title: Stimulation of Drosophila TrpL by capacitative Ca2+ entry.
abstract: Trp-like protein (TrpL, where Trp is transient receptor-potential protein) of Drosophila, a non-selective cation channel activated in photoreceptor cells by a phospholipase C-dependent mechanism, is thought to be a prototypical receptor-activated channel. Our previous studies showed that TrpL channels are not activated by depletion of internal Ca2+ stores when expressed in Sf9 cells. Using fura-2 to measure cation influx via TrpL, and cell-attached patch recordings to monitor TrpL single-channel activity directly, we have found a thapsigargin-induced increase in TrpL activity in the presence of extracellular bivalent cations, with Ca2+>Sr2+>> Ba2+. The increase in TrpL channel activity was blocked by concentrations of La3+ that completely inhibited endogenous capacitative Ca2+ entry (CCE), but have no effect on TrpL, suggesting that TrpL exhibits trans-stimulation by cation entry via CCE. TrpL has two putative calmodulin (CaM)-binding domains, designated CBS-1 and CBS-2. To determine which site may be required for stimulation of TrpL by the cytosolic free Ca2+ concentration ([Ca2+]i), a chimaeric construct was created in which the C-terminal domain of TrpL containing CBS-2 was attached to human TrpC1, a short homologue of Trp that is not activated by depletion of internal Ca2+ stores or by a rise in [Ca2+]i. This gain-of-function mutant, designated TrpC1-TrpL, exhibited trans-stimulation by Ca2+ entry via CCE. Examination of CaM binding in gel-overlay experiments showed that TrpL and the TrpC1-TrpL chimaera bound CaM, but TrpC1 or a truncated version of TrpL lacking CBS-2 did not. These results suggest that only CBS-2 binds CaM in native TrpL and that the C-terminal domain containing this site is important for trans-stimulation of TrpL by CCE.
authors.affiliation: Rammelkamp Center for Education and Research, MetroHealth Medical Center, and Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 2500 MetroHealth Drive, Cleveland, OH 44109-1998, USA.
authors.forename: M
authors.initials: M
authors.lastname: Estacion
authors.forename: W G
authors.initials: WG
authors.lastname: Sinkins
authors.forename: W P
authors.initials: WP
authors.lastname: Schilling
mesh_terms: D000818:Animals; D001464:Barium / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000187:drug effects; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D004721:Endoplasmic Reticulum / Q000187:drug effects; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D007811:Lanthanum / Q000378:metabolism; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D013324:Strontium / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D050051:Transient Receptor Potential Channels
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002413:Cations, Divalent; D029721:Drosophila Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D001464:Barium; D019284:Thapsigargin; D007811:Lanthanum; D000252:Calcium-Transporting ATPases; D002118:Calcium; D013324:Strontium