RECEPTOR-MEDIATED ACTIVATION OF RECOMBINANT TRPL EXPRESSED IN SF9 INSECT CELLS

The Drosophila proteins, Trp and Trpl, are suggested to be cation channels responsible for depolarization of the receptor potential associated with stimulation of insect photoreceptor cells by light. Consistent with this hypothesis, we recently showed that recombinant Trpl forms Ca2+- and Ba2+-permeable non-selective cation channels when expressed in Sf9 cells using the baculovirus expression vector. As Trpl may be activated in the photoreceptor cell after stimulation of phospholipase C, we hypothesized that a similar regulation of recombinant Trpl may be observed in the Sf9 cell after activation of heterologous membrane receptors linked to Ca2+-signal-transduction pathways. To test this hypothesis, Ca2+ signalling was examined in Fura-2-loaded Sf9 cells infected with baculovirus containing cDNA for the M5 muscarinic receptor alone (M5 cells) or in cells co-infected with both M5 and Trpl-containing baculoviruses (MS-Trpl cells). Addition of carbachol (100 mu M) to M5 cells produced an increase in cytosolic free Ca2+ concentration ([Ca2+](i)) (mean +/- S.D.; n = 17) from 101+/-20 to 762+/-178 nM which declined to a sustained elevated level of 384+/-102 nM after 3 min. The sustained component was eliminated by removal of extracellular Ca2+ or by addition of La3+ or Gd3+ (10 mu M). In MS-Trpl cells, basal [Ca2+](i) increased as a function of time after infection. To evaluate the contribution of Ca2+ influx to the overall profile observed, Ba2+ a Ca2+ surrogate that is not a substrate for the Ca2+ pump, was used. The increase in basal [Ca2+](i) seen in M5-Trpl cells was associated with an increase in basal Ba2+ influx. Addition of carbachol to MS-Trpl cells at 30-36 h after infection produced a large increase in [Ca2+](i) to a sustained value of 677+/-143 nM. This change in [Ca2+](i) was (1) blocked by atropine, (2) attenuated in the absence of extracellular Ca2+, and (3) relatively insensitive to La3+, but blocked by Gd3+ in the 0.1-1 mM range. In the presence of 10 mu M Gd3+ to block the endogenous-receptor-mediated Ca2+-influx pathway, carbachol produced a dramatic increase in Ba2+ influx in M5-Trpl cells. In sharp contrast, neither Ca2+ nor Ba2+ influx through Trpl was affected by thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+-ATPase pump. These results suggest that recombinant Trpl (1) is constitutively active under basal non-stimulated conditions, (2) can be further activated by a receptor-dependent mechanism and (3) is not affected by depletion of the internal Ca2+ stores by thapsigargin.