The COOH-terminal domain of Drosophila TRP channels confers thapsigargin sensitivity

Previous studies have shown that the Drosophila cation channels designated Trp and TrpI can be functionally expressed in Sf9 insect cells using baculovirus expression vectors. The trp gene encodes a Ca2+-permeable channel that is activated by thapsigargin, blocked by low micromolar Gd3+, and is relatively selective for Ca2+ versus Na+ and Ba2+. In contrast, trpl encodes a Ca2+-permeable cation channel that is constitutively active, not affected by thapsigarsn, blocked by high micromolar Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The region of lowest sequence identity between Trp and TrpI occurs in the COOH-terminal domain. To test the hypothesis that this region is responsible for the differential sensitivity of these channels to thapsigargin, chimeric constructs of Trp and TrpI were created in which the COOH terminal tail region of each protein was exchanged. The Trp construct with the TrpI COOH-tail was constitutively active, insensitive to thapsigarsn, but retained selectivity for Ca2+ over Na+ and Ba2+. In contrast, the TrpI construct with the Trp COOH-tail was not constitutively active, could be activated by thapsigargin, but remained nonselective with respect to Ca2+, Ba2+, and Na+. These results suggest that the COOH-terminal domain of TrpI plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin.