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.