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Ca2+ dependence of the Ca2+-selective TRPV6 channel Bodding M; Flockerzi VJ Biol Chem 2004[Aug]; 279 (35): 36546-52Microfluorimetry 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[MESH]|Biotinylation[MESH]|Blotting, Western[MESH]|Buffers[MESH]|Calcium Channels/*chemistry/metabolism[MESH]|Calcium/*chemistry/metabolism[MESH]|Cations[MESH]|Cell Line[MESH]|Cell Line, Tumor[MESH]|Cell Membrane/metabolism[MESH]|Chelating Agents/pharmacology[MESH]|Cytosol/metabolism[MESH]|Egtazic Acid/pharmacology[MESH]|Electrophysiology[MESH]|Fluorescent Dyes/pharmacology[MESH]|Fura-2/pharmacology[MESH]|Humans[MESH]|Mutagenesis, Site-Directed[MESH]|Mutation[MESH]|Patch-Clamp Techniques[MESH]|Protein Binding[MESH]|Rats[MESH]|Recombinant Proteins/chemistry[MESH]|TRPV Cation Channels[MESH]|Time Factors[MESH]|Transfection[MESH] |
