Biomedical subjects

Matthias Bödding

Publications and source records attributed to Matthias Bödding.

7 recordsLinked to original sources

PubMed2006

TRP proteins and cancer.

Cancer is the second most common cause of death in western countries. It is therefore of fundamental importance to improve the treatment of patients with malignant tumors. This goal can only be achieved if we get closer insight in the various mechanisms leading to tumor formation. Significant progress in the understanding of carcinogenesis has been made during the last couple of years. Ion channels contribute to the regulation of cell proliferation which has initially been shown for K+ channels. Meanwhile, other ion channels such as Cl-, Na+ and Ca2+ channels seem to influence cellular function like growth, migration and invasion. In addition, cation channels of the transient receptor potential (TRP) superfamily are implicated in cancer formation. Most recent data concerning TRP vanilloid (TRPV) type 6, TRP melastatin (TRPM) type 1 and 8 channels and their relevance for common human cancer types will be highlighted in this review. Furthermore, TRP channel structure and function will be discussed in the light of their possible importance as prognostic markers and targets for drug discovery.

Calcium Channels↗

PubMed2004

Voltage-dependent changes of TRPV6-mediated Ca2+ currents.

The physiological role and activation mechanism for most proteins of the transient receptor potential (TRP) family are unknown. This is also the case for the highly Ca(2+) selective transient receptor potential vanilloid type 6 (TRPV6) channel. Patch clamp experiments were performed on transiently transfected human embryonic kidney (HEK) cells to address this issue. Currents were recorded under various conditions of intracellular Ca(2+) buffering and monitored at the same voltage throughout. No TRPV6-mediated Ca(2+) entry was detected under in vivo Ca(2+) buffering conditions at a slightly negative holding potential; however, moderate depolarization resulted in current activation. Very similar results were obtained with different Ca(2+) chelators, either EGTA or BAPTA dialyzing the cell. TRPV6 channel activity showed a negative correlation with the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and was modulated by the membrane potential: Hyperpolarization decreases and depolarization increases TRPV6-mediated currents. Monovalent ions permeated TRPV6 channels in the absence of extracellular divalent cations. These currents were resistant to changes in the holding potential while the negative correlation to the [Ca(2+)](i) was conserved, indicating that the voltage-dependent current changes depend on blocking and unblocking the charge carrier Ca(2+) within the pore. In summary, these results suggest that the voltage dependence of TRPV6-mediated Ca(2+) influx is of physiological importance since it occurs at cytosolic Ca(2+) buffering and takes place within a physiologically relevant membrane potential range.

Biological Transport↗

PubMed2004

Ca2+ dependence of the Ca2+-selective TRPV6 channel.

Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca(2+)-sensing Ca(2+) channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)), which decreased to the same level such as in non-transfected cells if external Ca(2+) was chelated by EGTA. Whole cell recordings from non-transfected HEK cells and cells expressing human TRPV6 revealed the presence of a basal inward current in both types of cells when the internal solution contained 0.1 mm EGTA and 100 nm [Ca(2+)](i) or if the cytosolic Ca(2+) buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca(2+)-induced current inhibition, because TRPV6 is negatively regulated by internal Ca(2+). However, dialyzing solutions with high [Ca(2+)] such as 1 microm into TRPV6-expressing cells did not block the basal inward current, which was not different from the recordings from non-transfected cells. In contrast, dialyzing 0.5 mm EGTA into TRPV6-expressing cells readily activated Ca(2+) inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca(2+) permeation was detectable, indicating that divalent cations block TRPV6 channels from the extracellular side. Like human TRPV6, the truncated human TRPV6(Delta695-725), which lacks the C-terminal domain required for Ca(2+)-calmodulin binding, does not form constitutive active channels, whereas the human TRPV6(D542A), carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.

Animals↗

PubMed2003

Store-operated Ca2+ current and TRPV6 channels in lymph node prostate cancer cells.

The contribution of endogenous and recombinant transient receptor potential vanilloid type 6 (TRPV6) channels to Ca2+ entry across the plasma membrane was studied in the human lymph node prostate cancer cell line (LNCaP). LNCaP cells do express the TRPV6 gene, and Ca2+ entry currents in these cells were detected after active and passive Ca2+ store depletion by intracellular application of inositol 1,4,5-trisphosphate, Ca2+ chelators, and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. This store-operated Ca2+ current (ISOC) had biophysical properties similar to those of the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukemia cells such as the activation mechanism, inward rectification, and Ca2+ selectivity. These properties are also shared by the Ca2+-sensing Ca2+ current (ITRPV6) recorded after heterologous expression of TRPV6 cDNA in human embryonic kidney and rat basophilic leukemia cells (Bödding, M., Wissenbach, U., Flockerzi, V. (2002) J. Biol. Chem. 277, 36656-36664). TRPV6 cDNA transfection of LNCaP cells restored recombinant ITRPV6, which can be distinguished from ISOC by the mechanism of activation, the voltage dependence of monovalent currents in the absence of external divalent cations, and the changes in Ca2+ current densities due to different membrane potentials. In addition, ISOC was not affected by antiandrogen or 1,25-dihydroxyvitamin D3 treatment of LNCaP cells, which up-regulates TRPV6 gene expression, or by androgen treatment, which has the opposite effect. Therefore, native channels responsible for ISOC are different from those for recombinant ITRPV6 and do not appear to be affected if one of their assumed subunits, TRPV6, is up- or down-regulated, suggesting a rather rigid subunit composition in vivo.

Androgens↗

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↗