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.
Bödding
Matthias
M
Experimentelle und klinische Pharmakologie und Toxikologie, Universität des Saarlandes, D-66421 Homburg, Germany. matthias.boedding@uniklinik.saarland.de
Flockerzi
Veit
V
D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002021:Buffers; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism; D002412:Cations; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004594:Electrophysiology; D005456:Fluorescent Dyes / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D050916:TRPV Cation Channels; D013997:Time Factors; D014162:Transfection
D002021:Buffers; D015220:Calcium Channels; D002412:Cations; D002614:Chelating Agents; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C120776:TRPV6 channel; D004533:Egtazic Acid; D002118:Calcium; D016257:Fura-2