THAPSIGARGIN AND RECEPTOR-MEDIATED ACTIVATION OF DROSOPHILA TRPL CHANNELS STABLY EXPRESSED IN A DROSOPHILA S2 CELL-LINE

The Drosophila melanogaster genes, transient receptor potential (trp) and transient receptor potential-like (trpl) encode putative plasma me mbrane cation channels TRP and TRPL, respectively. We have stably co-e xpressed Drosophila TRPL with a Drosophila muscarinic acetylcholine re ceptor (DMI) in a Drosophila cell line (S2 cells). Basal Ca2+ levels m easured using Fura-2/AM in unstimulated S2-DM1-TRPL cells were low and indistinguishable from untransfected cells, indicating that the TRPL channels were not constitutively active in this expression system. Act ivation of DM1 receptor in S2-DM1-TRPL cells by 100 mu M carbamylcholi ne induced Ca2+ release from an intracellular Ca2+ pool followed by a Gd3+-insensitive Ca2+ influx. Pretreatment of S2-DM1-TRPL cells with 1 0 mu M atropine abolished Gd3+-insensitive Ca2+ influx triggered by ca rbamylcholine, but the response was not blocked by prior incubation wi th pertussis toxin. TRPL channels could also be reliably activated by bath application of 1 mu M thapsigargin for 10 min or 100 nM thapsigar gin for 60 min in Ca2+-free solution. In some cells, TRPL channels act ivated by thapsigargin could further be activated by carbamylcholine. The findings suggest that, when stably expressed in the S2 cell line, TRPL may be regulated by two distinct mechanisms: (i) store depletion; and (ii) stimulation of DM1 receptor via pertussis-toxin insensitive G-protein (or the subsequent activation of PLC), but without further r equirement for Ca2+ release.