The COOH-terminal domain of Drosophila TRP channels confers thapsigargin sensitivity.
Previous studies have shown that the Drosophila cation channels designated Trp and Trpl 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 thapsigargin, blocked by high micromolar Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The region of lowest sequence identity between Trp and Trpl 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 Trpl were created in which the COOH-terminal tail region of each protein was exchanged. The Trp construct with the Trpl COOH-tail was constitutively active, insensitive to thapsigargin, but retained selectivity for Ca2+ over Na+ and Ba2+. In contrast, the Trpl construct with the Trp COOH-tail was not constitutively active, could be activated by thapsigargin, but remained non-selective with respect to Ca2+, Ba2+, and Na+. These results suggest that the COOH-terminal domain of Trpl plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin.
Sinkins
W G
WG
Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109-1998, USA.
Vaca
L
L
Hu
Y
Y
Kunze
D L
DL
Schilling
W P
WP
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D004330:Drosophila; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D005682:Gadolinium / Q000494:pharmacology; D017344:Genes, Insect; D007301:Insect Hormones / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D019476:Insect Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D018411:Spodoptera; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014162:Transfection; D050051:Transient Receptor Potential Channels
D015220:Calcium Channels; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D007301:Insect Hormones; D019476:Insect Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013729:Terpenes; D050051:Transient Receptor Potential Channels; C063169:trp protein, Drosophila; D001464:Barium; D019284:Thapsigargin; C085706:FLAG peptide; D012964:Sodium; D005682:Gadolinium; D000252:Calcium-Transporting ATPases; D002118:Calcium