Stimulation of Drosophila TrpL by capacitative Ca2+ entry

• Published in: Biochemical journal Vol. 341 ( Pt 1); no. 1; pp. 41 - 49
• Main Authors: Estacion, M, Sinkins, W G, Schilling, W P
• Format: Journal Article
• Language: English
• Published: England 01.07.1999
• Subjects: Animals; Barium - metabolism; Biological Transport - drug effects; Calcium - metabolism; Calcium-Transporting ATPases - drug effects; Calmodulin - metabolism; Calmodulin-Binding Proteins - drug effects; Calmodulin-Binding Proteins - genetics; Calmodulin-Binding Proteins - metabolism; Cations, Divalent - metabolism; Drosophila; Drosophila Proteins; Endoplasmic Reticulum - drug effects; Humans; Ion Channel Gating; Ion Channels - drug effects; Ion Channels - genetics; Ion Channels - metabolism; Lanthanum - metabolism; Membrane Proteins - drug effects; Membrane Proteins - genetics; Membrane Proteins - metabolism; Patch-Clamp Techniques; Recombinant Fusion Proteins - drug effects; Recombinant Fusion Proteins - metabolism; Spodoptera - cytology; Spodoptera - virology; Strontium - metabolism; Thapsigargin - pharmacology; Transient Receptor Potential Channels
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/0264-6021:3410041

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.