THE COOH-TERMINAL DOMAIN OF DROSOPHILA TRP CHANNELS CONFERS THAPSIGARGIN SENSITIVITY

Previous studies have shown that the Drosophila cation channels design ated Trp and TrpI can be functionally expressed in Sf9 insect cells us ing baculovirus expression vectors. The trp gene encodes a Ca2+-permea ble channel that is activated by thapsigargin, blocked by low micromol ar Gd3+, and is relatively selective for Ca2+ versus Na+ and Ba2+. In contrast, trpl encodes a Ca2+-permeable cation channel that is constit utively active, not affected by thapsigarsn, blocked by high micromola r Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The reg ion of lowest sequence identity between Trp and TrpI occurs in the COO H-terminal domain. To test the hypothesis that this region is responsi ble for the differential sensitivity of these channels to thapsigargin , chimeric constructs of Trp and TrpI were created in which the COOH t erminal tail region of each protein was exchanged. The Trp construct w ith the TrpI COOH-tail was constitutively active, insensitive to thaps igarsn, but retained selectivity for Ca2+ over Na+ and Ba2+. In contra st, the TrpI construct with the Trp COOH-tail was not constitutively a ctive, could be activated by thapsigargin, but remained nonselective w ith respect to Ca2+, Ba2+, and Na+. These results suggest that the COO H-terminal domain of TrpI plays an important role in determining const itutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin.