling may induce deregulation of cell proliferation and suppression of cell death providing the basis for cancer development. In human prostate cancer, a correlation between the mRNA expression of the Ca(2+) channel TRPV6 and the staging of the cancer has been described. We have analyzed the influence of TRPV6 on cell proliferation within HEK-293 cells. We show that TRPV6 increases cell proliferation of HEK-293 cells in a Ca(2+) dependent manner. The increased proliferation correlates with slightly increased intracellular Ca(2+) levels without interfering with the intrinsic Ca(2+) dependence of HEK-293 cell proliferation. Low doses of econazole inhibit both, TRPV6 dependent Ca(2+) signals and cell proliferation while BTP2, a potent inhibitor of Ca(2+) signals and cell proliferation in T-cells, neither influences TRPV6 dependent Ca(2+) signals nor cell proliferation of HEK-293 cells. Our data demonstrate that TRPV6 increases the rate of Ca(2+) dependent cell proliferation which is a prerequisite for its potential role in tumor progression.

Anilides↗

PubMed2005

Transcriptional regulation and processing increase the functional variability of TRPM channels.

Mammalian TRP channels display heterogenous biophysical properties and are involved in a variety of signal transduction pathways. To carry out their diverse biological functions and to adapt these functions to changes of the environment, mechanisms to regulate their molecular structure are required. Transcriptional regulation and post-transcriptional RNA processing represent essential instruments to generate TRP channel variants with modified properties. TRP variants are expressed depending on the tissue and developmental state. They can show distinct biophysical properties and mechanisms of activation, and thereby determine channel function and malfunction in certain human diseases. In this review, we give an overview of the variants of a given TRP gene, with the focus on the TRPM subfamily, and discuss their relevance with respect to their function under physiological and pathological conditions.

Animals↗

PubMed2004

TRPV6 and prostate cancer: cancer growth beyond the prostate correlates with increased TRPV6 Ca2+ channel expression.

Life expectancy for patients suffering from prostate cancer is inversely correlated with the degree of extraprostatic metastasis. In order to find pharmacological tools to treat this aggressive growth it is important to define targets whose expression not only correlates with the malignancy of the cancerous cells, but that are also amenable to pharmacological intervention. In this review, we would like to focus on the potential role of a distinct class of ion channels that may be involved in this process.

Calcium Channels↗

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

Ca2+-selective transient receptor potential V channel architecture and function require a specific ankyrin repeat.

Transient receptor potential (TRP) proteins form cation-conducting ion channels with currently 28 known genes encoding TRP channel monomers in mammals. These monomers are thought to coassemble to form homo- or heterotetrameric channels, but the signals governing their assembly are unknown. Within the TRPV subgroup, TRPV5 and TRPV6 show exclusive calcium selectivity and play an important role in calcium uptake. To identify signals that mediate assembly of functional TRPV6, we screened domains for self-association using co-immunoprecipitation, sucrose gradient centrifugation, bacterial two-hybrid assays, and patch clamp analysis. Of the two identified interaction domains within the N-terminal region, we showed that the first domain encompassing the third ankyrin repeat is the stringent requirement for physical assembly of TRPV6 subunits and when transferred to an unrelated protein enables its interaction with TRPV6. Deletion of this repeat or mutation of critical residues within this repeat rendered nonfunctional channels that do not co-immunoprecipitate or form tetramers. Suppression of dominant-negative inhibitors of TRPV6-specific currents was achieved by deletion of ankyrin (ANK) 3. We propose that the third ANK repeat initiates a molecular zippering process that proceeds past the fifth ANK repeat and creates an intracellular anchor that is necessary for functional subunit assembly.

Amino Acid Sequence↗

PubMed2004

TRP channels as potential drug targets.

Calcium (Ca2+) is an ubiquitous intracellular signal that is responsible for a plethora of cellular processes including fertilization, secretion, contraction, neuronal signaling and learning. In addition, changes in intracellular Ca2+ have been known to influence cell proliferation and differentiation for more than three decades. Recent studies have indicated that members of the transient receptor potential (TRP) family of ion channels which respond to many different modes of stimulation both from within and outside the cell may be a primary mode of cation and Ca2+ entry into cells and may have roles in growth control. Moreover, changes in the expression of these channels may contribute to certain cancers. In the following, recent results concerning the expression and function of members of this family of ion channels are summarized.

Animals↗

PubMed2003

Expression of the Ca2+-selective cation channel TRPV6 in human prostate cancer: a novel prognostic marker for tumor progression.

Members of the TRP superfamily of cation channels have homeostatic and regulatory functions in cells and changes in their expression may contribute to malignant growth. Previously, we have demonstrated that the gene of the Ca2+-selective cation channel CaT-L or TRPV6 is not expressed in benign prostate tissues including benign prostate hyperplasia, but is upregulated in prostate cancer. Here, we report on the differential expression of TRPV6 mRNA in prostate tissue obtained from 140 patients with prostate cancer. Using in situ hybridization, TRPV6 transcripts were undetectable in benign prostate tissue, high-grade prostatic intraepithelial neoplasia (n=57), incidental adenocarcinoma and all tumors less than 2.3 cubic centimeter (cc). In prostatectomy specimens from 97 clinically organ-confined tumors, TRPV6 expression correlated significantly with the Gleason score (P=0.032), pathological stage (P<0.001) and extraprostatic extension (P=0.025). Lymph node metastasis (n=17) and androgen-insensitive tumors (n=27) revealed TRPV6 expression in 63 and 67% of cases, respectively. The latter, however, revealed markedly and significantly decreased levels when compared with untreated tumors (P=0.044). In summary, the data demonstrate that TRPV6 expression is associated with prostate cancer progression. Accordingly, TRPV6 represents a prognostic marker and, as a plasma membrane Ca2+ channel, a promising target for new therapeutic strategies to treat advanced prostate cancer.

Biomarkers, Tumor↗

PubMed2003

Voltage dependence of the Ca2+-activated cation channel TRPM4.

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials.

Action Potentials↗

PubMed2003

TRPC3 mediates T-cell receptor-dependent calcium entry in human T-lymphocytes.

Stimulation of the T-cell receptor (TCR) activates Ca2+ entry across the plasma membrane, which is a key triggering event for the T-cell-associated immune response. We show that TRPC3 channels are important for the TCR-dependent Ca2+ entry pathway. The TRPC3 gene was found to be damaged in human T-cell mutants defective in Ca2+ influx. Mutations of the TRPC3 gene were accompanied by changes of TRPC3 gene expression. Introduction of the complete human TRPC3 cDNA into those mutants rescued Ca2+ currents as well as TCR-dependent Ca2+ signals. Our data provide the initial step toward understanding the molecular nature of endogenous Ca2+ channels participating in T-cell activation and put forward TRPC3 as a new target for modulating the immune response.

Barium↗

PubMed2002

Pain perception in mice lacking the beta3 subunit of voltage-activated calcium channels.

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the alpha1B subunit (N-type). The accessory beta3 subunits of calcium channels are preferentially associated with the alpha1B subunit in neurones. Here we show that deletion of the beta3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of beta3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (alpha1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.

Amino Acid Sequence↗

PubMed2002

The recombinant human TRPV6 channel functions as Ca2+ sensor in human embryonic kidney and rat basophilic leukemia cells.

The activation mechanism of the recently cloned human transient receptor potential vanilloid type 6 (TRPV6) channel, originally termed Ca(2+) transporter-like protein and Ca(2+) transporter type 1, was investigated in whole-cell patch-clamp experiments using transiently transfected human embryonic kidney and rat basophilic leukemia cells. The TRPV6-mediated currents are highly Ca(2+)-selective, show a strong inward rectification, and reverse at positive potentials, which is similar to store-operated Ca(2+) entry in electrically nonexcitable cells. The gating of TRPV6 channels is strongly dependent on the cytosolic free Ca(2+) concentration; lowering the intracellular free Ca(2+) concentration results in Ca(2+) influx, and current amplitude correlates with the intracellular EGTA or BAPTA concentration. This is also the case for TRPV6-mediated currents in the absence of extracellular divalent cations; compared with endogenous currents in nontransfected rat basophilic leukemia cells, these TRPV6-mediated monovalent currents reveal differences in reversal potential, inward rectification, and slope at very negative potentials. Release of stored Ca(2+) by inositol 1,4,5-trisphosphate and/or the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin appears not to be involved in TRPV6 channel gating in both cell lines but, in rat basophilic leukemia cells, readily activates the endogenous Ca(2+) release-activated Ca(2+) current. In conclusion, TRPV6, expressed in human embryonic kidney cells and in rat basophilic leukemia cells, functions as a Ca(2+)-sensing Ca(2+) channel independently of procedures known to deplete Ca(2+) stores.

Animals↗

PubMed2002

Molecular determinants of permeation through the cation channel TRPV4.

We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.

Amino Acid Sequence↗

PubMedDate not supplied

The TRPV6 gene, cDNA and protein.

The mouse TRPV6 gene is localized on chromosome 6 and extends over 15.66kb. The encoded protein comprises 727 amino acid residues with a calculated relative molecular mass of 83,210Da. TRPV6 is glycosylated and both variants, the glycosylated and the de-glycosylated proteins, are recognized by various polyclonal and monoclonal antibodies, which were raised against TRPV6. Like human TRPV6, mouse TRPV6 binds calmodulin in the presence, but not in the absence of Ca2+. TRPV6 is abundantly expressed in mouse pancreas and placenta, and to a much lesser extend in mouse stomach and kidney. No transcript expression was detected in poly(A)+RNA isolated from heart, brain, intestine, esophagus or aortic endothelial cells.

Amino Acid Sequence↗