Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca2+

Abstract

Whole-cell current recordings were used to examine the involvement of intracellular Ca2+ in the modulation of recombinant transient-receptor-potential like (TRPL) channels of Drosophila photoreceptor cells. TRPL was stably transfected in Chinese hamster ovary (CHO) cells and the expression of a calmodulin-binding protein with a molecular mass that corresponded to TRPL was demonstrated using calmodulin overlays. In cells expressing TRPL, ionic currents that were prominently outwardly rectifying were detected prior to activation of intracellular signalling pathways. The outwardly rectifying currents reversed close to 0 mV and did not occur after removal of permeant cations from the intracellular space. This suggests that TRPL forms non-selective cationic channels that appear to be constitutively active in mammalian cell lines. The TRPL channel currents were enhanced by manoeuvres that activate the phospholipase C (PLC) signalling pathway. These included activation of membrane receptors by thrombin, activation of G proteins by cell dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and release of Ca2+ from intracellular stores by dialysis with inositol 1,4,5-trisphosphate (IP3). After complete depletion of Ca2+ stores, IP3 had no effect on TRPL currents, suggesting that IP3 does not activate recombinant TRPL channels directly. However, thapsigargin, which induces a rise of cytosolic Ca2+, increased TRPL channel currents. Cell dialysis with solutions containing various concentrations of Ca2+ enhanced TRPL currents in a dose-dependent manner (EC50=450 nM Ca2+). Conversely, chelation of cytosolic Ca2+ abolished TRPL channel currents. The present results indicate that the activity of recombinant TRPL channels expressed in mammalian cell lines is up-regulated by a rise of cytosolic Ca2+.

INSIGHTS ON TRP CHANNELS FROM IN VIVO STUDIES IN DROSOPHILA

“…In addition, they showed that 10 mM BAPTA blocked the constitutive opening of the TRPL channels in COS cells. An additional study found, in the same experimental system, that a rise in cellular Ca 2+ is required for activation of TRPL channels (84).…”

“…TRPL channels were expressed in several expression systems (60,78,(80)(81)(82)(83)(84). The main functional characteristics of the expressed channels are, in general, similar to those of the native Drosophila TRPL channels.…”

Expression of Drosophila Ca2+ permeable transient receptor potential-like channel protein in a prostate cancer cell line decreases cell survival

“…One way to distinguish between Na + and Ca 2+ as mediators of the effect of TRPL on apoptosis would be to test the effect of expression of a Ca 2+ -selective member of the TRP family such as TRPV5, TRPV6 or possibly TRPC5 or TRPV1. 14 However, while heterologously expressed TRPL is often constitutively active, [15][16][17] it is less clear whether TRPV1, TRPV5, TRPV6, or TRPC5 are constitutively active.…”

“…This constitutive activity is thought to be due to differences between the environment of the Drosophila photoreceptor cell and the host animal cell, which lead to altered regulation of the opening of the TRPL channels. [15][16][17] The constitutive activity of TRPL causes a sustained increase in [Ca 2+ ] cyt , which can kill the transfected cells. [15][16][17] The aim of the present study was to express the Drosophila TRPL nonselective cation channel in a prostate cancer cell line and to test whether this leads to apoptosis and decreased cell survival.…”

“…[15][16][17] The constitutive activity of TRPL causes a sustained increase in [Ca 2+ ] cyt , which can kill the transfected cells. [15][16][17] The aim of the present study was to express the Drosophila TRPL nonselective cation channel in a prostate cancer cell line and to test whether this leads to apoptosis and decreased cell survival. The experiments have been conducted using LNCaP cells, and a replication-deficient adenovirus to deliver TRPL cDNA into LNCaP cells.…”