Stimulation of Drosophila TrpL by capacitative Ca super(2+) entry

• Published in: Biochemical journal Vol. 341; no. 1; pp. 41 - 49
• Main Authors: Estacion, M, Sinkins, W G, Schilling, W P
• Format: Journal Article
• Language: English
• Published: 01.07.1999
• Subjects: calcium; Drosophila; phospholipase
• ISSN: 0264-6021

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 Ca super(2+) 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 Ca super(2+) > Sr super(2+) > Ba super(2+). The increase in TrpL channel activity was blocked by concentrations of La super(3+) that completely inhibited endogenous capacitative Ca super(2+) 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 Ca super(2+) concentration ([Ca super(2+)] sub(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 Ca super(2+) stores or by a rise in [Ca super(2+)] sub(i). This gain-of-function mutant, designated TrpC1-TrpL, exhibited trans-stimulation by Ca super(2+) 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.