Activation of recombinant trp by thapsigargin in Sf9 insect cells

Abstract

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin, whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store. Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for capacitative Ca2+ entry.

Depletion of intracellular calcium stores activates an outward potassium current in mast and RBL‐1 cells that is correlated with CRAC channel activation

“…Comparing the endogenous insect SOC channels with expressed trp channels in Sf9 cells, Vaca et al [29] showed that this SOC channel and the trp channel were clearly different regarding the permeation properties of Ca 2+, Ba 2+, Mg 2+, and Na +. Different permeation properties were also reported for store operated channels in a human epidermal cancer cell line (A431) [20], in vascular endothelium [28], in HT29 colonic carcinoma cells [16], and in MDCK cells [4]. Selectivity and permeation properties have so far been proven the best tools to distinguish different members of the SOC family.…”

Molecular cloning of a widely expressed human homologue for the Drosophila trp gene

“…Indeed, although the two Drosophila trp proteins are most related among the four, the functional roles of trp and trpl in Drosophila phototransduction are thought to be different [6,20]. When expressed in Sf9 cells, the Drosophila trp was activated by store depletion whereas the Drosophila trpl was not and channels formed by these two proteins also had different ion selectivities [10,11]. Part of these differences may result from the fact that at the C-terminus of the Drosophila trp, there is a 9-fold repeat of a very hydrophilic eight amino acid sequence (DKDKKPGD), which does not occur in trpl and the other two trp homologues which have shorter C-terminal tails.…”

“…4a) trp or trpl, the expressed protein was found to form Ca 2÷ permeant channels that functions in the IPJCa 2+ signaling pathway [10][11][12][13]. Therefore, it is reasonable to assume that the human trp may also play a role in the IPJCa 2+ signaling pathway.…”

“…This was demonstrated recently by expression of the cDNAs for trp and trpl in insect Sf9 cells using the baculovirus system. It was fount that trp forms a Ca 2÷ permeable cation channel which is activated by store depletion with thapsigargin [10] whereas trpl forms a Ca -'+ permeable non-selective cation channel which is not only constitutively active when over-expressed in SO cells but also can be up-regulated by receptor stimulation [11][12][13]. However, it was also noticed that neither trp nor trpl mimicked the endogenous Ca 2+ influx channel of the Sf9 cells, suggesting the existence of at least one other channel in insects involved in Ca 2+ entry [10].…”

“…It was fount that trp forms a Ca 2÷ permeable cation channel which is activated by store depletion with thapsigargin [10] whereas trpl forms a Ca -'+ permeable non-selective cation channel which is not only constitutively active when over-expressed in SO cells but also can be up-regulated by receptor stimulation [11][12][13]. However, it was also noticed that neither trp nor trpl mimicked the endogenous Ca 2+ influx channel of the Sf9 cells, suggesting the existence of at least one other channel in insects involved in Ca 2+ entry [10]. Because of the similarities between the IP3/Ca 2+ pathway in mammalian tissues and the phototransduction process in insects, it is expected that homologous genes of trp and/or trpl should exist in mammalian tissues, which may form one or several of the cation channels that participate in Ca 2+ entry following receptor stimulation.…”