Thapsigargin and receptor-mediated activation of Drosophila TRPL channels stably expressed in a Drosophila S2 cell line

Abstract

Listen

Translate article

The Drosophila melanogaster genes, transient receptor potential ( trp) and transient receptor potential-like ( trpl) encode putative plasma membrane cation channels TRP and TRPL, respectively. We have stably co-expressed Drosophila TRPL with a Drosophila muscarinic acetylcholine receptor (DM1) in a Drosophila cell line (S2 cells). Basal Ca 2+ levels measured 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. Activation of DM1 receptor in S2-DM1-TRPL cells by 100 μM carbamylcholine induced Ca 2+ release from an intracellular Ca 2+ pool followed by a Gd 3+-insensitive Ca 2+ influx. Pretreatment of S2-DM 1-TRPL cells with 10 μM atropine abolished Gd 3+-insensitive Ca 2+ influx triggered by carbamylcholine, but the response was not blocked by prior incubation with pertussis toxin. TRPL channels could also be reliably activated by bath application of 1 μM thapsigargin for 10 min or 100 nM thapsigargin for 60 min in Ca 2+-free solution. In some cells, TRPL channels activated 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 requirement for Ca 2+ release.