Inhibition des TRPC3-Kationenkanals durch Lanthanidionen

The lanthanide ions La3+ and Gd3+ block Ca2+-permeable cation channels and have been used as important tools to characterize channels of the transient receptor potential (TRP) family. However, widely different concentrations of La3+ and Gd3+ have reportedly been required for block of TRP3 channels in various expression systems. The present study provides a possible explanation for this discrepancy. After overexpression of TRP3 in Chinese hamster ovary cells, whole-cell currents through TRP3 were reversibly inhibited by La3+ with an EC50 of 4 µM. For comparison, the organic blocker SKF96365 required an EC50 of 8 µM. Gd3+ blocked with an EC50 of 0.1 µM, but this block was slow in onset and was not reversible after wash-out. When the two lanthanides were added to the cytosolic side of inside-out patches, block was achieved with considerably lower concentrations (EC50 for La3+, 0.02 µM; EC50 for Gd3+, 0.02 µM). Uptake of La3+ into the cytosol of Chinese hamster ovary cells was demonstrated with intracellular fura-2. We conclude that lanthanides block TRP3 more potently from the cytosolic than from the extracellular side of the plasma membrane and that uptake of lanthanides will largely affect the apparent EC50 values after extracellular application

Inhibition des TRPC3-Kationenkanals durch Lanthanidionen

The lanthanide ions La3+ and Gd3+ block Ca2+-permeable cation channels and have been used as important tools to characterize channels of the transient receptor potential (TRP) family. However, widely different concentrations of La3+ and Gd3+ have reportedly been required for block of TRP3 channels in various expression systems. The present study provides a possible explanation for this discrepancy. After overexpression of TRP3 in Chinese hamster ovary cells, whole-cell currents through TRP3 were reversibly inhibited by La3+ with an EC50 of 4 µM. For comparison, the organic blocker SKF96365 required an EC50 of 8 µM. Gd3+ blocked with an EC50 of 0.1 µM, but this block was slow in onset and was not reversible after wash-out. When the two lanthanides were added to the cytosolic side of inside-out patches, block was achieved with considerably lower concentrations (EC50 for La3+, 0.02 µM; EC50 for Gd3+, 0.02 µM). Uptake of La3+ into the cytosol of Chinese hamster ovary cells was demonstrated with intracellular fura-2. We conclude that lanthanides block TRP3 more potently from the cytosolic than from the extracellular side of the plasma membrane and that uptake of lanthanides will largely affect the apparent EC50 values after extracellular application

Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD

An early key event in the activation of neutrophil granulocytes is Ca(2+) influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase-PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca(2+) channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD(+). Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na(+) and Ca(2+), which is regulated by ADP-ribose and the redox state