Ca2+ dependence of the Ca2+selective TRPV6 channel

Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca2+-sensing Ca2+ channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca2+ concentration ([Ca2+](i)), which decreased to the same level such as in non-transfected cells if external Ca2+ 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 [Ca2+](i) or if the cytosolic Ca2+ buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca2+-induced current inhibition, because TRPV6 is negatively regulated by internal Ca2+. However, dialyzing solutions with high [Ca2+] such as 1 muM 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 Ca2+ inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca2+ 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 Ca2+-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.